WO2018136831A1 - Pseudoknot compositions and methods for inhibiting factor d - Google Patents

Pseudoknot compositions and methods for inhibiting factor d Download PDF

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Publication number
WO2018136831A1
WO2018136831A1 PCT/US2018/014579 US2018014579W WO2018136831A1 WO 2018136831 A1 WO2018136831 A1 WO 2018136831A1 US 2018014579 W US2018014579 W US 2018014579W WO 2018136831 A1 WO2018136831 A1 WO 2018136831A1
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rna
seq
aptamer
modifications
ome modified
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PCT/US2018/014579
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French (fr)
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Carl ERICKSON
Christopher P. Rusconi
Kevin G. Mclure
Matthew Levy
Arijit BHOWMICK
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Vitrisa Therapeutics, Inc.
Albert Einstein College Of Medicine, Inc.
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Publication of WO2018136831A1 publication Critical patent/WO2018136831A1/en

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag

Definitions

  • Visual impairment is a national and global health concern that has a negative impact on physical and mental health.
  • the number of people with visual impairment and blindness is increasing due to an overall aging population.
  • Visual impairment and blindness can be caused by any one of a large number of eye diseases and disorders affecting people of all ages.
  • age-related macular degeneration AMD is an eye disorder that is currently the leading cause of vision loss in people fifty years of age or older in industrialized countries. It is estimated that by 2020, the number of people with AMD could exceed 196 million and by 2040, that number is expected to rise to 288 million.
  • AMD is a degenerative eye disease that progresses from early stages to advanced stages of the disease. Risk factors for the disease include aging, lifestyle factors such as smoking, and genetics.
  • the clearest indicator of progression to AMD is the appearance of drusen, yellow-white deposits under the retina, and it is an important component of both forms of AMD: exudative ("wet”) and non-exudative (“dry”).
  • Wet AMD causes vision loss due to abnormal blood vessel growth in the choriocapillaris through Batch's membrane.
  • geographic atrophy The most advanced form of dry AMD, known as geographic atrophy, is generally more gradual and occurs when light-sensitive cells in the macula atrophy, blurring and eliminating vision in the affected eye. While there are currently some promising treatments for wet AMD, no FDA-approved treatment exists for dry AMD or geographic atrophy.
  • STGD childhood-onset Stargardt Disease
  • Stargardt 1 a genetic, rare juvenile macular dystrophy generally associated with loss of central vision in the first two decades of life.
  • STGD has a prevalence of approximately 1/20,000 affecting approximately 30,000 people in the US.
  • STGD affects many ages, with the childhood- onset population at highest risk and most need.
  • Patients with childhood-onset STGD tend to develop early severe visual acuity loss, significantly compromised retinal function, and rapid retinal pigment epithelial (RPE) cell atrophy with accompanying loss of retinal function.
  • the median ages of onset and the median age at baseline examination are 8.5 (range, 3-16) and 12 years (range, 7-16), respectively.
  • STGD is an autosomal recessive genetic disease or complex heterozygous disease, caused by mutations in the ABCA4 gene.
  • the ABCA4 gene encodes the photoreceptor protein ABCA4 Transporter, which is responsible for removal of bisretinoid fluorophores, which can include N-retinylidene-N- retinyethanolamine (A2E), all-trans-retinal and related photo-oxidation products of vitamin A aldehyde which together constitute lipofuscin, from photoreceptor cells.
  • ABCA4 Transporter which is responsible for removal of bisretinoid fluorophores, which can include N-retinylidene-N- retinyethanolamine (A2E), all-trans-retinal and related photo-oxidation products of vitamin A aldehyde which together constitute lipofuscin, from photoreceptor cells.
  • Accumulation of all- trans-retinal in photoreceptor cells is believed to damage RPE cells via oxidative stress, and trigger or promote complement-mediated damage to RPE cells, leading to retinal atrophy.
  • a related disease termed Stargardt-like macular dystrophy, also known as STGD3 is inherited in a dominant autosomal manner and is due to mutations in the ELOVL4 gene.
  • ELOVL4 encodes the ELOVL4 protein, ELOVL fatty acid elongase 4. Mutations in ELOVL4 protein associated with STGD lead to mis-folding and accumulation of ELOVL4 protein aggregates in retinal cells, which impact retinal cell function, eventually leading to cell death and retinal atrophy. No treatments exist for STGD or Stargardt-like disease.
  • an aptamer having a nucleic acid sequence, wherein the aptamer forms a pseudoknot secondary structure which specifically binds to complement Factor D (fD).
  • the aptamer of the present invention forms a pseudoknot secondary structure which binds to fD, preferably with high affinity and/or high specificity.
  • the aptamer has a nucleic acid sequence that does not comprise any one of SEQ ID NOs:475-534.
  • an aptamer of any of the preceding specifically binds to an exosite of fD.
  • an aptamer of any of the preceding has a nucleic acid sequence containing 30-90 nucleotides.
  • an aptamer of any of the preceding has an H-H type pseudoknot secondary structure. In some cases, an aptamer of any of the preceding has a pseudoknot structure comprising up to four loops and up to three base-paired stems. In some cases, an aptamer of any of the preceding has up to three base-paired stems, wherein each base-paired stem of the up to three base-paired stems comprises up to 15 base pairs. In some cases, an aptamer of any of the preceding has up to four loops, wherein each loop of the up to four loops comprises up to 12 nucleotides.
  • an aptamer of any of the preceding has a pseudoknot secondary structure comprising, in a 5' to 3' direction, a first base-paired stem, a first loop, a second base-paired stem, a second loop, a third loop joining the first base-paired stem with a third base-paired stem, the third base-paired stem, and a fourth loop.
  • the first base-paired stem has from 2 to 12 base pairs.
  • the first base-paired stem has from 3 to 9 base pairs.
  • the first base-paired stem has 5 base pairs.
  • the first base-paired stem has 7 base pairs.
  • the first base-paired stem has one or more mismatched base pairs.
  • the second base-paired stem has from 2 to 9 base pairs. In some cases, the second base-paired stem has 6 or 7 base pairs. In some cases, the third base-paired stem has from 2 to 6 base pairs. In some cases, the third base-paired stem has 3 or 4 base pairs. In some cases, the third base-paired stem comprises a nucleic acid sequence of 5 '-NMHG-3', where N is any nucleotide; M is A or C; and H is A, C, or U. In some cases, the first loop has from 1 to 5 nucleotides. In some cases, the first loop has from 2 to 5 nucleotides. In some cases, the first loop has 2 nucleotides.
  • the first loop comprises, in a 5' to 3 ' direction, GU. In some cases, the first loop comprises, in a 5' to 3' direction, GG. In some cases, the second loop has from 2 to 9 nucleotides. In some cases, the second loop has from 4 to 6 nucleotides. In some cases, the second loop comprises, in a 5' to 3 ' direction, AGUC. In some cases, the third loop has from 2 to 12 nucleotides. In some cases, the third loop has from 3 to 14 nucleotides. In some cases, the third loop comprises one or more non-nucleotidyl spacers. In some cases, the fourth loop has 0, 1, or 2 nucleotides. In some cases, the fourth loop has a single nucleotide. In some cases, the single nucleotide is G or U.
  • an aptamer of any of the preceding is an RNA aptamer or a modified RNA aptamer.
  • an aptamer of any of the preceding is a DNA aptamer or a modified DNA aptamer.
  • an aptamer of any of the preceding comprises at least one modified nucleotide.
  • an aptamer of any of the preceding comprises a nuclease- stabilized nucleic acid backbone.
  • an aptamer of any of the preceding specifically binds to fD with a K d of less than about 50nM.
  • an aptamer of any of the preceding inhibits fD in an alternative complement dependent hemolysis assay with an IC 50 of less than about 50nM.
  • an aptamer of any of the preceding is conjugated to a polyethylene glycol (PEG) molecule.
  • PEG polyethylene glycol
  • the PEG molecule has a molecular weight of 80 kDa or less.
  • an aptamer having a nucleic acid sequence comprising any one of SEQ ID NOs: 1-3, 10-474, and 543-556 or a nucleic acid sequence as described in Table 2, or having at least 80% sequence identity to any one of SEQ ID NOs: 1-3, 10-474, and 543- 556 or a nucleic acid sequence as described in Table 2.
  • an aptamer according to any of the preceding is provided for use in a method of therapy; for use in a method of treatment that benefits from modulating fD; for use in a method of treatment that benefits from inhibiting a function associated with fD; or for use in a method for the treatment of ocular diseases.
  • an aptamer according to any of the preceding is provided and a pharmaceutically acceptable carrier, excipient, or diluent
  • a method for modulating complement Factor D (fD) in a biological system comprising: administering to the biological system, an aptamer according to any of the preceding, thereby modulating fD in the biological system.
  • the modulating comprises inhibiting a function associated with fD.
  • FIG. 1 depicts aspects of the alternative complement pathway.
  • FIG. 2A and FIG. 2B depict modeling of the intravitreal (IVT) inhibition of Factor D by an anti-Factor D aptamer at various IVT concentrations over time.
  • FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D depict non-limiting examples of small molecule inhibitors of fD.
  • FIG. 4A, FIG. 4B, and FIG. 4C depict predicted secondary structure for a family of pseudoknot fD aptamers directed to the fD exosite according to an embodiment of the disclosure.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, and FIG. 5G depict predicted secondary structures of various fD aptamers according to an embodiment of the disclosure.
  • FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, and FIG. 6G depict predicted secondary structures of various fD aptamers according to an embodiment of the disclosure.
  • FIG. 7 depicts the amino acid sequence of human complement Factor D, chymotrypsin numbering scheme, and fD numbering scheme.
  • FIG. 8A, FIG. 8B, and FIG. 8C depict a non-limiting example of an aptamer library sequence that may be utilized to generate anti-Factor D aptamers according to an embodiment of the disclosure.
  • FIG. 9 depicts a non-limiting example of a method for selecting anti-Factor D aptamers according to an embodiment of the disclosure.
  • FIG. 10 depicts measurement of K d values of enriched libraries of anti-Factor D aptamers according to an embodiment of the disclosure.
  • FIG. 11 depicts binding analysis of anti-Factor D aptamers by flow cytometry according to an embodiment of the disclosure.
  • FIG. 12 depicts measurement of K d values of anti -Factor D aptamers according to an embodiment of the disclosure.
  • FIG. 13 depicts a competition binding assay according to an embodiment of the disclosure.
  • FIG. 14 depicts examples of data obtained from an alternative complement dependent hemolysis assay according to an embodiment of the disclosure.
  • FIG. 15 depicts examples of data obtained from a fD esterase activity assay according to an embodiment of the disclosure.
  • FIG. 16 depicts examples of data obtained from a competition binding assay according to an embodiment of the disclosure.
  • FIG. 17 depicts examples of data obtained from a direct binding assay according to an embodiment of the disclosure.
  • FIG. 18A and FIG. 18B depict examples of data obtained from an alternative complement dependent hemolysis assay according to an embodiment of the disclosure.
  • FIG. 19 depicts a non-limiting example of an aptamer minimization experiment in accordance with embodiments of the disclosure.
  • FIG. 20 depicts non-limiting examples of fD aptamers in accordance with embodiments of the disclosure.
  • FIG. 21 depicts a non-limiting example of results obtained from SPR complex assembly in accordance with embodiments of the disclosure.
  • FIG. 22 depicts a non-limiting example of dose-dependent inhibition of
  • the disclosure herein provides methods and compositions for the treatment of ocular diseases or disorders.
  • the methods and compositions include the use of an anti-fD pseudoknot aptamer for, e.g., the treatment of ocular diseases or disorders
  • the ocular disease is macular degeneration.
  • macular degeneration is age-related macular degeneration.
  • age-related macular degeneration is dry age-related macular degeneration.
  • dry age-related macular degeneration is advanced dry age- related macular degeneration (i.e., geographic atrophy).
  • the ocular disease is wet age-related macular degeneration.
  • the ocular disease is Stargardt disease.
  • the methods and compositions involve the inhibition of the alternative complement pathway. In some cases, the methods and compositions involve the inhibition of a function associated with Factor D (fD). In some cases, the methods and compositions involve the inhibition of a function associated with fD for the treatment of ocular diseases. In some cases, the methods and compositions involve the inhibition of a function associated with fD for the treatment of dry age-related macular degeneration or geographic atrophy. In some cases, the methods and compositions involve the inhibition of a function associated with fD for the treatment of wet age-related macular degeneration. In some cases, the methods and
  • compositions involve the inhibition of a function associated with fD for the treatment of Stargardt disease.
  • compositions may include oligonucleotides that selectively bind to and modulate an activity associated with fD.
  • the oligonucleotide may include oligonucleotides that selectively bind to and modulate an activity associated with fD.
  • compositions of the disclosure inhibit a function associated with fD.
  • the oligonucleotide compositions may bind directly to an exosite of fD or to a region of fD that includes the exosite site.
  • the oligonucleotides are aptamers, such as RNA aptamers or DNA aptamers.
  • the aptamers of the disclosure may have secondary structures.
  • the secondary structures may include a pseudoknot secondary structure which generally includes one or more loops and one or more stems.
  • aptamers having pseudoknot structures for modulating fD are described herein.
  • sequence identity refers to an exact nucleotide-to-nucleotide or amino acid- to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence.
  • Two or more sequences can be compared by determining their "percent identity .”
  • the percent identity of two sequences, whether nucleic acid or amino acid sequences is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health.
  • the BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol.
  • the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program.
  • the program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.
  • aptamer refers to an oligonucleotide and/or nucleic acid analogues that can bind to a specific target molecule. Aptamers can include RNA, DNA, modified RNA, modified DNA, any nucleic acid analogue, and/or combinations thereof.
  • Aptamers can be single-stranded oligonucleotides. In some cases, aptamers may comprise more than one nucleic acid strand (e.g., two or more nucleic acid strands). Without wishing to be bound by theory, aptamers are thought to bind to a three-dimensional structure of a target molecule. Aptamers may be monomelic (composed of a single unit) or multimeric (composed of multiple units). Multimeric aptamers can be homomeric (composed of multiple identical units) or heteromeric (composed of multiple non-identical units). Aptamers herein may be described by their primary structures which may refer to the linear nucleotide sequence of the aptamer.
  • aptamers herein will generally be described from the 5' end to the 3' end, unless otherwise stated. Additionally or alternatively, aptamers herein may be described by their secondary structures which may refer to the combination of single- stranded regions and base pairing interactions within the aptamer.
  • the term "pseudoknot” as used herein may refer to the secondary structure of an aptamer of the disclosure.
  • An aptamer having a pseudoknot secondary structure may have at least two stem-loop structures in which the stems are non-nested relative to each other.
  • An aptamer may have a secondary structure having at least two complementary regions of the same nucleic acid strand that base-pair to form a double helix (referred to herein as a "stem"). Generally, these complementary regions are complementary when read in the opposite direction.
  • the term “stem” as used herein may refer to either of the complementary nucleotide regions individually or may encompass a base-paired region containing both complementary regions, or a portion thereof.
  • the term “stem” may refer to the 5' side of the stem, that is, the stem sequence that is closer to the 5' end of the aptamer; additionally or alternatively, the term “stem” may refer to the 3 ' side of the stem, that is, the stem sequence that is closer to the 3' end of the aptamer. In some cases, the term “stem” may refer to the 5' side of the stem and the 3' side of the stem, collectively.
  • the term “base-paired stem” is generally used herein to refer to both complementary stem regions collectively. A base-paired stem may be perfectly complementary meaning that 100% of its base pairs are Watson-Crick base pairs.
  • a base-paired stem may also be “partially complementary.”
  • the term “partially complementary stem” refers to a base-paired stem that is not entirely made up of Watson-Crick base pairs but does contain base pairs (either Watson-Crick base pairs or G-U/U-G wobble base pairs) at each terminus.
  • a partially complementary stem contains both Watson-Crick base-pairs and G-U/U- G wobble base pairs.
  • a partially complementary stem is exclusively made up of G-U/U-G wobble base pairs.
  • a partially complementary stem may contain mis-matched base pairs and/or unpaired bases in the region between the base pairs at each terminus of the stem; but in such cases, the mis-matched base pairs and/or unpaired bases make up at most 50% of the positions between the base pairs at each terminus of the stem.
  • a stem as described herein may be referred to by the position, in a 5' to 3' direction on the aptamer, of the 5' side of the stem (i.e., the stem sequence closer to the 5' terminus of the aptamer), relative to the 5' side of additional stems present on the aptamer.
  • stem 1 may refer to the stem sequence that is closest to the 5' terminus of the aptamer, its complementary stem sequence, or both stem sequences collectively.
  • stem 2 may refer to the next stem sequence that is positioned 3 ' relative to SI, its complementary stem sequence, or both stem sequences collectively
  • Each additional stem may be referred to by its position, in a 5' to 3' direction, on the aptamer, as described above.
  • S3 may be positioned 3' relative to S2 on the aptamer
  • S4 may be positioned 3'relative to S3 on the aptamer
  • first stem is used to refer to a stem in the aptamer, irrespective of its location.
  • a first stem may be SI, S2, S3, S4 or any other stem in the aptamer.
  • a stem may be adjacent to an unpaired region.
  • An unpaired region may be present at a terminus of the aptamer or at an internal region of the aptamer.
  • loop generally refers to an internal unpaired region of an aptamer.
  • the term “loop” may refer to any unpaired region of an aptamer that is flanked on both the 5' end and the 3 ' end by a stem region.
  • a loop sequence may be adjacent to a single base-paired stem, such that the loop and stem structure together resemble a hairpin.
  • the primary sequence of the aptamer contains a first stem sequence adjacent to the 5' end of the loop sequence and a second stem sequence adjacent to the 3 ' end of the loop sequence; and the first and second stem sequences are complementary to each other.
  • each terminus of a loop is adjacent to first and second stem sequences that are not complementary.
  • a loop as described herein may be referred to by its position, in a 5' to 3' direction, on the aptamer.
  • loop 1 may refer to a loop sequence that is positioned most 5' on the aptamer.
  • loop 2 may refer to a loop sequence that is positioned 3 ' relative to LI
  • loop 3 may refer to a loop sequence that is positioned 3 ' relative to L2.
  • Each additional loop may be referred to by its position, in a 5' to 3 ' direction, on the aptamer, as described above.
  • L4 may be positioned 3 ' relative to L3 on the aptamer
  • L5 may be positioned 3'relative to L4 on the aptamer
  • first loop is used to refer to a loop in the aptamer, irrespective of its location.
  • a first loop may be LI, L2, L3, L4 or any other loop in the aptamer.
  • an aptamer when an aptamer includes more than one stem and/or more than one loop, the stems and loops are numbered consecutively in ascending order from the 5' end to the 3 ' end of the primary nucleotide sequence.
  • exosite may refer to a protein domain or region of a protein that is capable of binding to another protein.
  • the exosite may also be referred to herein as a "secondary binding site", for example, a binding site that is remote from or separate from a primary binding site (e.g., an active site).
  • primary and secondary binding sites may overlap. Binding of a molecule to an exosite may cause a physical change in the protein (e.g., a conformational change).
  • the activity of a protein may be dependent on occupation of the exosite.
  • the exosite may be distinct from an allosteric site.
  • the oligonucleotide compositions of the disclosure may bind to the exosite of fD or to part of the exosite of fD, or may bind to a region of fD that includes the exosite. In some cases, the oligonucleotide compositions of the disclosure may bind to the exosite of fD or to part of the exosite of fD, or may bind to a region of fD that includes the exosite. In some cases, the oligonucleotide compositions of the disclosure may block or occlude the exosite such that the natural substrate of fD is prevented from accessing the exosite. In such cases, the
  • oligonucleotide may block access to the exosite without directly binding the exosite (e.g., may bind to a region of fD other than the exosite in such a way that the exosite is sterically occluded).
  • catalytic cleft refers to a domain of an enzyme in which a substrate molecule binds to and undergoes a chemical reaction.
  • the active site may include amino acid residues that form temporary bonds with the substrate (e.g., a binding site) and amino acid residues that catalyze a reaction of that substrate (e.g., catalytic site).
  • the active site may be a groove or pocket (e.g., a cleft) of the enzyme which can be located in a deep tunnel within the enzyme or between the interfaces of multimeric enzymes.
  • epitope refers to the part of an antigen (e.g., a substance that stimulates an immune system to generate an antibody against) that is specifically recognized by the antibody.
  • the antigen is a protein or peptide and the epitope is a specific region of the protein or peptide that is recognized and bound by an antibody.
  • the aptamers described herein bind to a region of fD that is an epitope for an anti-fD antibody or antibody fragment thereof, wherein the anti-fD antibody inhibits a function associated with fD.
  • the aptamer binding region of fD overlaps with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the epitope for an anti-fD antibody or the binding site of another fD-inhibiting molecule.
  • a polypeptide can be any protein, peptide, protein fragment or component thereof.
  • a polypeptide can be a protein naturally occurring in nature or a protein that is ordinarily not found in nature.
  • a polypeptide can consist largely of the standard twenty protein-building amino acids or it can be modified to incorporate non-standard amino acids.
  • a polypeptide can be modified, typically by the host cell, by e.g., adding any number of biochemical functional groups, including phosphorylation, acetylation, acylation, formylation, alkylation, methylation, lipid addition (e.g.
  • Polypeptides can undergo structural changes in the host cell such as the formation of disulfide bridges or proteolytic cleavage.
  • the peptides described herein may be therapeutic peptides utilized for e.g., the treatment of a disease.
  • subject and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • the complement system is a part of the innate immune system that enhances the ability of antibodies and phagocytic cells to clear pathogens from an organism. Although the system is not adaptable and does not change over the course of an individual's lifetime, it can be recruited and brought into action by the adaptive immune system.
  • the complement system consists of a number of small proteins found in the blood, in general synthesized by the liver, and normally circulating as inactive precursors (pro-proteins). When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end result of this complement activation or complement fixation cascade is massive amplification of the response and activation of the cell-killing membrane attack complex. Over 30 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane receptors.
  • the alternative complement pathway is a rapid, antibody-independent route for complement system activation and amplification.
  • the alternative pathway comprises several components: C3, Factor B (fB), and fD.
  • fB Factor B
  • Activation of the alternative pathway occurs when C3b, a proteolytic cleavage form of C3, is bound to an activating surface agent such as a bacterium.
  • fB is then bound to C3b, and cleaved by fD to yield the C3 convertase C3bBb.
  • Amplification of C3 convertase activity occurs as additional C3b is produced and deposited.
  • the amplification response is further aided by the binding of the positive regulator protein properdin (Factor P), which stabilizes the active convertase against degradation, extending its half-life from 1-2 minutes to 18 minutes.
  • the C3 convertase further assembles into a C5 convertase (C3b3bBb).
  • This complex subsequently cleaves complement component C5 into two components: the C5a polypeptide (9 kDa) and the C5b polypeptide (170 kDa).
  • the C5a polypeptide binds to a 7 transmembrane G- protein coupled receptor, which was originally associated with leukocytes and is now known to be expressed on a variety of tissues including hepatocytes and neurons.
  • the C5a molecule is the primary chemotactic component of the human complement system and can trigger a variety of biological responses including leukocyte chemotaxis, smooth muscle contraction, activation of intracellular signal transduction pathways, neutrophil-endothelial adhesion, cytokine and lipid mediator release and oxidant formation.
  • the alternative complement pathway is believed to play a role in the pathogenesis of a variety of ischemic, inflammatory and autoimmune diseases including age-related macular degeneration, geographic atrophy, Stargardt disease, systemic lupus erythematosus, rheumatoid arthritis, and asthma.
  • components of the alternative complement pathway may be important targets for the treatment of these diseases.
  • AMD Age-related macular degeneration
  • AMD is a chronic and progressive eye disease that is the leading cause of irreparable vision loss in the United States, Europe, and Japan.
  • AMD is characterized by the progressive deterioration of the central portion of the retina referred to as the macula.
  • the clearest indicator of progression to AMD is the appearance of drusen, yellow- white deposits under the retina, which are plaques of material that are derived from the metabolic waste products of retinal cells.
  • the appearance of drusen is an important component of both forms of AMD: exudative ("wet") and non-exudative ("dry”).
  • drusen The presence of numerous, intermediate-to-large drusen is associated with the greatest risk of progression to late-stage disease, characterized by geographic atrophy and/or neovascularization.
  • geographic atrophy The majority of patients with wet AMD experience severe vision loss in the affected eye within months to two years after diagnosis of the disease, although vision loss can occur within hours or days.
  • Dry AMD is more gradual and occurs when light-sensitive cells in the macula slowly atrophy, gradually blurring central vision in the affected eye. Vision loss is exacerbated by the formation and accumulation of drusen and sometimes the deterioration of the retina, although without abnormal blood vessel growth and bleeding.
  • Geographic atrophy is a term used to refer to advanced dry AMD.
  • FIG. 1 depicts a potential role for the alternative complement pathway in the pathogenesis of geographic atrophy.
  • multiple factors may lead to activation of the alternative complement pathway, including the appearance of drusen in the eye, immune dysfunction, and genetic differences that predispose individuals to complement activation.
  • amplification of C3 convertase activity may occur as additional C3b is produced and deposited.
  • C3 convertase activity may lead to inflammation and opsonization.
  • the C3 convertase may further assemble into a C5 convertase (C3b3bBb) which may lead to cell death through formation of the Membrane Attack Complex.
  • the oligonucleotide compositions of the disclosure may be used to treat AMD. In some cases, the oligonucleotide compositions of the disclosure may be used to treat wet AMD. In some cases, the oligonucleotide compositions of the disclosure may be used to treat geographic atrophy. In some cases, the oligonucleotide compositions of the disclosure may be used to stop, slow, or reverse the progression of wet AMD or geographic atrophy. In some cases, the oligonucleotide compositions of the disclosure may be used to treat symptoms associated with wet AMD or geographic atrophy.
  • STGD Stargardt Disease
  • ABCA4 Transporter which is responsible for removal of bisretinoid fluorophores, which can include N-retinylidene-N- retinyethanolamine (A2E), all-trans-retinal and related photo-oxidation products of vitamin A aldehyde which together constitute lipofuscin from photoreceptor cells. Accumulation of all- trans-retinal in photoreceptor cells is believed to damage RPE cells via oxidative stress, and trigger or promote complement-mediated damage to RPE cells, leading to retinal atrophy.
  • A2E N-retinylidene-N- retinyethanolamine
  • STGD is characterized by the progressive deterioration of the central portion of the retina referred to as the macula, generally beginning in the first two decades of life.
  • the clearest indicator of progression of STGD is the appearance of drusen, yellow-white deposits under the retina, which are plaques of material that are derived from the metabolic waste products of retinal cells, including all-trans-retinal and other vitamin A-related metabolites.
  • the onset of STGD is typically between the ages of 6-20 years, with early symptoms including difficulties in reading and adjusting to light. Patients with childhood-onset STGD tend to develop early severe visual acuity loss, significantly compromised retinal function, and rapid retinal pigment epithelial (RPE) cell atrophy with accompanying loss of retinal function.
  • RPE retinal pigment epithelial
  • the median ages of onset and the median age at baseline examination are 8.5 (range, 3-16) and 12 years (range, 7- 16), respectively. Patients with adult-onset disease are more likely to preserve visual acuity for a longer time and show slighter retinal dysfunction. Accumulation of all-trans-retinal in photoreceptor cells leads to inflammation, oxidative stress, deposition of auto-fluorescent lipofuscin pigments in the retinal pigment epithelium and retinal atrophy. Lipofuscin deposits (drusen), and oxidative products, trigger the alternative complement pathway into an
  • ELOVL4 encodes the ELOVL4 protein, ELOVL fatty acid elongase 4.
  • ELOVL4 protein associated with STGD leads to mis-folding and accumulation of ELOVL4 protein aggregates in retinal cells, which impact retinal cell function, eventually leading to cell death and retinal atrophy.
  • Complement pathway activation is also thought to play a role in Stargardt-like disease, and therefore inhibitors of complement, particularly complement factor D, are anticipated to stop or slow the progression of vision loss in individuals with Stargardt-like disease.
  • the oligonucleotide compositions of the disclosure may be used to treat Stargardt or Stargardt-like disease. In some cases, the oligonucleotide compositions of the disclosure may be used to stop, slow, or reverse the progression of Stargardt or Stargardt-like disease. In some cases, the oligonucleotide compositions of the disclosure may be used to treat symptoms associated with Stargardt or Stargardt-like disease.
  • the methods and compositions described herein utilize one or more aptamers for the treatment of an ocular disease. In some cases, the methods and compositions described herein utilize one or more aptamers for modulating an activity associated with fD.
  • aptamer refers to oligonucleotide molecules that bind to a target (e.g., a protein) with high affinity and specificity through non-Watson-Crick base pairing interactions.
  • a target e.g., a protein
  • the aptamers described herein are non-naturally occurring oligonucleotides (i.e., synthetically produced) that are isolated and used for the treatment of a disorder or a disease.
  • Aptamers can bind to essentially any target molecule including, without limitation, proteins, oligonucleotides, carbohydrates, lipids, small molecules, and even bacterial cells.
  • the aptamers described herein are oligonucleotides that bind to proteins of the alternative complement pathway. Whereas many naturally occurring oligonucleotides, such as mRNA, encode information in their linear base sequences, aptamers generally do not encode information in their linear base sequences. Further, aptamers can be distinguished from naturally occurring oligonucleotides in that binding of aptamers to target molecules is dependent upon secondary and tertiary structures of the aptamer.
  • Aptamers may be suitable as therapeutic agents and may be preferable to other therapeutic agents because: 1) aptamers may be fast and economical to produce because aptamers can be developed entirely by in vitro processes; 2) aptamers may have low toxicity and may lack an immunogenic response; 3) aptamers may have high specificity and affinity for their targets; 4) aptamers may have good solubility; 5) aptamers may have tunable pharmacokinetic properties; 6) aptamers may be amenable to site-specific conjugation of PEG and other carriers; and 7) aptamers may be stable at ambient temperatures.
  • Aptamers as described herein may include any number of modifications that can affect the function or affinity of the aptamer.
  • aptamers may be unmodified or they may contain modified nucleotides to improve stability, nuclease resistance or delivery characteristics.
  • modifications may include chemical substitutions at the sugar and/or phosphate and/or base positions, for example, at the 2' position of ribose, the 5 position of pyrimidines, and the 8 position of purines, various 2'-modified pyrimidines and modifications with 2'-amino (2'- H 2 ), 2'-fluoro (2'-F), and/or 2'-0-methyl (2'-OMe) substituents.
  • aptamers described herein comprise a 2'-OMe and/or a 2'F modification to increase in vivo stability.
  • the aptamers described herein contain modified nucleotides to improve the affinity and specificity of the aptamers for a specific epitope, exosite or active site. Examples of modified nucleotides include those modified with guanidine, indole, amine, phenol, hydroxymethyl, or boronic acid.
  • pyrimidine nucleotide triphosphate analogs or CE-phosphoramidites may be modified at the 5 position to generate, for example, 5- benzylaminocarbonyl-2'-deoxyuridine (BndU); 5-[N-(phenyl-3-propyl)carboxamide]-2'- deoxyuridine (PPdU); 5-(N-thiophenylmethylcarboxyamide)-2'-deoxyuridine (ThdU); 5-(N-4- fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU); 5-(N-(l -naphthylmethyl)carboxamide)- 2'-deoxyuridine (NapdU); 5 -(N-2-naphthylmethylcarboxyamide)-2'-deoxyuridine (2NapdU); 5- ( -l-naphthylethylcarboxyamide)-2'-deoxyuridine ( EdU); 5-(N-2- naph
  • Modifications of the aptamers contemplated in this disclosure include, without limitation, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and functionality to the nucleic acid aptamer bases or to the nucleic acid aptamer as a whole. Modifications to generate
  • oligonucleotide populations that are resistant to nucleases can also include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof.
  • modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate, phosphorodithioate, or alkyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases isocytidine and isoguanosine.
  • Modifications can also include 3' and 5' modifications such as capping, e.g., addition of a 3'-3'- dT cap to increase exonuclease resistance.
  • Aptamers of the disclosure may generally comprise nucleotides having ribose in the ⁇ -D- ribofuranose configuration. In some cases, 100% of the nucleotides present in the aptamer have ribose in the ⁇ -D-ribofuranose configuration. In some cases, at least 50% of the nucleotides present in the aptamer have ribose in the ⁇ -D-ribofuranose configuration.
  • At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%), at least 90%, or 100% of the nucleotides present in the aptamer have ribose in the ⁇ -D- ribofuranose configuration.
  • the length of the aptamer can be variable. In some cases, the length of the aptamer is less than 100 nucleotides. In some cases, the length of the aptamer is greater than 10 nucleotides. In some cases, the length of the aptamer is between 10 and 90 nucleotides.
  • the aptamer can be, without limitation, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, or about 90 nucleotides in length.
  • a polyethylene glycol (PEG) polymer chain is covalently bound to the aptamer, referred to herein as PEGylation.
  • PEGylation may increase the half-life and stability of the aptamer in physiological conditions.
  • the PEG polymer is covalently bound to the 5' end of the aptamer.
  • the PEG polymer is covalently bound to the 3' end of the aptamer.
  • the PEG polymer is covalently bound to specific site on a nucleobase within the aptamer, including the 5-position of a pyrimidine or 8-position of a purine.
  • an aptamer described herein may be conjugated to a PEG having the general formula, H-(0-CH 2 -CH 2 ) n -OH.
  • an aptamer described herein may be conjugated to a methoxy-PEG (mPEG) of the general formula, CH 3 0-(CH 2 -CH 2 -0)n-H.
  • the aptamer is conjugated to a linear chain PEG or mPEG.
  • the linear chain PEG or mPEG may have an average molecular weight of up to about 30 kD.
  • Multiple linear chain PEGs or mPEGs can be linked to a common reactive group to form multi-arm or branched PEGs or mPEGs.
  • more than one PEG or mPEG can be linked together through an amino acid linker (e.g., lysine) or another linker, such as glycerine.
  • the aptamer is conjugated to a branched PEG or branched mPEG.
  • Branched PEGs or mPEGs may be referred to by their total mass (e.g., two linked 20kD mPEGs have a total molecular weight of 40kD).
  • Branched PEGs or mPEGs may have more than two arms.
  • Multi-arm branched PEGs or mPEGs may be referred to by their total mass (e.g. four linked 10 kD mPEGs have a total molecular weight of 40 kD).
  • an aptamer of the present disclosure is conjugated to a PEG polymer having a total molecular weight from about 5 kD to about 200 kD, for example, about 5 kD, about 10 kD, about 20 kD, about 30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, about 80 kD, about 90 kD, about 100 kD, about 1 10 kD, about 120 kD, about 130 kD, about 140 kD, about 150 kD, about 160 kD, about 170 kD, about 180 kD, about 190 kD, or about 200 kD.
  • the aptamer is conjugated to a PEG having a total molecular weight from about
  • the reagent that may be used to generate PEGylated aptamers is a branched PEG N-Hydroxysuccinimide (mPEG- HS) having the general formula:
  • the branched PEGs can be linked through any appropriate reagent, such as an amino acid (e.g., lysine or glycine residues).
  • the reagent used to generate PEGylated aptamers is [N 2 - (monomethoxy 20K polyethylene glycol carbamoyl)-N 6 -(monomethoxy 20K polyethylene glycol carbamoyl)] -lysine N-hydroxysuccinimide having the formula:
  • PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed or single-arm linear PEG.
  • the reagent used to generate PEGylated aptamers has the formula:
  • X is N-hydroxysuccinimide and the PEG arms are of different molecular weights
  • a 40 kD PEG of this architecture may be composed of 2 arms of 5 kD and 4 arms of7.5 kD.
  • Such PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed PEG or a single-arm linear PEG.
  • the reagent that may be used to generate PEGylated aptamers is a non- branched mPEG-Succinimidyl Propionate (mPEG-SPA), having the general formula:
  • the reactive ester may be -0-CH 2 - CH 2 -CO 2 -NHS.
  • the reagent that may be used to generate PEGylated aptamers may include a branched PEG linked through glycerol, such as the SunbrightTM series from NOF Corporation, Japan.
  • Non-limitin examples of these reagents include:
  • the reagents may include a non-branched mPEG Succinimidyl alpha- methylbutanoate (mPEG-SMB) having the general formula:
  • the reactive ester may be 0-CH 2 .CH 2- CH(CH 3 )-C0 2 -NHS.
  • the PEG reagents may include nitrophenyl carbonate-linked PEGs, having the general formula:
  • the reagents used to generate PEGylated aptamers may include PEG with thiol-reactive groups that can be used with a thiol-modified linker.
  • PEG PEG with thiol-reactive groups that can be used with a thiol-modified linker.
  • One non-limiting example may include reagents having the following general structure: where mPEG is about 10 kD, about 20 kD or about 30 kD.
  • Another non-limiting example may include reagents having the following general structure:
  • Branched PEGs with thiol reactive groups that can be used with a thiol-modified linker, as described above, may include reagents in which the branched PEG has a total molecular weight of about 40 kD or about 60 kD (e.g., where each mPEG is about 20 kD or about 30 kD).
  • the reagents used to generated PEGylated aptamers may include reagents having the following structure:
  • the reaction is carried out between about pH 6 and about pH 10, or between about pH 7 and pH 9 or about pH 8.
  • the reagents used to generate PEGylated aptamers may include reagents having the following structure:
  • the reagents used to generate PEGylated aptamers may include reagents having the following structure:
  • the aptamer is associated with a single PEG molecule. In other cases, the aptamer is associated with two or more PEG molecules.
  • the aptamers described herein may be bound or conjugated to one or more molecules having desired biological properties. Any number of molecules can be bound or conjugated to aptamers, non-limiting examples including antibodies, peptides, proteins, carbohydrates, enzymes, polymers, drugs, small molecules, gold nanoparticles, radiolabels, fluorescent labels, dyes, haptens (e.g., biotin), other aptamers, or nucleic acids (e.g., siRNA). In some cases, aptamers may be conjugated to molecules that increase the stability, the solubility or the bioavailability of the aptamer. Non-limiting examples include polyethylene glycol (PEG) polymers, carbohydrates and fatty acids.
  • PEG polyethylene glycol
  • molecules that improve the transport or delivery of the aptamer may be used, such as cell penetration peptides.
  • cell penetration peptides can include peptides derived from Tat, penetratin, polyarginine peptide Argg sequence, Transportan, VP22 protein from Herpes Simplex Virus (HSV), antimicrobial peptides such as Buforin I and SynB, polyproline sweet arrow peptide molecules, Pep-1 and MPG.
  • the aptamer is conjugated to a lipophilic compound such as cholesterol, dialkyl glycerol, diacyl glycerol, or a non-immunogenic, high molecular weight compound or polymer such as polyethylene glycol (PEG) or other water-soluble pharmaceutically acceptable polymers including, but not limited to, polyaminoamines
  • a lipophilic compound such as cholesterol, dialkyl glycerol, diacyl glycerol, or a non-immunogenic, high molecular weight compound or polymer such as polyethylene glycol (PEG) or other water-soluble pharmaceutically acceptable polymers including, but not limited to, polyaminoamines
  • PAMAM polysaccharides such as dextran, or polyoxazolines (POZ).
  • the molecule to be conjugated can be covalently bonded or can be associated through non-covalent interactions with the aptamer of interest.
  • the molecule to be conjugated is covalently attached to the aptamer.
  • the covalent attachment may occur at a variety of positions on the aptamer, for example, to the exocyclic amino group on the base, the 5- position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5' or 3 ' terminus.
  • the covalent attachment is to the 5' or 3' hydroxyl group of the aptamer.
  • the aptamer can be attached to another molecule directly or with the use of a spacer or linker.
  • a lipophilic compound or a non-immunogenic, high molecular weight compound can be attached to the aptamer using a linker or a spacer.
  • linkers and attachment chemistries are known in the art.
  • 6- (trifluoroacetamido)hexanol (2-cyanoethyl-N,N-diisopropyl)phosphoramidite can be used to add a hexylamino linker to the 5' end of the synthesized aptamer.
  • linker phosphoramidites may include: TFA-amino C4 CED phosphoramidite having the structure:
  • 5'-amino modifier 5 having the structure:
  • DMT 4,4'-Dimethoxytrityl and 5' thiol-modifier C6 having the structure:
  • the 5'-thiol modified linker may be used, for example, with PEG-maleimides, PEG- vinylsulfone, PEG-iodoacetamide and PEG-orthopyridyl-disulfide.
  • the aptamer may be bonded to the 5'-thiol through a maleimide or vinyl sulfone functionality.
  • the aptamer formulated according to the present disclosure may also be modified by encapsulation within a liposome.
  • the aptamer formulated according to the present disclosure may also be modified by encapsulation within a micelle.
  • Liposomes and micelles may be comprised of any lipids, and in some cases the lipids may be phospholipids, including phosphatidylcholine.
  • the aptamers described herein are designed to inhibit a function associated with an alternative complement pathway enzyme.
  • an anti-fD aptamer is used to inhibit a function associated with fD (e.g., inhibit the catalytic activity of fD).
  • the aptamers described herein are designed to prevent an interaction or binding of two or more proteins of the alternative complement pathway.
  • an aptamer binds to fD and prevents binding of the complex C3bBb to fD.
  • the aptamers described herein may bind to a region of fD that is recognized by an antibody or antibody fragment thereof that inhibits a function associated with fD.
  • the antibody or antibody fragment thereof that inhibits a function associated with fD has an amino acid sequence of heavy chain variable region of: EVQLVQSGPELKKPGASVKVSCKASGYTFTNYGMNWVRQA
  • DKTHT (SEQ ID NO: 7) and an amino acid sequence of light chain variable region of:
  • FIG. 2 depicts modeling of the intravitreal (IVT) inhibition of Factor D by an anti -Factor
  • FIG. 2A and FIG. 2B demonstrate IVT inhibition of Factor D at various IVT concentrations of an anti-Factor D aptamer.
  • Effective inhibition of TVT Factor D inhibition was modeled using a standard 2 compartment model, assuming reported IVT half-lives for Fabs (7 days, LUCENTIS ® ) and PEGylated aptamers (10 days, MACUGEN ® ) and 1 : 1 inhibition of Factor D by each therapy at the relevant IVT concentrations (IC 50 data). As depicted in FIG.
  • FIG. 2A depicts the predicted IVT drug concentration (nM) of a PEGylated aptamer (dotted line) and an anti-Factor D antibody (solid line) over the number of weeks post IVT injection.
  • the aptamers described herein may bind to a region of fD that is recognized by a small molecule inhibitor that inhibits a function associated with fD, non-limiting examples including dichloroisocoumarin or any one of the compounds depicted in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D.
  • the aptamers described herein may bind to a region of fD that is recognized by a peptide inhibitor that inhibits a function associated with fD.
  • an aptamer of the disclosure comprises one of the following sequences described in Table 1 or Table 2.
  • GAGGCAUUAGUCAGCCGAAGUCUGGUGUCU NO: 43 CAGUUUGUUUACGGUCGGCUGCGU
  • GAGUCAUAAGUCCACCGAAGUCUUUUGGCU NO: 54 CUGUUUUCUCCAGGUCGGUGGCUG
  • GAGGCAUUAGGCCGGCGAAGUUUAAUGGCU NO: 207 CAGGAAUCCUAUGUUCGGGGGCAU
  • GAGAUU AGGCC ACC GGAGUCUAAUGCCUC G NO: 215 GACGUAUUCAGUUCGGUGGCUG
  • CAGUCAUUAGGGCGUAGAAGUCUAAUGUCU NO: 230 AGAGUGUUCUCCGUUCUGCGCCGG

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Abstract

The application discloses methods and compositions for the inhibition of the alternative complement pathway. The methods and compositions involve the use of aptamers for inhibiting complement Factor D. The application further provides anti-Factor D aptamers for the treatment of dry age-related macular degeneration, geographic atrophy, wet age-related macular degeneration or Stargardt disease. In some cases, the compositions include aptamers having a pseudoknot secondary structure for the inhibition of fD.

Description

PSEUDOKNOT COMPOSITIONS AND METHODS FOR INHIBITING FACTOR D
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application Nos. 62/448,872, filed January 20, 2017, and 62/536,401, filed July 24, 2017, which applications are incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] Visual impairment is a national and global health concern that has a negative impact on physical and mental health. The number of people with visual impairment and blindness is increasing due to an overall aging population. Visual impairment and blindness can be caused by any one of a large number of eye diseases and disorders affecting people of all ages. In one example, age-related macular degeneration (AMD) is an eye disorder that is currently the leading cause of vision loss in people fifty years of age or older in industrialized countries. It is estimated that by 2020, the number of people with AMD could exceed 196 million and by 2040, that number is expected to rise to 288 million. AMD is a degenerative eye disease that progresses from early stages to advanced stages of the disease. Risk factors for the disease include aging, lifestyle factors such as smoking, and genetics. The clearest indicator of progression to AMD is the appearance of drusen, yellow-white deposits under the retina, and it is an important component of both forms of AMD: exudative ("wet") and non-exudative ("dry"). Wet AMD causes vision loss due to abnormal blood vessel growth in the choriocapillaris through Batch's membrane. The most advanced form of dry AMD, known as geographic atrophy, is generally more gradual and occurs when light-sensitive cells in the macula atrophy, blurring and eliminating vision in the affected eye. While there are currently some promising treatments for wet AMD, no FDA-approved treatment exists for dry AMD or geographic atrophy.
[0003] A second example is childhood-onset Stargardt Disease ("STGD"), also known as Stargardt 1, a genetic, rare juvenile macular dystrophy generally associated with loss of central vision in the first two decades of life. STGD has a prevalence of approximately 1/20,000 affecting approximately 30,000 people in the US. STGD affects many ages, with the childhood- onset population at highest risk and most need. Patients with childhood-onset STGD tend to develop early severe visual acuity loss, significantly compromised retinal function, and rapid retinal pigment epithelial (RPE) cell atrophy with accompanying loss of retinal function. The median ages of onset and the median age at baseline examination are 8.5 (range, 3-16) and 12 years (range, 7-16), respectively. Patients with adult-onset disease are more likely to preserve visual acuity for a longer time and show slighter retinal dysfunction. STGD is an autosomal recessive genetic disease or complex heterozygous disease, caused by mutations in the ABCA4 gene. The ABCA4 gene encodes the photoreceptor protein ABCA4 Transporter, which is responsible for removal of bisretinoid fluorophores, which can include N-retinylidene-N- retinyethanolamine (A2E), all-trans-retinal and related photo-oxidation products of vitamin A aldehyde which together constitute lipofuscin, from photoreceptor cells. Accumulation of all- trans-retinal in photoreceptor cells is believed to damage RPE cells via oxidative stress, and trigger or promote complement-mediated damage to RPE cells, leading to retinal atrophy. A related disease termed Stargardt-like macular dystrophy, also known as STGD3, is inherited in a dominant autosomal manner and is due to mutations in the ELOVL4 gene. ELOVL4 encodes the ELOVL4 protein, ELOVL fatty acid elongase 4. Mutations in ELOVL4 protein associated with STGD lead to mis-folding and accumulation of ELOVL4 protein aggregates in retinal cells, which impact retinal cell function, eventually leading to cell death and retinal atrophy. No treatments exist for STGD or Stargardt-like disease.
SUMMARY OF THE INVENTION
[0004] In one aspect, an aptamer is provided having a nucleic acid sequence, wherein the aptamer forms a pseudoknot secondary structure which specifically binds to complement Factor D (fD). The aptamer of the present invention forms a pseudoknot secondary structure which binds to fD, preferably with high affinity and/or high specificity. In some cases, the aptamer has a nucleic acid sequence that does not comprise any one of SEQ ID NOs:475-534. In some cases, an aptamer of any of the preceding specifically binds to an exosite of fD. In some cases, an aptamer of any of the preceding has a nucleic acid sequence containing 30-90 nucleotides. In some cases, an aptamer of any of the preceding has an H-H type pseudoknot secondary structure. In some cases, an aptamer of any of the preceding has a pseudoknot structure comprising up to four loops and up to three base-paired stems. In some cases, an aptamer of any of the preceding has up to three base-paired stems, wherein each base-paired stem of the up to three base-paired stems comprises up to 15 base pairs. In some cases, an aptamer of any of the preceding has up to four loops, wherein each loop of the up to four loops comprises up to 12 nucleotides.
[0005] In some cases, an aptamer of any of the preceding has a pseudoknot secondary structure comprising, in a 5' to 3' direction, a first base-paired stem, a first loop, a second base-paired stem, a second loop, a third loop joining the first base-paired stem with a third base-paired stem, the third base-paired stem, and a fourth loop. In some cases, the first base-paired stem has from 2 to 12 base pairs. In some cases, the first base-paired stem has from 3 to 9 base pairs. In some cases, the first base-paired stem has 5 base pairs. In some cases, the first base-paired stem has 7 base pairs. In some cases, the first base-paired stem has one or more mismatched base pairs. In some cases, the second base-paired stem has from 2 to 9 base pairs. In some cases, the second base-paired stem has 6 or 7 base pairs. In some cases, the third base-paired stem has from 2 to 6 base pairs. In some cases, the third base-paired stem has 3 or 4 base pairs. In some cases, the third base-paired stem comprises a nucleic acid sequence of 5 '-NMHG-3', where N is any nucleotide; M is A or C; and H is A, C, or U. In some cases, the first loop has from 1 to 5 nucleotides. In some cases, the first loop has from 2 to 5 nucleotides. In some cases, the first loop has 2 nucleotides. In some cases, the first loop comprises, in a 5' to 3 ' direction, GU. In some cases, the first loop comprises, in a 5' to 3' direction, GG. In some cases, the second loop has from 2 to 9 nucleotides. In some cases, the second loop has from 4 to 6 nucleotides. In some cases, the second loop comprises, in a 5' to 3 ' direction, AGUC. In some cases, the third loop has from 2 to 12 nucleotides. In some cases, the third loop has from 3 to 14 nucleotides. In some cases, the third loop comprises one or more non-nucleotidyl spacers. In some cases, the fourth loop has 0, 1, or 2 nucleotides. In some cases, the fourth loop has a single nucleotide. In some cases, the single nucleotide is G or U.
[0006] In some cases, an aptamer of any of the preceding is an RNA aptamer or a modified RNA aptamer. In some cases, an aptamer of any of the preceding is a DNA aptamer or a modified DNA aptamer. In some cases, an aptamer of any of the preceding comprises at least one modified nucleotide. In some cases, an aptamer of any of the preceding comprises a nuclease- stabilized nucleic acid backbone. In some cases, an aptamer of any of the preceding specifically binds to fD with a Kd of less than about 50nM. In some cases, an aptamer of any of the preceding inhibits fD in an alternative complement dependent hemolysis assay with an IC50 of less than about 50nM. In some cases, an aptamer of any of the preceding is conjugated to a polyethylene glycol (PEG) molecule. In some cases, the PEG molecule has a molecular weight of 80 kDa or less.
[0007] In another aspect, an aptamer is provided having a nucleic acid sequence comprising any one of SEQ ID NOs: 1-3, 10-474, and 543-556 or a nucleic acid sequence as described in Table 2, or having at least 80% sequence identity to any one of SEQ ID NOs: 1-3, 10-474, and 543- 556 or a nucleic acid sequence as described in Table 2.
[0008] In another aspect, an aptamer according to any of the preceding is provided for use in a method of therapy; for use in a method of treatment that benefits from modulating fD; for use in a method of treatment that benefits from inhibiting a function associated with fD; or for use in a method for the treatment of ocular diseases.
[0009] In yet another aspect, an aptamer according to any of the preceding is provided and a pharmaceutically acceptable carrier, excipient, or diluent
[0010] In yet another aspect, a method is provided for modulating complement Factor D (fD) in a biological system, the method comprising: administering to the biological system, an aptamer according to any of the preceding, thereby modulating fD in the biological system. In some cases, the modulating comprises inhibiting a function associated with fD.
INCORPORATION BY REFERENCE
[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0013] FIG. 1 depicts aspects of the alternative complement pathway.
[0014] FIG. 2A and FIG. 2B depict modeling of the intravitreal (IVT) inhibition of Factor D by an anti-Factor D aptamer at various IVT concentrations over time.
[0015] FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D depict non-limiting examples of small molecule inhibitors of fD.
[0016] FIG. 4A, FIG. 4B, and FIG. 4C depict predicted secondary structure for a family of pseudoknot fD aptamers directed to the fD exosite according to an embodiment of the disclosure.
[0017] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, and FIG. 5G depict predicted secondary structures of various fD aptamers according to an embodiment of the disclosure.
[0018] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, and FIG. 6G depict predicted secondary structures of various fD aptamers according to an embodiment of the disclosure.
[0019] FIG. 7 depicts the amino acid sequence of human complement Factor D, chymotrypsin numbering scheme, and fD numbering scheme. [0020] FIG. 8A, FIG. 8B, and FIG. 8C depict a non-limiting example of an aptamer library sequence that may be utilized to generate anti-Factor D aptamers according to an embodiment of the disclosure.
[0021] FIG. 9 depicts a non-limiting example of a method for selecting anti-Factor D aptamers according to an embodiment of the disclosure.
[0022] FIG. 10 depicts measurement of Kd values of enriched libraries of anti-Factor D aptamers according to an embodiment of the disclosure.
[0023] FIG. 11 depicts binding analysis of anti-Factor D aptamers by flow cytometry according to an embodiment of the disclosure.
[0024] FIG. 12 depicts measurement of Kd values of anti -Factor D aptamers according to an embodiment of the disclosure.
[0025] FIG. 13 depicts a competition binding assay according to an embodiment of the disclosure.
[0026] FIG. 14 depicts examples of data obtained from an alternative complement dependent hemolysis assay according to an embodiment of the disclosure.
[0027] FIG. 15 depicts examples of data obtained from a fD esterase activity assay according to an embodiment of the disclosure.
[0028] FIG. 16 depicts examples of data obtained from a competition binding assay according to an embodiment of the disclosure.
[0029] FIG. 17 depicts examples of data obtained from a direct binding assay according to an embodiment of the disclosure.
[0030] FIG. 18A and FIG. 18B depict examples of data obtained from an alternative complement dependent hemolysis assay according to an embodiment of the disclosure.
[0031] FIG. 19 depicts a non-limiting example of an aptamer minimization experiment in accordance with embodiments of the disclosure.
[0032] FIG. 20 depicts non-limiting examples of fD aptamers in accordance with embodiments of the disclosure.
[0033] FIG. 21 depicts a non-limiting example of results obtained from SPR complex assembly in accordance with embodiments of the disclosure.
[0034] FIG. 22 depicts a non-limiting example of dose-dependent inhibition of
C3bB inactivated fD complex assembly with a fD aptamer in accordance with embodiments of the disclosure. DETAILED DESCRIPTION OF THE INVENTION
[0035] The disclosure herein provides methods and compositions for the treatment of ocular diseases or disorders. In some cases, the methods and compositions include the use of an anti-fD pseudoknot aptamer for, e.g., the treatment of ocular diseases or disorders In some cases, the ocular disease is macular degeneration. In some cases, macular degeneration is age-related macular degeneration. In some cases, age-related macular degeneration is dry age-related macular degeneration. In some cases, dry age-related macular degeneration is advanced dry age- related macular degeneration (i.e., geographic atrophy). In some cases, the ocular disease is wet age-related macular degeneration. In some cases, the ocular disease is Stargardt disease. In some cases, the methods and compositions involve the inhibition of the alternative complement pathway. In some cases, the methods and compositions involve the inhibition of a function associated with Factor D (fD). In some cases, the methods and compositions involve the inhibition of a function associated with fD for the treatment of ocular diseases. In some cases, the methods and compositions involve the inhibition of a function associated with fD for the treatment of dry age-related macular degeneration or geographic atrophy. In some cases, the methods and compositions involve the inhibition of a function associated with fD for the treatment of wet age-related macular degeneration. In some cases, the methods and
compositions involve the inhibition of a function associated with fD for the treatment of Stargardt disease.
[0036] In various aspects, the compositions may include oligonucleotides that selectively bind to and modulate an activity associated with fD. In some instances, the oligonucleotide
compositions of the disclosure inhibit a function associated with fD. In some cases, the oligonucleotide compositions may bind directly to an exosite of fD or to a region of fD that includes the exosite site. In some cases, the oligonucleotides are aptamers, such as RNA aptamers or DNA aptamers. In particular examples, the aptamers of the disclosure may have secondary structures. The secondary structures may include a pseudoknot secondary structure which generally includes one or more loops and one or more stems. Various examples of aptamers having pseudoknot structures for modulating fD are described herein.
[0037] The practice of some embodiments disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988)
Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).
[0038] In general, "sequence identity" refers to an exact nucleotide-to-nucleotide or amino acid- to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity ." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215 :403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25 :3389- 3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.
[0039] The term "aptamer" as used herein refers to an oligonucleotide and/or nucleic acid analogues that can bind to a specific target molecule. Aptamers can include RNA, DNA, modified RNA, modified DNA, any nucleic acid analogue, and/or combinations thereof.
Aptamers can be single-stranded oligonucleotides. In some cases, aptamers may comprise more than one nucleic acid strand (e.g., two or more nucleic acid strands). Without wishing to be bound by theory, aptamers are thought to bind to a three-dimensional structure of a target molecule. Aptamers may be monomelic (composed of a single unit) or multimeric (composed of multiple units). Multimeric aptamers can be homomeric (composed of multiple identical units) or heteromeric (composed of multiple non-identical units). Aptamers herein may be described by their primary structures which may refer to the linear nucleotide sequence of the aptamer. Aptamer sequences herein will generally be described from the 5' end to the 3' end, unless otherwise stated. Additionally or alternatively, aptamers herein may be described by their secondary structures which may refer to the combination of single- stranded regions and base pairing interactions within the aptamer.
[0040] The term "pseudoknot" as used herein may refer to the secondary structure of an aptamer of the disclosure. An aptamer having a pseudoknot secondary structure may have at least two stem-loop structures in which the stems are non-nested relative to each other. An aptamer may have a secondary structure having at least two complementary regions of the same nucleic acid strand that base-pair to form a double helix (referred to herein as a "stem"). Generally, these complementary regions are complementary when read in the opposite direction. The term "stem" as used herein may refer to either of the complementary nucleotide regions individually or may encompass a base-paired region containing both complementary regions, or a portion thereof. For example, the term "stem" may refer to the 5' side of the stem, that is, the stem sequence that is closer to the 5' end of the aptamer; additionally or alternatively, the term "stem" may refer to the 3 ' side of the stem, that is, the stem sequence that is closer to the 3' end of the aptamer. In some cases, the term "stem" may refer to the 5' side of the stem and the 3' side of the stem, collectively. The term "base-paired stem" is generally used herein to refer to both complementary stem regions collectively. A base-paired stem may be perfectly complementary meaning that 100% of its base pairs are Watson-Crick base pairs. A base-paired stem may also be "partially complementary." As used herein, the term "partially complementary stem" refers to a base-paired stem that is not entirely made up of Watson-Crick base pairs but does contain base pairs (either Watson-Crick base pairs or G-U/U-G wobble base pairs) at each terminus. In some cases, a partially complementary stem contains both Watson-Crick base-pairs and G-U/U- G wobble base pairs. In other cases, a partially complementary stem is exclusively made up of G-U/U-G wobble base pairs. A partially complementary stem may contain mis-matched base pairs and/or unpaired bases in the region between the base pairs at each terminus of the stem; but in such cases, the mis-matched base pairs and/or unpaired bases make up at most 50% of the positions between the base pairs at each terminus of the stem.
[0041] A stem as described herein may be referred to by the position, in a 5' to 3' direction on the aptamer, of the 5' side of the stem (i.e., the stem sequence closer to the 5' terminus of the aptamer), relative to the 5' side of additional stems present on the aptamer. For example, stem 1 (SI) may refer to the stem sequence that is closest to the 5' terminus of the aptamer, its complementary stem sequence, or both stem sequences collectively. Similarly, stem 2 (S2) may refer to the next stem sequence that is positioned 3 ' relative to SI, its complementary stem sequence, or both stem sequences collectively Each additional stem may be referred to by its position, in a 5' to 3' direction, on the aptamer, as described above. For example, S3 may be positioned 3' relative to S2 on the aptamer, S4 may be positioned 3'relative to S3 on the aptamer, and so on In some cases, the term "first stem" is used to refer to a stem in the aptamer, irrespective of its location. For example, a first stem may be SI, S2, S3, S4 or any other stem in the aptamer.
[0042] A stem may be adjacent to an unpaired region. An unpaired region may be present at a terminus of the aptamer or at an internal region of the aptamer.
[0043] As used herein, the term "loop" generally refers to an internal unpaired region of an aptamer. The term "loop" may refer to any unpaired region of an aptamer that is flanked on both the 5' end and the 3 ' end by a stem region. In some cases, a loop sequence may be adjacent to a single base-paired stem, such that the loop and stem structure together resemble a hairpin. In such cases, generally the primary sequence of the aptamer contains a first stem sequence adjacent to the 5' end of the loop sequence and a second stem sequence adjacent to the 3 ' end of the loop sequence; and the first and second stem sequences are complementary to each other. In some cases, each terminus of a loop is adjacent to first and second stem sequences that are not complementary.
[0044] A loop as described herein may be referred to by its position, in a 5' to 3' direction, on the aptamer. For example, loop 1 (LI) may refer to a loop sequence that is positioned most 5' on the aptamer. Similarly, loop 2 (L2) may refer to a loop sequence that is positioned 3 ' relative to LI, and loop 3 (L3) may refer to a loop sequence that is positioned 3 ' relative to L2. Each additional loop may be referred to by its position, in a 5' to 3 ' direction, on the aptamer, as described above. For example, L4 may be positioned 3 ' relative to L3 on the aptamer, L5 may be positioned 3'relative to L4 on the aptamer, and so on. In some cases, the term "first loop" is used to refer to a loop in the aptamer, irrespective of its location. For example, a first loop may be LI, L2, L3, L4 or any other loop in the aptamer.
[0045] Unless otherwise stated, when an aptamer includes more than one stem and/or more than one loop, the stems and loops are numbered consecutively in ascending order from the 5' end to the 3 ' end of the primary nucleotide sequence.
[0046] The term "exosite" as used herein may refer to a protein domain or region of a protein that is capable of binding to another protein. The exosite may also be referred to herein as a "secondary binding site", for example, a binding site that is remote from or separate from a primary binding site (e.g., an active site). In some cases, the primary and secondary binding sites may overlap. Binding of a molecule to an exosite may cause a physical change in the protein (e.g., a conformational change). In some cases, the activity of a protein may be dependent on occupation of the exosite. In some examples, the exosite may be distinct from an allosteric site. In some cases, the oligonucleotide compositions of the disclosure may bind to the exosite of fD or to part of the exosite of fD, or may bind to a region of fD that includes the exosite. In some cases, the oligonucleotide compositions of the disclosure may bind to the exosite of fD or to part of the exosite of fD, or may bind to a region of fD that includes the exosite. In some cases, the oligonucleotide compositions of the disclosure may block or occlude the exosite such that the natural substrate of fD is prevented from accessing the exosite. In such cases, the
oligonucleotide may block access to the exosite without directly binding the exosite (e.g., may bind to a region of fD other than the exosite in such a way that the exosite is sterically occluded).
[0047] The term "catalytic cleft" or "active site" as used herein refers to a domain of an enzyme in which a substrate molecule binds to and undergoes a chemical reaction. The active site may include amino acid residues that form temporary bonds with the substrate (e.g., a binding site) and amino acid residues that catalyze a reaction of that substrate (e.g., catalytic site). The active site may be a groove or pocket (e.g., a cleft) of the enzyme which can be located in a deep tunnel within the enzyme or between the interfaces of multimeric enzymes.
[0048] The term "epitope" as used herein refers to the part of an antigen (e.g., a substance that stimulates an immune system to generate an antibody against) that is specifically recognized by the antibody. In some cases, the antigen is a protein or peptide and the epitope is a specific region of the protein or peptide that is recognized and bound by an antibody. In some cases, the aptamers described herein bind to a region of fD that is an epitope for an anti-fD antibody or antibody fragment thereof, wherein the anti-fD antibody inhibits a function associated with fD. In some cases, the aptamer binding region of fD overlaps with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the epitope for an anti-fD antibody or the binding site of another fD-inhibiting molecule.
[0049] The terms "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polypeptide can be any protein, peptide, protein fragment or component thereof. A polypeptide can be a protein naturally occurring in nature or a protein that is ordinarily not found in nature. A polypeptide can consist largely of the standard twenty protein-building amino acids or it can be modified to incorporate non-standard amino acids. A polypeptide can be modified, typically by the host cell, by e.g., adding any number of biochemical functional groups, including phosphorylation, acetylation, acylation, formylation, alkylation, methylation, lipid addition (e.g. palmitoylation, myristoylation, prenylation, etc) and carbohydrate addition (e.g. N-linked and O-linked glycosylation, etc). Polypeptides can undergo structural changes in the host cell such as the formation of disulfide bridges or proteolytic cleavage. The peptides described herein may be therapeutic peptides utilized for e.g., the treatment of a disease.
[0050] The terms "subject" and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
The Complement System and the Alternative Complement Pathway
[0051] The complement system is a part of the innate immune system that enhances the ability of antibodies and phagocytic cells to clear pathogens from an organism. Although the system is not adaptable and does not change over the course of an individual's lifetime, it can be recruited and brought into action by the adaptive immune system.
[0052] The complement system consists of a number of small proteins found in the blood, in general synthesized by the liver, and normally circulating as inactive precursors (pro-proteins). When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end result of this complement activation or complement fixation cascade is massive amplification of the response and activation of the cell-killing membrane attack complex. Over 30 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane receptors.
[0053] The alternative complement pathway is a rapid, antibody-independent route for complement system activation and amplification. The alternative pathway comprises several components: C3, Factor B (fB), and fD. Activation of the alternative pathway occurs when C3b, a proteolytic cleavage form of C3, is bound to an activating surface agent such as a bacterium. fB is then bound to C3b, and cleaved by fD to yield the C3 convertase C3bBb.
Amplification of C3 convertase activity occurs as additional C3b is produced and deposited. The amplification response is further aided by the binding of the positive regulator protein properdin (Factor P), which stabilizes the active convertase against degradation, extending its half-life from 1-2 minutes to 18 minutes.
[0054] The C3 convertase further assembles into a C5 convertase (C3b3bBb). This complex subsequently cleaves complement component C5 into two components: the C5a polypeptide (9 kDa) and the C5b polypeptide (170 kDa). The C5a polypeptide binds to a 7 transmembrane G- protein coupled receptor, which was originally associated with leukocytes and is now known to be expressed on a variety of tissues including hepatocytes and neurons. The C5a molecule is the primary chemotactic component of the human complement system and can trigger a variety of biological responses including leukocyte chemotaxis, smooth muscle contraction, activation of intracellular signal transduction pathways, neutrophil-endothelial adhesion, cytokine and lipid mediator release and oxidant formation.
[0055] The alternative complement pathway is believed to play a role in the pathogenesis of a variety of ischemic, inflammatory and autoimmune diseases including age-related macular degeneration, geographic atrophy, Stargardt disease, systemic lupus erythematosus, rheumatoid arthritis, and asthma. Thus, components of the alternative complement pathway may be important targets for the treatment of these diseases.
Age-related macular degeneration
[0056] Age-related macular degeneration ("AMD") is a chronic and progressive eye disease that is the leading cause of irreparable vision loss in the United States, Europe, and Japan. AMD is characterized by the progressive deterioration of the central portion of the retina referred to as the macula. The clearest indicator of progression to AMD is the appearance of drusen, yellow- white deposits under the retina, which are plaques of material that are derived from the metabolic waste products of retinal cells. The appearance of drusen is an important component of both forms of AMD: exudative ("wet") and non-exudative ("dry"). The presence of numerous, intermediate-to-large drusen is associated with the greatest risk of progression to late-stage disease, characterized by geographic atrophy and/or neovascularization. The majority of patients with wet AMD experience severe vision loss in the affected eye within months to two years after diagnosis of the disease, although vision loss can occur within hours or days. Dry AMD is more gradual and occurs when light-sensitive cells in the macula slowly atrophy, gradually blurring central vision in the affected eye. Vision loss is exacerbated by the formation and accumulation of drusen and sometimes the deterioration of the retina, although without abnormal blood vessel growth and bleeding. Geographic atrophy is a term used to refer to advanced dry AMD.
Geographic atrophy is characterized by an "island" of atrophied photoreceptors cells. It is believed that the alternative complement pathway may play a role in the pathogenesis of AMD.
[0057] For example, FIG. 1 depicts a potential role for the alternative complement pathway in the pathogenesis of geographic atrophy. In this example, multiple factors may lead to activation of the alternative complement pathway, including the appearance of drusen in the eye, immune dysfunction, and genetic differences that predispose individuals to complement activation. As described above, amplification of C3 convertase activity may occur as additional C3b is produced and deposited. C3 convertase activity may lead to inflammation and opsonization. The C3 convertase may further assemble into a C5 convertase (C3b3bBb) which may lead to cell death through formation of the Membrane Attack Complex.
[0058] In some aspects, the oligonucleotide compositions of the disclosure may be used to treat AMD. In some cases, the oligonucleotide compositions of the disclosure may be used to treat wet AMD. In some cases, the oligonucleotide compositions of the disclosure may be used to treat geographic atrophy. In some cases, the oligonucleotide compositions of the disclosure may be used to stop, slow, or reverse the progression of wet AMD or geographic atrophy. In some cases, the oligonucleotide compositions of the disclosure may be used to treat symptoms associated with wet AMD or geographic atrophy.
Stargardt Disease
[0059] Stargardt Disease ("STGD") is a rare, genetic, macular dystrophy with an incidence of 1/20,000, affecting approximately 30,000 individuals in the United States. STGD is an autosomal recessive or complex heterozygous genetic disease caused by mutations in the ABCA4 gene. The ABCA4 gene encodes the photoreceptor protein ABCA4 Transporter, which is responsible for removal of bisretinoid fluorophores, which can include N-retinylidene-N- retinyethanolamine (A2E), all-trans-retinal and related photo-oxidation products of vitamin A aldehyde which together constitute lipofuscin from photoreceptor cells. Accumulation of all- trans-retinal in photoreceptor cells is believed to damage RPE cells via oxidative stress, and trigger or promote complement-mediated damage to RPE cells, leading to retinal atrophy.
STGD is characterized by the progressive deterioration of the central portion of the retina referred to as the macula, generally beginning in the first two decades of life. The clearest indicator of progression of STGD is the appearance of drusen, yellow-white deposits under the retina, which are plaques of material that are derived from the metabolic waste products of retinal cells, including all-trans-retinal and other vitamin A-related metabolites. The onset of STGD is typically between the ages of 6-20 years, with early symptoms including difficulties in reading and adjusting to light. Patients with childhood-onset STGD tend to develop early severe visual acuity loss, significantly compromised retinal function, and rapid retinal pigment epithelial (RPE) cell atrophy with accompanying loss of retinal function. The median ages of onset and the median age at baseline examination are 8.5 (range, 3-16) and 12 years (range, 7- 16), respectively. Patients with adult-onset disease are more likely to preserve visual acuity for a longer time and show slighter retinal dysfunction. Accumulation of all-trans-retinal in photoreceptor cells leads to inflammation, oxidative stress, deposition of auto-fluorescent lipofuscin pigments in the retinal pigment epithelium and retinal atrophy. Lipofuscin deposits (drusen), and oxidative products, trigger the alternative complement pathway into an
inflammatory response leading to cell death. Data supporting the role of alternative complement in STGD include human cell models, genetic mouse models and the accumulation of complement factors in humans in drusen during disease progression Therefore, inhibitors of complement, particularly complement factor D, are anticipated to stop or slow the progression of vision loss in individuals with STGD. A related disease termed Stargardt-like macular dystrophy, also known as STGD3, is inherited in a dominant autosomal manner and is due to mutations in the ELOVL4 gene. ELOVL4 encodes the ELOVL4 protein, ELOVL fatty acid elongase 4. Mutations in ELOVL4 protein associated with STGD lead to mis-folding and accumulation of ELOVL4 protein aggregates in retinal cells, which impact retinal cell function, eventually leading to cell death and retinal atrophy. Complement pathway activation is also thought to play a role in Stargardt-like disease, and therefore inhibitors of complement, particularly complement factor D, are anticipated to stop or slow the progression of vision loss in individuals with Stargardt-like disease.
[0060] In some aspects, the oligonucleotide compositions of the disclosure may be used to treat Stargardt or Stargardt-like disease. In some cases, the oligonucleotide compositions of the disclosure may be used to stop, slow, or reverse the progression of Stargardt or Stargardt-like disease. In some cases, the oligonucleotide compositions of the disclosure may be used to treat symptoms associated with Stargardt or Stargardt-like disease.
Aptamers
[0061] In some cases, the methods and compositions described herein utilize one or more aptamers for the treatment of an ocular disease. In some cases, the methods and compositions described herein utilize one or more aptamers for modulating an activity associated with fD. The term aptamer as used herein refers to oligonucleotide molecules that bind to a target (e.g., a protein) with high affinity and specificity through non-Watson-Crick base pairing interactions. Generally, the aptamers described herein are non-naturally occurring oligonucleotides (i.e., synthetically produced) that are isolated and used for the treatment of a disorder or a disease. Aptamers can bind to essentially any target molecule including, without limitation, proteins, oligonucleotides, carbohydrates, lipids, small molecules, and even bacterial cells. The aptamers described herein are oligonucleotides that bind to proteins of the alternative complement pathway. Whereas many naturally occurring oligonucleotides, such as mRNA, encode information in their linear base sequences, aptamers generally do not encode information in their linear base sequences. Further, aptamers can be distinguished from naturally occurring oligonucleotides in that binding of aptamers to target molecules is dependent upon secondary and tertiary structures of the aptamer.
[0062] Aptamers may be suitable as therapeutic agents and may be preferable to other therapeutic agents because: 1) aptamers may be fast and economical to produce because aptamers can be developed entirely by in vitro processes; 2) aptamers may have low toxicity and may lack an immunogenic response; 3) aptamers may have high specificity and affinity for their targets; 4) aptamers may have good solubility; 5) aptamers may have tunable pharmacokinetic properties; 6) aptamers may be amenable to site-specific conjugation of PEG and other carriers; and 7) aptamers may be stable at ambient temperatures.
[0063] Aptamers as described herein may include any number of modifications that can affect the function or affinity of the aptamer. For example, aptamers may be unmodified or they may contain modified nucleotides to improve stability, nuclease resistance or delivery characteristics. Examples of such modifications may include chemical substitutions at the sugar and/or phosphate and/or base positions, for example, at the 2' position of ribose, the 5 position of pyrimidines, and the 8 position of purines, various 2'-modified pyrimidines and modifications with 2'-amino (2'- H2), 2'-fluoro (2'-F), and/or 2'-0-methyl (2'-OMe) substituents. In some cases, aptamers described herein comprise a 2'-OMe and/or a 2'F modification to increase in vivo stability. In some cases, the aptamers described herein contain modified nucleotides to improve the affinity and specificity of the aptamers for a specific epitope, exosite or active site. Examples of modified nucleotides include those modified with guanidine, indole, amine, phenol, hydroxymethyl, or boronic acid. In other cases, pyrimidine nucleotide triphosphate analogs or CE-phosphoramidites may be modified at the 5 position to generate, for example, 5- benzylaminocarbonyl-2'-deoxyuridine (BndU); 5-[N-(phenyl-3-propyl)carboxamide]-2'- deoxyuridine (PPdU); 5-(N-thiophenylmethylcarboxyamide)-2'-deoxyuridine (ThdU); 5-(N-4- fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU); 5-(N-(l -naphthylmethyl)carboxamide)- 2'-deoxyuridine (NapdU); 5 -(N-2-naphthylmethylcarboxyamide)-2'-deoxyuridine (2NapdU); 5- ( -l-naphthylethylcarboxyamide)-2'-deoxyuridine ( EdU); 5-(N-2- naphthylethylcarboxyamide)-2'-deoxyuridine (2NEdU); 5-( -tryptaminocarboxyamide)-2'- deoxyuridine (TrpdU); 5-isobutylaminocarbonyl-2'-deoxyuridine (IbdU); 5-(N- tyrosylcarboxyamide)-2'-deoxyuridine (TyrdU); 5-(N-isobutylaminocarbonyl-2'-deoxyuridine (iBudU); 5-( -benzylcarboxyamide)-2'-0-methyluridine, 5-(N-benzylcarboxyamide)-2'- fluorouridine, 5-(N-phenethylcarboxyamide)-2'-deoxyuridine (PEdU), 5-(N-3,4- methylenedioxybenzylcarboxyamide)-2'-deoxyuridine (MBndU), 5-(N- imidizolylethylcarboxyamide)-2'-deoxyuridine (ImdU), 5-(N-isobutylcarboxyamide)-2'-0- methyluridine, 5-(N-isobutylcarboxyamide)-2'-fluorouridine, 5-(N— R-threoninylcarboxyamide)- 2'-deoxyuridine (ThrdU), 5-(N-tryptaminocarboxyamide)-2'-0-methyluridine, 5-(N- tryptaminocarboxyamide)-2'-fluorouridine, 5-(N-[l-(3- trimethylamonium)propyl]carboxyamide)-2'-deoxyuridine chloride, 5-(N- naphthylmethylcarboxyamide)-2'-0-methyluridine, 5-(N-naphthylmethylcarboxyamide)-2'- fluorouridine, 5-(N-[l-(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine), 5-(N-2- naphthylmethylcarboxyamide)-2'-0-methyluridine, 5-(N-2-naphthylmethylcarboxyamide)-2'- fluorouridine, 5-(N-l-naphthylethylcarboxyamide)-2'-0-methyluridine, 5-(N-l- naphthylethylcarboxyamide)-2'-fluorouridine, 5-( -2-naphthylethylcarboxyamide)-2'-0- methyluridine, 5-(N-2-naphthylethylcarboxyamide)-2'-fluorouridine, 5-(N-3- benzofuranylethylcarboxyamide)-2'-deoxyuridine (BFdU), 5-(N-3- benzofuranylethylcarboxyamide)-2'-0-methyluridine, 5-(N-3-benzofuranylethylcarboxyamide)- 2'-fluorouridine, 5-(N-3-benzothiophenylethylcarboxyamide)-2'-deoxyuridine (BTdU), 5-(N-3- benzothiophenylethylcarboxyamide)-2'-0-m ethyl uridine, 5-(N-3- benzothiophenylethylcarboxyamide)-2'-fluorouridine; 5-[N-(l-morpholino-2- ethyl)carboxamide]-2'-deoxyuridine (MOEdu); R-tetrahydrofuranylmethyl-2'-deoxyuridine (RTMdU); 3-methoxybenzyl-2'-deoxyuridine (3MBndU); 4-methoxybenzyl-2'-deoxyuridine (4MBndU); 3,4-dimethoxybenzyl-2'-deoxyuridine (3,4DMBndU); S-tetrahydrofuranylmethyl- 2'-deoxyuridine (STMdU); 3,4-methylenedioxyphenyl-2-ethyl-2'-deoxyuridine (MPEdU); 4- pyridinylmethyl-2'-deoxyuridine (PyrdU); or l-benzimidazol-2-ethyl-2'-deoxyuridine (BidU); 5- (amino-l-propenyl)-2'-deoxyuridine; 5-(indole-3-acetamido-l-propenyl)-2'-deoxyuridine; or 5- (4-pivaloylbenzamido-l-propenyl)-2'-deoxyuridine.
[0064] Modifications of the aptamers contemplated in this disclosure include, without limitation, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and functionality to the nucleic acid aptamer bases or to the nucleic acid aptamer as a whole. Modifications to generate
oligonucleotide populations that are resistant to nucleases can also include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof. Such
modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate, phosphorodithioate, or alkyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases isocytidine and isoguanosine.
Modifications can also include 3' and 5' modifications such as capping, e.g., addition of a 3'-3'- dT cap to increase exonuclease resistance.
[0065] Aptamers of the disclosure may generally comprise nucleotides having ribose in the β-D- ribofuranose configuration. In some cases, 100% of the nucleotides present in the aptamer have ribose in the β-D-ribofuranose configuration. In some cases, at least 50% of the nucleotides present in the aptamer have ribose in the β-D-ribofuranose configuration. In some cases, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%), at least 90%, or 100% of the nucleotides present in the aptamer have ribose in the β-D- ribofuranose configuration.
[0066] The length of the aptamer can be variable. In some cases, the length of the aptamer is less than 100 nucleotides. In some cases, the length of the aptamer is greater than 10 nucleotides. In some cases, the length of the aptamer is between 10 and 90 nucleotides. The aptamer can be, without limitation, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, or about 90 nucleotides in length.
[0067] In some instances, a polyethylene glycol (PEG) polymer chain is covalently bound to the aptamer, referred to herein as PEGylation. Without wishing to be bound by theory, PEGylation may increase the half-life and stability of the aptamer in physiological conditions. In some cases, the PEG polymer is covalently bound to the 5' end of the aptamer. In some cases, the PEG polymer is covalently bound to the 3' end of the aptamer. In some cases, the PEG polymer is covalently bound to specific site on a nucleobase within the aptamer, including the 5-position of a pyrimidine or 8-position of a purine.
[0068] In some cases, an aptamer described herein may be conjugated to a PEG having the general formula, H-(0-CH2-CH2)n-OH. In some cases, an aptamer described herein may be conjugated to a methoxy-PEG (mPEG) of the general formula, CH30-(CH2-CH2-0)n-H. In some cases, the aptamer is conjugated to a linear chain PEG or mPEG. The linear chain PEG or mPEG may have an average molecular weight of up to about 30 kD. Multiple linear chain PEGs or mPEGs can be linked to a common reactive group to form multi-arm or branched PEGs or mPEGs. For example, more than one PEG or mPEG can be linked together through an amino acid linker (e.g., lysine) or another linker, such as glycerine. In some cases, the aptamer is conjugated to a branched PEG or branched mPEG. Branched PEGs or mPEGs may be referred to by their total mass (e.g., two linked 20kD mPEGs have a total molecular weight of 40kD). Branched PEGs or mPEGs may have more than two arms. Multi-arm branched PEGs or mPEGs may be referred to by their total mass (e.g. four linked 10 kD mPEGs have a total molecular weight of 40 kD). In some cases, an aptamer of the present disclosure is conjugated to a PEG polymer having a total molecular weight from about 5 kD to about 200 kD, for example, about 5 kD, about 10 kD, about 20 kD, about 30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, about 80 kD, about 90 kD, about 100 kD, about 1 10 kD, about 120 kD, about 130 kD, about 140 kD, about 150 kD, about 160 kD, about 170 kD, about 180 kD, about 190 kD, or about 200 kD. In one non-limiting example, the aptamer is conjugated to a PEG having a total molecular weight of about 40 kD.
[0069] In some cases, the reagent that may be used to generate PEGylated aptamers is a branched PEG N-Hydroxysuccinimide (mPEG- HS) having the general formula:
Figure imgf000019_0001
with a 20 kD, 40 kD or 60 kD total molecular weight (e.g., where each mPEG is about lOkD, 20 kD or about 30 kD). As described above, the branched PEGs can be linked through any appropriate reagent, such as an amino acid (e.g., lysine or glycine residues).
[0070] In one non-limiting example, the reagent used to generate PEGylated aptamers is [N2- (monomethoxy 20K polyethylene glycol carbamoyl)-N6-(monomethoxy 20K polyethylene glycol carbamoyl)] -lysine N-hydroxysuccinimide having the formula:
Figure imgf000019_0002
[0071] In yet another non-limiting example, the reagent used to generate PEGylated aptamers
B7C OK;¾C«,£$^C;H-;
has the formula: where X is N-hydroxysuccinimide and the PEG arms are of approximately equivalent molecular weight. Such PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed or single-arm linear PEG.
[0072] In some examples, the reagent used to generate PEGylated aptamers has the formula:
Figure imgf000020_0001
where X is N-hydroxysuccinimide and the PEG arms are of different molecular weights, for example, a 40 kD PEG of this architecture may be composed of 2 arms of 5 kD and 4 arms of7.5 kD. Such PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed PEG or a single-arm linear PEG.
[0073] In some cases, the reagent that may be used to generate PEGylated aptamers is a non- branched mPEG-Succinimidyl Propionate (mPEG-SPA), having the general formula:
Figure imgf000020_0002
where mPEG is about 20 kD or about 30 kD. In one example, the reactive ester may be -0-CH2- CH2-CO2-NHS.
[0074] In some instances, the reagent that may be used to generate PEGylated aptamers may include a branched PEG linked through glycerol, such as the Sunbright™ series from NOF Corporation, Japan. Non-limitin examples of these reagents include:
Figure imgf000020_0003
(SUNBRIGHT® GL2-
400GS2);
Figure imgf000021_0001
(SU BRIGHT® GL2-400HS); and
Figure imgf000021_0002
[0075] In another example, the reagents may include a non-branched mPEG Succinimidyl alpha- methylbutanoate (mPEG-SMB) having the general formula:
Figure imgf000021_0003
where mPEG is between 10 and 30 kD. In one example, the reactive ester may be 0-CH2.CH2- CH(CH3)-C02-NHS.
[0076] In other instances, the PEG reagents may include nitrophenyl carbonate-linked PEGs, having the general formula:
Figure imgf000021_0004
[0077] Compounds including nitrophenyl carbonate can be conjugated to primary amine containing linkers.
[0078] In some cases, the reagents used to generate PEGylated aptamers may include PEG with thiol-reactive groups that can be used with a thiol-modified linker. One non-limiting example may include reagents having the following general structure:
Figure imgf000022_0001
where mPEG is about 10 kD, about 20 kD or about 30 kD. Another non-limiting example may include reagents having the following general structure:
Figure imgf000022_0002
where each mPEG is about 10 kD, about 20 kD, or about 30 kD and the total molecular weight is about 20 kD, about 40 kD, or about 60 kD, respectively. Branched PEGs with thiol reactive groups that can be used with a thiol-modified linker, as described above, may include reagents in which the branched PEG has a total molecular weight of about 40 kD or about 60 kD (e.g., where each mPEG is about 20 kD or about 30 kD).
[0079] In some cases, the reagents used to generated PEGylated aptamers may include reagents having the following structure:
Figure imgf000022_0003
In some cases, the reaction is carried out between about pH 6 and about pH 10, or between about pH 7 and pH 9 or about pH 8.
[0080] In some cases, the reagents used to generate PEGylated aptamers may include reagents having the following structure:
Figure imgf000023_0001
[0081] In some cases, the reagents used to generate PEGylated aptamers may include reagents having the following structure:
Figure imgf000023_0002
[0082] In some cases, the aptamer is associated with a single PEG molecule. In other cases, the aptamer is associated with two or more PEG molecules.
[0083] In some cases, the aptamers described herein may be bound or conjugated to one or more molecules having desired biological properties. Any number of molecules can be bound or conjugated to aptamers, non-limiting examples including antibodies, peptides, proteins, carbohydrates, enzymes, polymers, drugs, small molecules, gold nanoparticles, radiolabels, fluorescent labels, dyes, haptens (e.g., biotin), other aptamers, or nucleic acids (e.g., siRNA). In some cases, aptamers may be conjugated to molecules that increase the stability, the solubility or the bioavailability of the aptamer. Non-limiting examples include polyethylene glycol (PEG) polymers, carbohydrates and fatty acids. In some cases, molecules that improve the transport or delivery of the aptamer may be used, such as cell penetration peptides. Non-limiting examples of cell penetration peptides can include peptides derived from Tat, penetratin, polyarginine peptide Argg sequence, Transportan, VP22 protein from Herpes Simplex Virus (HSV), antimicrobial peptides such as Buforin I and SynB, polyproline sweet arrow peptide molecules, Pep-1 and MPG. In some embodiments, the aptamer is conjugated to a lipophilic compound such as cholesterol, dialkyl glycerol, diacyl glycerol, or a non-immunogenic, high molecular weight compound or polymer such as polyethylene glycol (PEG) or other water-soluble pharmaceutically acceptable polymers including, but not limited to, polyaminoamines
(PAMAM) and polysaccharides such as dextran, or polyoxazolines (POZ).
[0084] The molecule to be conjugated can be covalently bonded or can be associated through non-covalent interactions with the aptamer of interest. In one example, the molecule to be conjugated is covalently attached to the aptamer. The covalent attachment may occur at a variety of positions on the aptamer, for example, to the exocyclic amino group on the base, the 5- position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5' or 3 ' terminus. In one example, the covalent attachment is to the 5' or 3' hydroxyl group of the aptamer.
[0085] In some cases, the aptamer can be attached to another molecule directly or with the use of a spacer or linker. For example, a lipophilic compound or a non-immunogenic, high molecular weight compound can be attached to the aptamer using a linker or a spacer. Various linkers and attachment chemistries are known in the art. In a non-limiting example, 6- (trifluoroacetamido)hexanol (2-cyanoethyl-N,N-diisopropyl)phosphoramidite can be used to add a hexylamino linker to the 5' end of the synthesized aptamer. This linker, as with the other amino linkers provided herein, once the group protecting the amine has been removed, can be reacted with PEG-NHS esters to produce covalently linked PEG-aptamers. Other non-limiting examples of linker phosphoramidites may include: TFA-amino C4 CED phosphoramidite having the structure:
Figure imgf000024_0001
5'-amino modifier C3 TFA having the structure:
Figure imgf000024_0002
MT amino modifier C6 CED phosphoramidite having the structure:
Figure imgf000024_0003
5'-amino modifier 5 having the structure:
Figure imgf000025_0001
MMT: 4-Monomethoxytrityl
5'-amino modifier C12 having the structure:
MMT: 4-Monomethoxytrityl
5' thiol-modifier C6 having the structure:
Figure imgf000025_0003
5' thiol-modifier C6 having the structure:
Figure imgf000025_0004
DMT: 4,4'-Dimethoxytrityl and 5' thiol-modifier C6 having the structure:
Figure imgf000026_0001
DMT: 4,4'-Dimethoxytrityl
[0086] The 5'-thiol modified linker may be used, for example, with PEG-maleimides, PEG- vinylsulfone, PEG-iodoacetamide and PEG-orthopyridyl-disulfide. In one example, the aptamer may be bonded to the 5'-thiol through a maleimide or vinyl sulfone functionality.
[0087] In some cases, the aptamer formulated according to the present disclosure may also be modified by encapsulation within a liposome. In other cases, the aptamer formulated according to the present disclosure may also be modified by encapsulation within a micelle. Liposomes and micelles may be comprised of any lipids, and in some cases the lipids may be phospholipids, including phosphatidylcholine.
[0088] In some cases, the aptamers described herein are designed to inhibit a function associated with an alternative complement pathway enzyme. In one example, an anti-fD aptamer is used to inhibit a function associated with fD (e.g., inhibit the catalytic activity of fD). In other cases, the aptamers described herein are designed to prevent an interaction or binding of two or more proteins of the alternative complement pathway. In one example, an aptamer binds to fD and prevents binding of the complex C3bBb to fD. The aptamers described herein may bind to a region of fD that is recognized by an antibody or antibody fragment thereof that inhibits a function associated with fD. In some cases, the antibody or antibody fragment thereof that inhibits a function associated with fD has an amino acid sequence of heavy chain variable region of: EVQLVQSGPELKKPGASVKVSCKASGYTFTNYGMNWVRQA
PGQGLEWMGWINTYTGETTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCER
GGVN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALT SGVHTFP AVLQ S SGLYSLS S VVTVP S S SLGTQT YICNVNHKP SNTKVDKKVEPKSC
DKTHT (SEQ ID NO: 7) and an amino acid sequence of light chain variable region of:
DIQVTQSPSSLSASVGDRVTITCITSTDIDDDMNWYQQKPGKVPKLLISGGNTLRPGVPS
RF S GS GS GTDFTLTIS SLQPED V AT YYCLQ SD SLP YTF GQGTK VEIKRT VAAP S VFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTL SK AD YEKHK VYACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 8).
[0089] FIG. 2 depicts modeling of the intravitreal (IVT) inhibition of Factor D by an anti -Factor
D aptamer at various IVT concentrations. FIG. 2A and FIG. 2B demonstrate IVT inhibition of Factor D at various IVT concentrations of an anti-Factor D aptamer. Effective inhibition of TVT Factor D inhibition was modeled using a standard 2 compartment model, assuming reported IVT half-lives for Fabs (7 days, LUCENTIS®) and PEGylated aptamers (10 days, MACUGEN®) and 1 : 1 inhibition of Factor D by each therapy at the relevant IVT concentrations (IC50 data). As depicted in FIG. 2A, effective inhibition curves after IVT injection are shown for an anti -Factor D Fab (dashed line), an anti -Factor D aptamer VT-001 (solid line), and the intercept with the serum level of Factor D (dotted line) can be visualized as a surrogate for loss of clinically relevant Factor D inhibition. FIG. 2B depicts the predicted IVT drug concentration (nM) of a PEGylated aptamer (dotted line) and an anti-Factor D antibody (solid line) over the number of weeks post IVT injection.
[0090] The aptamers described herein may bind to a region of fD that is recognized by a small molecule inhibitor that inhibits a function associated with fD, non-limiting examples including dichloroisocoumarin or any one of the compounds depicted in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D. The aptamers described herein may bind to a region of fD that is recognized by a peptide inhibitor that inhibits a function associated with fD.
[0091] In some cases, an aptamer of the disclosure comprises one of the following sequences described in Table 1 or Table 2.
Table 1. fD Aptamer Sequences
SEQ ID Backbone Sequence 5' to 3'
NO.
SEQ ID RNA GGGAGUGUGUACGAGGCAUUAGGCCGCCAC CCAAACUGCAGUCCUCGUAAGUCUGCCUGG NO: 1
CGGCUUUGAUACUUGAUCGCCCUAGAAGC
SEQ ID RNA GGGAGUGUGUACGAGGCAUUAGUCCGCCGA AGUCUUUUGGCUCGGUUUUUUCAAGGUCGG NO: 2
CGGCUUUGAUACUUGAUCGCCCUAGAAGC
SEQ ID RNA GGGAGUGUGUACGAGGCAUUAGGCCGCCAC CUCGUUUGAUUGCGGUUGUUCGGCCGCGGG
NO: 3
CGGCUUUGAUACUUGAUCGCCCUAGAAGC
SEQ ID RNA GGGAGUGUGUACGAGGCAUUAGGCCGCCGA AGUCUAAUGUCCUCGGCGCUGAAAGUUC- NO: 10
GGCGGCUUUGAUACUUGAUCGCCCUAGAAG C
SEQ ID RNA GGGAGUGUGUACGAGGCAUUAGGCCGCCGA AGUCUAAUGGCUCGGGUGUUCUAAGUUC- NO: 11
GGCGGCUUUGAUACUUGAUCGCCCUAGAAG C
SEQ ID RNA GAGGCAUUAGUCCGCCGAAGUCUUUUGGCU CGGUUUUUUCAAGGUCGGCGGCUUU NO: 12 SEQ ID RNA GAGGCAUUAGGCCGCCGAAGUCUAAUGGCU
CGGGUGUUCUAAGUUCGGCGGCUU NO: 13
SEQ ID RNA GAGGC AUU AGGC CGC C G AAGUCU A AUGUC C UCGGCGCUGAAAGUUCGGCGGCUUU NO: 14
SEQ ID RNA GAGGUAUAAGUCCGCGGAAGUCUUUUGUCU CGGGUAUAUGCAGGUCCGCGGCUGU
NO: 15
SEQ ID RNA
GGUAGUAGUCCACCGAAGUCUACUGGCUCG NO: 16 GUUAUAUACAGGUCGGUGCUGU
SEQ ID RNA
GAGCAAUUAGUACUCCGAAGUCUAUUUGCC NO: 17 CGGUUCACACAUGGUCGGAGCAU
SEQ ID RNA
GAGGCAUAAGUCCGCCGAAGUCUUAUGGCU NO: 18 CGGUUUAUUUAUGGUCGGCGGCAU
SEQ ID RNA
GGGCAUGACUACGCCGAAGUCUCUUGUCUC
NO: 19 GGCUUUCGUAUGUUCGGCGUCAU
SEQ ID RNA
GAGGCAUCAGUCCGCCGAAGUCUUGUGCCU NO: 20 UGGUUUGUUAUGGUCGGCGGCAU
SEQ ID RNA
CAUAAGUCCACCGAAGUCUUAUGGAUCGGA NO: 21 UUUUUCAUGAUCGGUGGCAU
SEQ ID RNA
GCAUCAGUCCUCCGAAGUCUUUUGGUUCGG NO: 22 AUUUUGUCUGGUCGGGGGCAG
SEQ ID RNA
GAGGCAUUAGUCUGCCGAAGUCUUUUGGCU
NO: 23 CGGUUUAUUCAUGUUCGGCAGCAU
SEQ ID RNA
GAGCCAUUGGUCCACCGAAGUCCUAUGACU NO: 24 CGUUUAAUUAAUGUUCGGCGGCAUU
SEQ ID RNA
GAGCCAUUGGUCCACCGAAGUCCUAUGACU NO: 25 CGUUUAAUUAAUGUUCGGCGGCAUU
SEQ ID RNA
GAUGCAUUGGUCCGCCGAAGUCCAAUGUAU NO: 26 CCGUUUCCUCAUGUUCGGCGGCAU
SEQ ID RNA
UUAGUCCGCUGAAGUCUUUUGGCUCGGGUU
NO: 27 UUUGAUGUUCGGCGGCAUU
SEQ ID RNA
CCAUUAGUCGGGGGAAGUUUUUUGGCUGGA NO: 28 UUAUUUCACGGUCCCCCGCGU
SEQ ID RNA
AUUAGUCCGCCGAAGUCUUUUGGUUACGUU
NO: 29 UAUACACGGUCGGCGGCGU SEQ ID RNA
GUGCCAUUAGUCCGCCGGAGUCUAUUGGGU
NO: 30 ACGUCAUUUCAUGGUCGGCGGCAU
SEQ ID RNA
GCCAAUAGUCCGUCGAAGUCUUUUGGCCCU NO: 31
GUUAUUUUAUGGUCGGCGGCAU
SEQ ID RNA
GCCAUUAGUCCGCCGAAGUCUAUUGGCCGG
NO: 32 UUGCUUAAUGGUCGGCGGCAUU
SEQ ID RNA
GUGCCAUUAGUCCGCGGAAGUCUACUGUCA
NO: 33 CGGUAUCUUGAUGGUCCGCGGCAUU
SEQ ID RNA
UUAGUCCGCCGAAGUCUUUUAGCUCGUUUU
NO: 34 CUUCAUGGUCGGCGGCAU
SEQ ID RNA
GAUGUCAACGUCCGUCGAAGUCUUUGGCAU NO: 35 CGGUUUUUUCAUGUUCGGCGGCAU
SEQ ID RNA
UAAGUCCGCCGAAGUCUUUUGCUUAAGCUC
NO: 36 CCGCAUGGUCGGCGGCAU
SEQ ID RNA
GACGCAUUAGUCCGCCGAAGUCUCUUGCGU NO: 37 CAGUUUUUUUCAUGGUCGGCGGCAU
SEQ ID RNA
UUAGUCCGCAUAAGUCUUUUGGAUCGCUUU NO: 38 GUUCAUGUUGUGCGGCAU
SEQ ID RNA
GAGGAAGUUGUUCGCAGAAGUCAACUGUCU
NO: 39 CGGAAUUUUCAAGGUCUGCGGCUU
SEQ ID RNA
AAGGCAUGUGUCCGCAGAAGUCAUAUGGAC
NO: 40 UUGAUUUUUUCAUGGUCUGCGGCAU
SEQ ID RNA
GAUACAUUAGUGCGCUGAAGUCUAAUGAAU NO: 41
CAGUUUUUUCACCGUCGGCGGGUG
SEQ ID RNA
AUGCAUUAGUCGGCCGAAGUCUGUUGUCUC
NO: 42 GGUGUUUUCACGGUCGGCCGCGU
SEQ ID RNA
GAGGCAUUAGUCAGCCGAAGUCUGGUGUCU NO: 43 CAGUUUGUUUACGGUCGGCUGCGU
SEQ ID RNA
GAGACAUUUGUCCGCCGAAGUCAUCUGUCU NO: 44 CGGUUUGUUCACGGUCGGCGGCGU
SEQ ID RNA
GUGGCAUCAGUCCGACGAAGUCUUUUGCCA NO: 45 UUUUAUGUUCAAGGUCGUCGGCUU
SEQ ID RNA
CAGGCAUUAGUCAGCCGAAGUCUUUUGCCU
NO: 46 GGGAUUUUCGAAGGUCGGCUGCUUU SEQ ID RNA
GGGCAAUGGUCCGCCGAAGUCCAUUGUCCG
NO: 47 GGAAUGUUGAUGAUCGGC GGCUUU
SEQ ID RNA
GGGGC A AUGGUC C AC C GA AGUCC GUUGGCU NO: 48 CCGUAUUUUCAAGGUCGGUGGCUU
SEQ ID RNA
GGCAAUAGUCCGCCGAAGUCUUUUGCCACG
NO: 49 UAUUCUUCAAGGUCGGCGGCUU
SEQ ID RNA
GGGCACUGGUCCGCCGAAGUCCUUUGACUC
NO: 50 GGUUUAUUCAUGGUCGGCGGCAU
SEQ ID RNA
GAGGCAAUGGUCCGCCCAAGUCCUUUGCCU NO: 51 CAGUUUAUUCAAGGUGGGCGGCUU
SEQ ID RNA
GAGUCAUGAGUCCGCCGAAGUCUCAUGGCU NO: 52 CGGUUUUCUGCAGGUCGGCGGCUGU
SEQ ID RNA
UUAGUACGCCGAAGUCUUAUGGCUCUAUUC
NO: 53 CAGGUCGGCGUCUG
SEQ ID RNA
GAGUCAUAAGUCCACCGAAGUCUUUUGGCU NO: 54 CUGUUUUCUCCAGGUCGGUGGCUG
SEQ ID RNA
AUAAGUCUGCCGAAGUCUUUUGUCAGUGUU NO: 55 UAUUCCGGGUCGGCAGCCG
SEQ ID RNA
UUAGUCCGGCGGAGUCUAUUGUUUCGGUUU NO: 56 UUUCCAGGUCGCCGGCUG
SEQ ID RNA
GAGGAAUUAGUCCGCAGAAGUCUCUUUCCU
NO: 57 CGGUUGGUUCCAGGUCUGCGGCUG
SEQ ID RNA
GAGGCAUGAGUCCGCCGAAGUCUCAUGUUU NO: 58 CGGUUUCCUCAAGGUCUGCGGCUU
SEQ ID RNA
GAGUGACUAGUCCGGCCAAGUCUAUUCGCU
NO: 59 CGGUUUCUUUACAGUGGCCGGGGU
SEQ ID RNA
GAGCCUUUAGUCCGUCGAAGUCUUUUAGCU
NO: 60 CGGAUUUAUCAUGGUCGGCGGCAU
SEQ ID RNA
UAGGCAUUAGUCAGCCGAAGUCUUUUGCCU
NO: 61 GGAUUUAUUUCGUGGUCGGCUGCAC
SEQ ID RNA
GGGACAUCAGUCCGACGAAGUCUGAUGGCU
NO: 62 CGGCUUACUCAUGUUCGUCGGCAU
SEQ ID RNA
CAGGCAGUAGUCCACCGAAGUCUACUGGCU
NO: 63 CGGUUAUAUACAGGUCGGUGGUUG SEQ ID RNA
AGGC AGUAGUC C AC CGAAGUCUACUGGCU
NO: 64 CGGUUAUAUCAGGUCGGUGGCUG
SEQ ID RNA
GAGCCAUAAGUCCACCGAAGUCUUUUGGCA NO: 65 CGUUUGGUUAAUGGUCGGUGGCAUU
SEQ ID RNA
GAGACAUAAGUCAGCCGAAGUCUUCUGGCA
NO: 66 CGUUUGGUUAAUGUUCGGUGGCAUU
SEQ ID RNA
CCAUUAGUCCGCCGAAGUCUAUUGGGUACG
NO: 67 UCAUUUUAUGGUCGGCGGCAU
SEQ ID RNA
GCCAUUAGUACGGCGAAGUCUCUUGGUGCG
NO: 68 UCCUUUUUAUGGUCGGCGUCAU
SEQ ID RNA
GAAGCAAGAGUUCGCCGAAGUCUCUUGCCU
NO: 69 CGGUAUAUCACUGUCGGCGUGUG
SEQ ID RNA
GACGCAUACGUACGCCGAAGUCAUAUGGUU
NO: 70 CGGUAUUUUCACUGUCGGCGGGUG
SEQ ID RNA
GAGGCAUUAGUCAGCCGAAGUCUAUUGGCU NO: 71 CGGUUUGUACAAGGUCGGCUGCUGU
SEQ ID RNA
GAGGCAUGAGUCCACCGAAGUCUCAUGUCU
NO: 72 CGGUAUGAUCAUGGUCGGUGGCAU
SEQ ID RNA
GAGGCAUGAGUCCACCGAAGUCUCAUGUCU NO: 73 CGGUAUGAUCAUGUCGGUGGCAUU
SEQ ID RNA
GAGGCUUAGUCCACCGAAGUCUUUUGCCUC NO: 74 GGUUUGUUGAUGGUCGGUGGCAUU
SEQ ID RNA
AGGCAUUAGUCCGCAGAAGUCUUUUGCCUC NO: 75 GGUUUUUUUGAUGGUGUGCGUCAUU
SEQ ID RNA
GAGGCAUUAGUCCGCCGAAGUCUUUUGCCU
NO: 76 CGGUAUUUUCAUGGUCGGCGGCAU
SEQ ID RNA
GAGACAUCAGUCCACCGAAGUCUUCUGCCU NO: 77 CGGUUUGUUCAUGGUGGGUGGCAU
SEQ ID RNA
GGCAAUUGUCAGCCGAAGUCAUUUGCCACG
NO: 78 UUCCUUUCAUGGUCGGCUGCAU
SEQ ID RNA
GAGGCAUUAGUCCGCCGAAGUCUUUUGGCU
NO: 79 CGGUUUUUUUGAUGGUCGGCGGCAUU
SEQ ID RNA
GAGGCAGCUGUCCGCCGAAGUCAUUUGGCU
NO: 80 CGGUUUUAUGAUGGUGGCGGCAUU SEQ ID RNA
GAGGCAUCGGGCAGCCGAAGUCCUUUGGCU NO: 81 CGGUAUUUUUGCUGAUCGGCUGCAGU
SEQ ID RNA
AGCCAUCAGUCCUCCGAAGUCCUUUUGCUC NO: 82 GGCAUUUUGACGGUAGGAGGCAUU
SEQ ID RNA
GAGUCGAUAGUCCACCGAAGUCUCUCGGCU
NO: 83 CGGUUUGGUUCUGGUCGGUUGCAG
SEQ ID RNA
GAGGCAAAAGUCCACCGAAGUCUUUUGGUU NO: 84 CGAUUCUUUCAUGGUCGGUGGCAU
SEQ ID RNA
GCCACUAGUCGACCGAAGUCUUUUGGCUUG NO: 85 GUUAUUUCACGGUCGGUCGCGU
SEQ ID RNA
GAGACAACAUUAGCCGAAUUCUUUUGUCAC NO: 86 GGUUUUUUCAUGGUCGGCUGCAU
SEQ ID RNA
GGCAAUAGUUAGCUGGAGUCUAUUCGCCCU
NO: 87 GUUAUGUACAGUUCGGCUGCUGU
SEQ ID RNA
GAGUCAUAAGUUAGCCGAAGUCUUUUGGCU NO: 88 CGAGGACGUAUAGGUCGGCUGCUGU
SEQ ID RNA
GACGCACGAGUCAGCCGAAGUCUCCUGUGU
NO: 89 CGGUCCUUACAUGGUCGGCUGCAU
SEQ ID RNA
AGCUAUUAUUCAGCCAAAGUCUAUUAGCUC
NO: 90 CGUUCAUUCAAGGUCGGCUGCUU
SEQ ID RNA
GGCAUUAAGUCAGCCGAAGUCUUAUGGGUC
NO: 91 CUUUGCUCACGGUCGGCUGCGU
SEQ ID RNA
GAGACAUAAGUCCGUCCAAGUCUUGUGUCU
NO: 92 UCGUUUGUUCACGGUGGGCGGCGU
SEQ ID RNA
GGCAUCAGUCCACGGAAGUCUGUUGGUUCG
NO: 93 AUUCCUUUAUGGUCCGUUCAU
SEQ ID RNA
GUGACAUAUGUCCGCCGAUGUCAUAUGUCU NO: 94 CGAGUCCUUGAAGGUCGGCGUCAUU
SEQ ID RNA
GAGACAUUUGUACGCGGAAGUCAUCUGUCU
NO: 95 CGGUUACUUAAUGGUCUGCGGCAUU
SEQ ID RNA
AGCCUUAGGUACACCGAAGUCUUAUGGCUG
NO: 96 GGUUUUAUCAUGGUCGGUGUCAU
SEQ ID RNA
CAGCCAUUGGCACACCGAAGUCCUUUGGAU
NO: 97 GGUUCUAUUCACGGUCGGUGUCGU SEQ ID RNA
GUGGCAUUGGUACGCCGAAGUCUAAUGUCA
NO: 98 CGCCUUAUUCACGGUCGGCGUCGU
SEQ ID RNA
GAGACAUUGGUACGCCGAAGUCCUCUGGCU
NO: 99 CGGUUUGUUCACGGUCGGCGUCGU
SEQ ID RNA
GUGGCAUAAGUUCGCCGAAGUCUUAUGGCU
NO: 100 CGGUUUUGUCAUGGUCGGUGGCAU
SEQ ID RNA
GUGGCAUUAGUGCGCCGAAGUCUAAUGGCU NO: 101 CGGUGUUUUCAUGGUCCGCGGCAU
SEQ ID RNA
GAUGCAUUAGUCGGCCGAAGUCUCUGCUUC NO: 102 GUCUGUUUAUGGUCGGCCGCAU
SEQ ID RNA
GCAUCAGUCCGCAGAAGUCUGUUGCUUCGG NO: 103 UUUUUUCAUGGUCUGCGGCAU
SEQ ID RNA
GAAGCAUUAGUCCGCCGAAGUUAUUGGUUC
NO: 104 GGAUGUUGAAUGGUCGGGGGCAUU
SEQ ID RNA
GAACCAUGAGUCCGCCGAAGUCUUAUGGCU NO: 105 CGUUUGUUGGUUGGUCGGCGGCAAU
SEQ ID RNA
GAGCCAUAAGUCUGCAGAAGUCUUAUGGGU NO: 106 UGGUGUUUUGAUGGUCUGCGGCAUU
SEQ ID RNA
GAGACAUUAGUCCGCCGAAGUCUUUUGUCU NO: 107 CGGUUUUUUACAUGUUCGGCGGCAU
SEQ ID RNA
GAGCCAUUAGUCCGUCGAAGUCUAUUGGCU
NO: 108 CGGUUUGUACAUGUUCGGCGGCAU
SEQ ID RNA
GAGGAAAUAGUCCGACGAAGUCUAUUGCCU NO: 109 CGUUUCCCUCAUGUUCGUCGGCAU
SEQ ID RNA
GAGGCAUUAGUCCGCGGAAGUGUAUUGUCU NO: 110 CGUUUCCUUCAAGUUCAGUGGCUU
SEQ ID RNA
AGUUACUACUCCGCCGAAGUCUUUUGGCUG NO: 111 GGAUCAUUCAUGGUCGGCGGCAU
SEQ ID RNA
GCCACUAGUCUGCCGAAGUCUUUUGGCGCG
NO: 112 GUAUAUUCAUGGUCGGCAGCAU
SEQ ID RNA
CAAUAGUUCGCCGAAGUCUUUUCGCGCUGU NO: 113 UAUUUCAUGGUCGGCGGCAU
SEQ ID RNA
GAGCCUUAAGUCCGCGUAAGUCUUUUGCCU NO: 114 CUGUCUAUUCAUGGUCGGCGGCAU SEQ ID RNA
GAGUCAUAGGUCCGCCGAAGUCCUUUGCUC NO: 115 UGUUCCUUCAUGGUGGCGGCAU
SEQ ID RNA
GAGGCAUAAGUCCGCCGAAGUCUUUUGGCU NO: 116 CGGUUCAUUCAUGGUCGGCGGCAU
SEQ ID RNA
GAGUCAUAAGUCCGCCGAAGUCUUUUGACU NO: 117 CGUGUUUUUCAUGGUCGGCGGCUU
SEQ ID RNA
GAGCCAUUAGUCCGCCGAAGUCUAUUCGCU NO: 118 CGGUUUUUCAAGGUCGGCGGCUU
SEQ ID RNA
GAGCCAUUAGUCCACCGAAGUCUUAUGGCC NO: 119 CGGUUUUAUCCAGGUCGGUGGCUG
SEQ ID RNA
GGGGCAUAAGUCCACCGAAGUCUUUUGGCC NO: 120 CGGGAUUUUGCAGGUCGGUGGCUGU
SEQ ID RNA
GACGCAUUGGUCCACCGAAGUCCUCUGCCU
NO: 121 CGGUCCUGUAUAGUUCGGUGGUUGU
SEQ ID RNA
GGGGCAUUGGUACACCGAAGUCCACUGGUA NO: 122 CCGUCUUUUACAGGUCGGUGUCUGU
SEQ ID RNA
GAGGCAUAAGUCCACCGAGUCUUAUGGCUC
NO: 123 GGUACUUUCAUGGUCGGUGGCUG
SEQ ID RNA
GUGGCAUUGGUCCGAGGAAGUCCAUUGUCA NO: 124 CGGUUUAUACCAGGUCCUCGGCUG
SEQ ID RNA
AGUCAUUUGUCCGCGGAAGUCAUUUGGCUA
NO: 125 CGUUGUUAUCAGGUCCGUGGCUG
SEQ ID RNA
UUGCCAUAAGUCCGUCGAAGUCUUCUGGCU NO: 126 AGUUAAUAUGUAGGUCGGCGGUUGU
SEQ ID RNA
UAGCCAUUAGUCCGGCGAAGUCUUCUGGCU NO: 127 AGGUUAUUAACGGGUCGUCGGCUGU
SEQ ID RNA
GAGGCAUUAGUCCGUAGAAGUCUAAUGGCA NO: 128 CGAAUAUUUCCAGGUCUACGGCUG
SEQ ID RNA
GAGGCAUAAGUCGCAGAAGUCUUAUGUCAC
NO: 129 GGUGUCAUCCAGGUCUGCGGCUG
SEQ ID RNA
AGUCAUUAGUCCGCAGAAGUCUAUUGUCUU NO: 130 GGAUUUUUCAGGUCUGCGGCUG
SEQ ID RNA
GAGUCAUUAGUCCGCAGAAGUCUGAUGGUU NO: 131 CGGUUUUUGGCGGGUCUGCGGCCGU SEQ ID RNA
AGGCAGUAGUCCGACCAAGUCUCCUGUCUG NO: 132 UUUGUUUUCAGGUGGUCGGCUG
SEQ ID RNA
GGGCAAUAGUCCGACGAAGUCUUUUGUCCC NO: 133 GGUUUUAUCCAGGUCGUCGGGUG
SEQ ID RNA
GGGGC AUU AGUC CGC C GA AGUCU A AUGGC C
NO: 134 CAGUUUGUUCCAGUUCGGCGGCUGU
SEQ ID RNA
GAGCCAUUAGUCCGCCGAAGUCUUUUGGCU NO: 135 CGGUUGAUUGCAGUUCGGCGGCUGU
SEQ ID RNA
GGCAUUAGUCCGCCGAAGUCUUUUGCCUUG NO: 136 GUAUUCUACAGGUCGGCGGCUGU
SEQ ID RNA
GGGGCAUUAGGCCGCUGAAGUCUAAUCGCC NO: 137 CUGAUGUUCAAUGUUCGGCGGCAU
SEQ ID RNA
GGCAUUAGGCCGUCGAAGUCUAAUGCUUAC
NO: 138 AGGGAUCUAUGUUCGGCGGCAU
SEQ ID RNA
AGGCAUUAGGCCGUCGAAGUCUAAUGCUUA NO: 139 CAGGGAUCUAUGUUCGGCGGCAU
SEQ ID RNA
AGUAGGCCGCUGAAGUCUACUUGACUGGGA NO: 140 GAUCUAUGUUCGGCGGCAU
SEQ ID RNA
AGUAGGCCGCUGAAGUCUACUUGACUGGGA NO: 141 GAUCUAUGUUCGGCGGCAU
SEQ ID RNA
GAGCCAGUAGGUCGCCGAUGUCUUCUGGCU NO: 142 GGGGAUUCAUACGUUCGGCGGCGU
SEQ ID RNA
C AUUAGGC AGC CGA AGUCU AAUGGCUC GGG NO: 143 UAUACUACGUUCGGCUGCGU
SEQ ID RNA
AGUAGGCCGACGGGUCUAGUGCUUGGGCUG
NO: 144 UUUUCAGUUCGUCGGCUG
SEQ ID RNA
GGGGCAUUGGUCAGACGAAGUCCAUUGCCU NO: 145 CGGGUAACCUCAGGUCGUCUGCUG
SEQ ID RNA
GAGCCAUAAGGCCACCGAAGUCUAAUGGCU
NO: 146 CGGGUACUCUCAGUUCGGCGGCUG
SEQ ID RNA
GAGUCAUUAGGCCGGCGAAGUCUAAUGGCU NO: 147 CGGGUAUUCUCAGUUCGGCGGCUG
SEQ ID RNA
GGGCCAUUAGGGCUGCGAAGUCUAAUGGCU NO: 148 CGAGUGUUGUCAGUUCGCCGCCUG SEQ ID RNA
GAGACAUUAGGCCGACGAAGUCUAAUGGCU NO: 149 CUUAUGGUCUCAGUUCGUCGGCUG
SEQ ID RNA
GAGCAUUAGGCCGCCGAAGUCUAAUGUCUC NO: 150 GGGAGUUCUCAGUUCGGUGGCUG
SEQ ID RNA
GUGGCAUUAGGCCGUCGAAGUCUAAUGUCU NO: 151 CGGCGGUUUCCAGUUCGGCGGCUG
SEQ ID RNA
AUUAGGCUGCAGAGUCUAAUGGCUUGUGUG NO: 152 UUUCCAGUUCUGCAGCUG
SEQ ID RNA
AGACAUUAGGCCGUCGAAGUCUAAUGUCUA
NO: 153 CGGUGUUCUAAGUUCGGCGGCUU
SEQ ID RNA
GAGACGUUAGCCCGCCGAAGUCUAAUGUCU NO: 154 CGGGUCUUGUCAGUUCGGCGGCUG
SEQ ID RNA
GAGGCAUUAGUCCGCCGAAGUCUAAUGGCU
NO: 155 CGUGUUUUCUAAGUUCGGCGGCUU
SEQ ID RNA
AGGCAUUCGUCCGCCGGAGUCGAAUCGCCU NO: 156 GGGUAUACUCUGUUCGGCGGCAG
SEQ ID RNA
AUUGGGCCGCCGGAGUCCAAUGCCUCGGAA NO: 157 GUCCAAUGUUCGGCGACAUU
SEQ ID RNA
GAUGCAUUAGGCCGGCGAAGUCUAAUGCUU NO: 158 CGGUUGUUCUAUGUUCGUCGGCAU
SEQ ID RNA
GGCACAUAAGGUCCUCGAAGUCUUAUGUGU
NO: 159 CGGCUGUUCUAUGUUCGGGGACAU
SEQ ID RNA
GUGUCAAAAGGCCGUCGAAGUCUUUUGGCU NO: 160 CUGGUUUUGUAUGUUCGGCGGCAU
SEQ ID RNA
GUGUCAUUAGGCUACCGAAGUCUAAUGGCU NO: 161 CGGAUAUUCUAUGCUCGGUGGCAU
SEQ ID RNA
GUGGCAUUAGGCCACCGAAGUCUAAUGGCU NO: 162 CGGAUUUUCAAUGUUCGGUGGCAUU
SEQ ID RNA
UGGCAUUAGGCCGUCGAAGUCUAAUGGCUA
NO: 163 GGAUCUUCUAUGUUCGGCGGCAU
SEQ ID RNA
GCUAUUAGGCC GCC GGAGUCUAAU AGCUAG NO: 164 GUUUUACCAUGUUCGGCGGCAU
SEQ ID RNA
GUGCCAUUGGGCCGGCGAAGUCUAAUGCCU NO: 165 CGGGUGUUCUAUGUUCGGCGGCAU SEQ ID RNA
GUGGCAUUAGGCCGUCGAAGUCUAAUGUCC NO: 166 CAGGUGUCCUAUGUUCGGCGGCAU
SEQ ID RNA
GUGGCAUUAGGCCGUCGAAGUCUAAUGUCC NO: 167 CAGGUGUCCUAUGUUCGGCGGCAU
SEQ ID RNA
GUGGCAUUAGGCCGUCGAAGUCUAAUGUCC
NO: 168 CAGGUGUCCUAUGUUCGGCGGCAU
SEQ ID RNA
GAGGCAUUAGGCCGUCGAAGUCUAAUGUCU NO: 169 CGAGUGUGAUAUGUUCGGCGGCAU
SEQ ID RNA
GAGGCAUUAGGCCGACGGAGUCUAAUGGCU NO: 170 CGGGUUUCCCAUGUUCGUCGGCAU
SEQ ID RNA
GAGGCAUAGGCCGACGGAGUCUAAUGGCUC NO: 171 GGGUUUCCCAUGUUCGUCGUCAU
SEQ ID RNA
GAGGCAUUAGGCCGACGGAGUCUAAUGGCU
NO: 172 CGGGUUUCCCAUGUUCGUCGGCAU
SEQ ID RNA
GAGGCAUUAGGUCGACGGAGUCUAAUGGCU NO: 173 CGGGUUUCCCAUGUUCGUCGGCAU
SEQ ID RNA
AGGCAUUAGGCCGACGGGGUCUAAUGGCUA
NO: 174 GGGUUUCACAUGUUCGUCGGCAU
SEQ ID RNA
GUGGCAUUAGGCCGACGGAGUCUAAUGGCU NO: 175 CGGUUUUCCCAUGUUCGUCGGCAU
SEQ ID RNA
GGCAUUAGGCCGACGGAGUCCAAUGGUUCG
NO: 176 GGUUUCCCAUGUUCGUAGGCAU
SEQ ID RNA
GAGGCAUUAGGCCGUCGGAGUCUAAUGGUU NO: 177 CGGGUUUCCCAUGUUCGUCGGCAU
SEQ ID RNA
GAGCCGUUAGGCCGACGGAGUCUAAUGGCU NO: 178 CGGGUGUCCCAUGUUCGUCGGCAU
SEQ ID RNA
GAGGCAUUAGGCCGACGGAGUCUAAUGGCU NO: 179 CGUGUUUCCCAUGUUCGUCGGCAU
SEQ ID RNA
GAGUCAUUAGGCCGAUGGAGUCUAAUGGCU
NO: 180 CGGGUUUCCCAUGUUCGUCGGCAU
SEQ ID RNA
GUGUCAUUAGGCCACCGGAGUCUAAUGGCA NO: 181 CUGGUGUCUGCAGUUCGGUGGCUGU
SEQ ID RNA
GGGUC AU A AGGC C AC C GGAGUCUU AUGGC C NO: 182 CUGGAAGUCUAUGUUCGGUGGCAU SEQ ID RNA
GUGUCAUUAGGCCACCGGAGUCUAAUGGCU NO: 183 CGGGUAAUCUAUGUUCGGUGGCAU
SEQ ID RNA
GUGUUAUUAGGCCACCGGAGUCUAAUGGCA NO: 184 CUGUUGUCUGCGUUCGGUGGCUGU
SEQ ID RNA
GAGUCAUUAGGCCGCCGGAGUCUAAUGGCU
NO: 185 CGUGUGGUCUACGUUCGGCGGCGU
SEQ ID RNA
GAGCCAUUAGGCCGCCGGAGUCUAGUGGUU NO: 186 CGCGUAUUCAAUGUUCGGCGGCAUU
SEQ ID RNA
GAGGCAUUAGGCCGCCGGAGUCUAAUGGUU NO: 187 CGUGUGUACUAUGUUCGGCGGCAU
SEQ ID RNA
GAGGCAUUAGGCCGCCGGAGUCUAAUGGCU NO: 188 CGUGUGUCCUAUGUUCGGCGGCAU
SEQ ID RNA
GGC AUUAGGCC GCC GGAGUCUAAUGGC C GU
NO: 189 GUUUCCUAUGUUCGGCGGCAU
SEQ ID RNA
GGCAUUAGGCCGACGAAGUCUAAUGUCUGG NO: 190 GGGGUUGUCUGUUCGUCGGCAG
SEQ ID RNA
GAGGCAUUAGGUCGCAGAAGUCUAAUGCGU NO: 191 GGGGGAUUCUUUGUUCGGCGGCAG
SEQ ID RNA
UAGGCAUUAGGCCGACGGAGUCUAAUGGCU NO: 192 GGGUUACUGUAUGUUCGUCGGCAU
SEQ ID RNA
GAGGCAUUAGUCCGCCGGAGUCUAAUACCU
NO: 193 CGUGUGUCUUACGUUCGGCGGCGU
SEQ ID RNA
AGGGAUUAGGCCGCCGGAGUCUAACCCCUA NO: 194 GAGUGUCUUAUGUUCGGCGGCAU
SEQ ID RNA
GUGGCAUUAGGCAGUCUAAGUCUAAUGCUU
NO: 195 CGGUAGUUUACGUUCGGCUGCGU
SEQ ID RNA
GAGGCAUUAGGCCACCUAAGUCUAAUGUUU NO: 196 CGCUUGAUGUAUGUUCGGCGGUAU
SEQ ID RNA
GAGGCAUUAGGCCGUCGAAGUCUAAUGUCU
NO: 197 CGGGUGUGUUAUGUUCGGCGGCAU
SEQ ID RNA
GAUGCGUUAGGCCGCCGGAGUCUAACGAAU NO: 198 CGGGUCUUGUAUGUUCGGCGGCAU
SEQ ID RNA
GAUGCAUCAGGCCGGCGAAGUCUAAUGCAU
NO: 199 CGAGUGUUCUAUGUUCGACGGCAU SEQ ID RNA
GAUGCAUUAGGCCGCCGGAGUCUAAUGCAU
NO: 200 CGGUUGUCCCAUGUUCGGCGGCAU
SEQ ID RNA
AGUGCAUUAGGCCGUCGAAGUCUAAUGCAU NO: 201 UGGUUGUCCUAUGUUCGGUGGCAU
SEQ ID RNA
GGGGUAUUAGGCCGUCGAAGUGUAAUGCCU
NO: 202 UGAGUUACCAUGUCGGCUGCAU
SEQ ID RNA
GUGGCAUUAGGCCGUUGGAGUCUAAUACCA
NO: 203 GGAUUGUCCGAUGCUCGGCUGCAUU
SEQ ID RNA
GAGGC AUUAGGC CGCUGAAGUCUAAUAC CU
NO: 204 CGACAGUUCUAUGUUCGGUGGCAU
SEQ ID RNA
GAGGCAUUAGGCCGUCGAAGUCUAAUGGCU NO: 205 CGUUAGUUCUAUGUUCUGCGGCAU
SEQ ID RNA
GAGGCAUUAGGCCGUCGAAGUCUAUUGGCU
NO: 206 CGGGAAUUCUAUGUUCGGCGGCAU
SEQ ID RNA
GAGGCAUUAGGCCGGCGAAGUUUAAUGGCU NO: 207 CAGGAAUCCUAUGUUCGGGGGCAU
SEQ ID RNA
GAGGCAUUAGGCCGCCGGAGUUAAUGGCUC
NO: 208 GGAUGUUGAUGUUCGGCGGCAUU
SEQ ID RNA
GAGGCAUUAGGCCGCCGGAGUCUAAUGGCU
NO: 209 CGGAUGUCUGAUGUUCGGCGGCAUU
SEQ ID RNA
GAGGCAUUAGGCCGACGGAGUCUAAUGGCU
NO: 210 CGUGUUCACUGAGUUCGUCGGCUC
SEQ ID RNA
GGGUCAUUAGGCCGGCGGAGUCUAAUGACG NO: 211 CGGUUGUACUCAUUUCGCCGGAUG
SEQ ID RNA
GUGUCAUUAGGCCGGCGGAGUCUAAUGGCU NO: 212 CGGGUGUUAUCAGUUCGCCGGCUG
SEQ ID RNA
GGGCAUUAGGCUGGCGGAGUCUAAUCCCUC NO: 213 GGUUGUUAUCUGUUCGCCAGCAG
SEQ ID RNA
GAGACAUUAGGCCAUCGGAGUCUAAUGCCU
NO: 214 CGGACGUACUCAGUGCGGUGGCUG
SEQ ID RNA
GAGAUU AGGCC ACC GGAGUCUAAUGCCUC G NO: 215 GACGUAUUCAGUUCGGUGGCUG
SEQ ID RNA
GAGUCAUUGGCCACCGGAGUCUAAUGUCUC NO: 216 GGACGUACUCAGUUCGGUGGCUG SEQ ID RNA
GUACCAUUAGGCCACCGAAGUCUAAUGGUU NO: 217 CGAGUGUUAUCAGUUCGGUGGCU
SEQ ID RNA
GUACCAUUAGGCCACCGAAGUCUAAUGCUU NO: 218 CGAGUGUUAUCAGUUCGGUGCUG
SEQ ID RNA
GAGCCAAUAGGCCACCGGAGUCUAUUGGCU
NO: 219 GGGUUGUCCUCAGUUCGGUGGCUG
SEQ ID RNA
GAGCCAAUAGGCUACCGGAGUCUAUUGGCU NO: 220 GGGUUGUCUCAGUUCGGUGGCUG
SEQ ID RNA
UAGCCAAUAGGCCAUCGGAGUCUAUUGGCU NO: 221 GGGUUGUCCUCAGUUCGGUGGCUG
SEQ ID RNA
GAGCCAAUAGGCCACCGGAGUCUAUUGGCU NO: 222 GGGUUCUCUUCAGUUCGGUGGCUG
SEQ ID RNA
GAGCCAAUAGGUCACCGGAGUCUAUUGUCU
NO: 223 GGGUUGUCCUCAGUUCGGUGGCUG
SEQ ID RNA
GAGGCAUUAGGCCUCUGAAGUCUAAUGGCU NO: 224 CGUAAUUUCUCAGUUCGGUGGCUG
SEQ ID RNA
GAUGCAUUAGUCCGCCGAAGUCUAAUGCGU
NO: 225 CGGGUCUUCUCAGUUCGGCGGCUG
SEQ ID RNA
GGUGCAUUAGUCCGCCGGAGUCUAAUGUAU NO: 226 CGGGUCGUCUCAGUUCGGCGGCUG
SEQ ID RNA
GUGGCAUUAGGCUGCCGAAGUCUAAUGUCA
NO: 227 CGGUUUAUCUCAGUUCGGCAGCUG
SEQ ID RNA
GUGGCAUUAGGCUGCCGAAGUCUAAUGUCA NO: 228 CGGGUGAUUUCAGUUCGGCAGCUG
SEQ ID RNA
GUGGUAUUAGGCCGUCGAAGUCUAAUGCCU
NO: 229 CGGUUGUUCGCAGUUCGGGCUGU
SEQ ID RNA
CAGUCAUUAGGGCGUAGAAGUCUAAUGUCU NO: 230 AGAGUGUUCUCCGUUCUGCGCCGG
SEQ ID RNA
CAAGCAGUAGGCCGACGAAGUCUACUGUCU
NO: 231 UGGAUGUUGUCAGUUCGGCGGCUG
SEQ ID RNA
GAGGCAUUAGGCCGACGGAGUCUAUUGCCU NO: 232 CGGGUGUUCUCAGUUCGUCGGCUG
SEQ ID RNA
GAGGCAUUAGGCCGACGGAGUCUAAUGUCC
NO: 233 CGGACUUCCCAGUUCGGCAGCUG SEQ ID RNA
GAGUCAUUAGGCCGACGGAGUCUAAUGCCU
NO: 234 CGGAACUUUACAGUUCGUCGGCUGU
SEQ ID RNA
GAGGCACUAGGCCGCCGAAGUCUAUUGGCU NO: 235 CGGGUGUUCUCAUUUCGGCGGAUG
SEQ ID RNA GGGAAGAGCUAGCGCUACAAGGCAUGUGUC CGCAGAAGUCAUAUGGACUUGAUUUUUUCA
NO: 236
UGGUCUGCGGCAUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGAAGUUGUU CGCAGAAGUCAACUGUCUCGGAAUUUUCAA NO: 237
GGUCUGCGGCUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGUGACUAGUC CGGCCAAGUCUAUUCGCUCGGUUUCUUUAC NO: 238
AGUGGCCGGGGUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGGCAGUAGUC CGACCAAGUCUCCUGUCUGUUUGUUUUCAG NO: 239
GUGGUCGGCUGUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGACAUAAGUC CGUCCAAGUCUUGUGUCUUCGUUUGUUACG NO: 240
GUGGGCGGCGUUGAUACUUGAUCGCCCUAG AAGCC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAUACAUUAGUG CGCUGAAGUCUAAUGAAUCAGUUUUUUCCC NO: 241
GUCGGCGGGUGUGAUACUUGAUCGCCCUAG AAGCA
SEQ ID RNA GGGAAGAGCUAGC GCUACGAUGCAUUAGUC GGCCGAAGUCUCUGCUUCGUCUGUUUAUGG NO: 242
UCGGCCGCAUUGAUACUUGAUCGCCCUAGA AGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGCCAUUAGUC CGCCGAAGUCUAUUCGCUCGGUUUUUCAAG
NO: 243
GUCGGCGGCUUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGUCAUGAGUC CGCCGAAGUCUCAUGGCUCGGUUUUCUGCA NO: 244
GGUCGGCGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGUCAUUAGUC CGCAGAAGUCUGAUGGUUCGGUUUUUGCGG NO: 245
GUCUGCGGCCGUGAUACUUGAUCGCCCUAG AAGCG
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGCCAUUAGUC CGCCGAAGUCUUUUGGCUCGGUUGAUUCAG NO: 246
UUCGGCGGCUGUGAUACUUGAUCGCCCUAG AAGCG SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUUAGUC
CGUAGAAGUCUAAUGGCACGAAUAUUUCCA
NO: 247
GGUCUACGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUAAGUC GCAGAAGUCUUAUGUCACGGUGUCAUCAGG NO: 248
UCUGCGGCUGUGAUACUUGAUCGCCCUAGA AGCC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGGAAUUAGUC CGCAGAAGUCUCUUUCCUCGGUUGGUUCCA NO: 249
GGUCUGCGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACCAGUCAUUAGUC CGCAGAAGUCUAUUGUCUUGGAUUUUUCAG NO: 250
GUCUGCGGCUGUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGGUAU AAGUC CGCGGAAGUCUUUUGUCUCGGGUAUAUCAG NO: 251
GUCCGCGGCUGUGAUACUUGAUCGCCCUAG AAGCG
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGUCAUAAGUU AGC CGAAGUCUUUUGGCUC GAGGACGUAUA NO: 252
GGUCGGCUGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGUCAUUUGUC CGCGGAAGUCAUUUGGCUACGUUGUUAUCA NO: 253
GGUCCGUGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACUUGCCAU AAGUC CGUCGAAGUCUUCUGGCUAGUUAAUAUGUA
NO: 254
GGUCGGCGGUUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACUAGCCAUUAGUC CGGCGAAGUCUUCUGGCUAGGUUAUUAACG NO: 255
GGUCGUCGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACC GGGC AAUAGUC CGACGAAGUCUUUUGUCCCGGUUUUAUCCA NO: 256
GGUCGUCGGGUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUUAGUC CGGCGGAGUCUAUUGUUUCGGUUUUUUCCA NO: 257
GGUCGCCGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGUGGCAUUGGUC CGAGGAAGUCCAUUGUCACGGUUUAUACCA NO: 258
GGUCCUCGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUUAGUA
CGCCGAAGUCUUAUGGCUCUAUUCCAGGUC NO: 259
GGCGUCUGUGAUACUUGAUCGCCCUAGAAG C
SEQ ID RNA GGGAAGAGCUAGCGCUACGGGGCAUUAGUC CGCCGAAGUCUAAUGGCCCAGUUUGUUCCA
NO: 260
GUUCGGCGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUAAGUC UGCCGAAGUCUUUUGUCAGUGUUUAUUCGG NO: 261
GUCGGCAGCCGUGAUACUUGAUCGCCCUAG AAGCC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGUCAUAAGUC CACCGAAGUCUUUUGGCUCUGUUUUCUCCA
NO: 262
GGUCGGUGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCCAUUAGUC CACCGAAGUCUUAUGGCCCGGUUUUAUCCA
NO: 263
GGUCGGUGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGGGGCAUAAGUC CACCGAAGUCUUUUGGCCCGGGAUUUUGCA
NO: 264
GGUCGGUGGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUAAGUC CACCGAGUCUUAUGGCUCGGUACUUUAUGG
NO: 265
UCGGUGGCUGUGAUACUUGAUCGCCCUAGA AGCC
SEQ ID RNA GGGAAGAGCUAGCGCUACCAGGUAGUAGUC CACCGAAGUCUACUGGCUCGGUUAUAUACA NO: 266
GGUCGGUGCUGUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACCAGGCAGUAGUC CACCGAAGUCUACUGGCUCGGUUAUAUACA
NO: 267
GGUCGGUGGUUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACCAGGCAGUAGUC CACCGAAGUCUACUGGCUCGGUUAUAUCAG NO: 268
GUCGGUGGCUGUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGGGGCAUUGGUA CACCGAAGUCCACUGGUACCGUCUUUUACA
NO: 269
GGUCGGUGUCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAC GC AUUGGUC CACCGAAGUCCUCUGCCUCGGUCCUGUAUA NO: 270
GUUCGGUGGUUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGUGGCAUCAGUC CGACGAAGUCUUUUGCCAUUUUAUGUUCAA NO: 271
GGUCGUCGGCUUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGUGGCAUUAGUC CGCCGAAGUCUUUUGCCUUGGUAUUCUACA NO: 272 GGUCGGCGGCUGUGAUACUUGAUCGCCCUA
GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGGGGCAAUGGUC CGCCGAAGUCCAUUGUCCGGGAAUGUUGAU NO: 273
GAUCGGCGGCUUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGGGGCAAUGGUC CACCGAAGUCCGUUGGCUCCGUAUUUUCAA
NO: 274
GGUCGGUGGCUUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGGGGCAAUAGUC CGCCGAAGUCUUUUGCCACGUAUUCUUCAA NO: 275
GGUCGGCGGCUUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAAUGGUC CGCCCAAGUCCUUUGCCUCAGUUUAUUCAA NO: 276
GGUGGGCGGCUUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGGGGCACUGGUC CGCCGAAGUCCUUUGACUCGGUUUAUUCAU NO: 277
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAAGCAAGAGUU CGCCGAAGUCUCUUGCCUCGGUAUAUCACU NO: 278
GUCGGCGUGUGUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUAAGUC CGCCGAAGUCUUAUGGCUCGGUUUAUUUAU NO: 279
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUAAGUC CGCCGAAGUCUUUUGGCUCGGUUCAUUCAU NO: 280
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGUUACUACUC CGCCGAAGUCUUUUGGCUGGGAUCAUUCAU NO: 281
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAUGUCAACGUC CGUCGAAGUCUUUGGCAUCGGUUUUUUCAU NO: 282
GUUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGUCAUAGGUC CGCCGAAGUCCUUUGCUCUGUUCCUUCAUG NO: 283
GUGGCGGCAUUGAUACUUGAUCGCCCUAGA AGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAUGCAUAAGUC CGCCGAAGUCUUUUGCUUAAGCUCCCGCAU NO: 284
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCCUUAAGUC
CGCGUAAGUCUUUUGCCUCUGUCUAUUCAU NO: 285
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCCAAUAGUC CGUCGAAGUCUUUUGGCCCUGUUAUUUUAU NO: 286
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGACAAUAGUU CGCCGAAGUCUUUUCGCGCUGUUAUUUCAU NO: 287
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUUAGUC UGCCGAAGUCUUUUGGCUCGGUUUAUUCAU NO: 288
GUUCGGCAGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCCACUAGUC UGCCGAAGUCUUUUGGCGCGGUAUAUUCAU
NO: 289
GGUCGGCAGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGUC GAUAGUC CACCGAAGUCUCUCGGCUCGGUUUGGUUCU
NO: 290
GGUCGGUUGCAGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGCCUUUAGUC CGUCGAAGUCUUUUAGCUCGGAUUUAUCAU NO: 291
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGCCUUUAGUC CGUCGAAGUCUUUUAGCUCGGAUUUAUCAU
NO: 292
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGCCAUUAGUC CGCCGAAGUCUUUUAGCUCGUUUUCUUCAU
NO: 293
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGACAUUAGUC CGCCGAAGUCUUUUGUCUCGGUUUUUUACA
NO: 294
UGUUCGGCGGCAUUGAUACUUGAUCGCCCU AGAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGCCAUUAGUC CGUCGAAGUCUAUUGGCUCGGUUUGUACAU NO: 295
GUUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGCCAUAAGUC CACCGAAGUCUUUUGGCACGUUUGGUUAAU
NO: 296
GGUCGGUGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGACAUAAGUC AGCCGAAGUCUUCUGGCACGUUUGGUUAAU NO: 297
GUUCGGUGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCCAUUAGUC CGCCGAAGUCUAUUGGCCGGUUGCUUAAUG
NO: 298
GUCGGCGGCAUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUCAGUC CACGGAAGUCUGUUGGUUCGAUUCCUUUAU
NO: 299
GGUCCGUUCAUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAAAAGUC CACCGAAGUCUUUUGGUUCGAUUCUUUCAU
NO: 300
GGUCGGUGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACAAGCCUUAGGUA CACCGAAGUCUUAUGGCUGGGUUUUAUCAU NO: 301
GGUCGGUGUCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACCAGCCAUUGGCA CACCGAAGUCCUUUGGAUGGUUCUAUUCAC
NO: 302
GGUCGGUGUCGUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCAAUUAGUA CUCCGAAGUCUAUUUGCCCGGUUCACACAU
NO: 303
GGUCGGAGCAUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGACAUCAGUC CACCGAAGUCUUCUGCCUCGGUUUGUUCAU NO: 304
GGUGGGUGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCUUAGUCC ACCGAAGUCUUUUGCCUCGGUUUGUUGAUG
NO: 305
GUCGGUGGCAUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUCAGUC CGCCGAAGUCUUGUGCCUUGGUUUGUUAUG
NO: 306
GUCGGCGGCAUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUUAGUC CGCUGAAGUCUUUUGGCUCGGGUUUUUGAU NO: 307
GUUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUUAGUC CGCCGAAGUCUUUUGGCUCGGUUUUUUUGA NO: 308
UGGUCGGCGGCAUUGAUACUUGAUCGCCCU AGAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAUGCAUUAGUC CGCAUAAGUCUUUUGGAUCGCUUUGUUCAU
NO: 309
GUUGUGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGUUGCAUCAGUC CGCAGAAGUCUGUUGCUUCGGUUUUUUCAU NO: 310 GGUCUGCGGCAUUGAUACUUGAUCGCCCUA
GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACAAGGCAUUAGUC CGCAGAAGUCUUUUGCCUCGGUUUUUUUGA NO: 311
UGGUGUGCGUCAUUGAUACUUGAUCGCCCU AGAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCCAUAAGUC UGCAGAAGUCUUAUGGGUUGGUGUUUUGAU
NO: 312
GGUCUGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAC GC AUUAGUC CGCCGAAGUCUCUUGCGUCAGUUUUUUUCA NO: 313
UGGUCGGCGGCAUUGAUACUUGAUCGCCCU AGAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGGCAUUAGUC CGCCGAAGUCUUUUGCCUCGGUAUUUUCAU NO: 314
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGGCAUAAGUC CACCGAAGUCUUAUGGAUCGGAUUUUUCAU NO: 315
GAUCGGUGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGUGGCAUAAGUU CGCCGAAGUCUUAUGGCUCGGUUUUGUCAU NO: 316
GGUCGGUGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGUCAUAAGUC CGCCGAAGUCUUUUGACUCGUGUUUUUCAU NO: 317
GGUCGGCGGCUUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGGCAGCUGUC CGCCGAAGUCAUUUGGCUCGGUUUUAUGAU NO: 318
GGUGGCGGCAUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGUGACAUAUGUC CGCCGAUGUCAUAUGUCUCGAGUCCUUGAA NO: 319
GGUCGGCGUCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACUAGCCAUCAGUC CUCCGAAGUCCUUUUGCUCGGCAUUUUGAC NO: 320
GGUAGGAGGC AUUGAUACUUGAUCGC CCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGGCAUCGGGC AGCCGAAGUCCUUUGGCUCGGUAUUUUUGC NO: 321
UGAUCGGCUGCAGUGAUACUUGAUCGCCCU AGAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAAGCAUCAGUC CUCCGAAGUCUUUUGGUUCGGAUUUUGUCU
NO: 322
GGUCGGGGGCAGUGAUACUUGAUCGCCCUA GAAGC SEQ ID RNA GGGAAGAGCUAGCGCUACGAAGCAUUAGUC
CGCCGAAGUUAUUGGUUCGGAUGUUGAAUG
NO: 323
GUCGGGGGCAUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAACCAUGAGUC CGCCGAAGUCUUAUGGCUCGUUUGUUGGUU NO: 324
GGUCGGCGGCAAUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGGGGCAUGACUA CGCCGAAGUCUCUUGUCUCGGCUUUCGUAU NO: 325
GUUCGGCGUCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGGGACAUCAGUC CGACGAAGUCUGAUGGCUCGGCUUACUCAU
NO: 326
GUUCGUCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGGUACAUCAGUC CGACGAAGUCUGAUGGCUCGGCUUACUCAU
NO: 327
GUUCGUCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUGAGUC CACCGAAGUCUCAUGUCUCGGUAUGAUCAU NO: 328
GGUCGGUGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUGAGUC CACCGAAGUCUCAUGUCUCGGUAUGAUCAU NO: 329
GUCGGUGGCAUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUGAGUC CGCCGAAGUCUCAUGUUUCGGUUUCCUCAA
NO: 330
GGUCUGCGGCUUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGAAAUAGUC CGACGAAGUCUAUUGCCUCGUUUCCCUCAU NO: 331
GUUCGUCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGGCAUUAGUC CGCGGAAGUGUAUUGUCUCGUUUCCUUCAA NO: 332
GUUCAGUGGCUUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCCAUUGGUC CACCGAAGUCCUAUGACUCGUUUAAUUAAU NO: 333
GUUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCCAUUGGUC CACCGAAGUCCUAUGACUCGUUUAAUUAAU
NO: 334
GUUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAUGCAUUGGUC CGCCGAAGUCCAAUGUAUCCGUUUCCUCAU NO: 335
GUUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGACAUUUGUC CGCCGAAGUCAUCUGUCUCGGUUUGUUCAC
NO: 336
GGUCGGCGGCGUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGACAUUGGUA CGCCGAAGUCCUCUGGCUCGGUUUGUUCAC NO: 337
GGUCGGCGUCGUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGACAUUUGUA CGCGGAAGUCAUCUGUCUCGGUUACUUAAU NO: 338
GGUCUGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGUGCCAUUAGUC CGCCGGAGUCUAUUGGGUACGUCAUUUCAU
NO: 339
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGUGCCAUUAGUC CGCCGAAGUCUAUUGGGUACGUCAUUUUAU NO: 340
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGAGCCAUUAGUC CGCCGAAGUCUUUUGGUUACGUUUAUACAC NO: 341
GGUCGGCGGCGUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGUGGCAUUGGUA CGCCGAAGUCUAAUGUCACGCCUUAUUCAC NO: 342
GGUCGGCGUCGUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGUGCCAUUAGUC CGCGGAAGUCUACUGUCACGGUAUCUUGAU
NO: 343
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACGUGGCAUUAGUG CGCCGAAGUCUAAUGGCUCGGUGUUUUCAU NO: 344
GGUCGGCGGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACUAGGCAUUAGUC AGCCGAAGUCUUUUGCCUGGAUUUAUUUCG NO: 345
UGGUCGGCUGCACUGAUACUUGAUCGCCCU AGAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACUAGGCAUUAGUC AGCCGAAGUCUUUUGCCUGGAUUUAUUUCG NO: 346
UGGUCGGCUGCACUGAUACUUGAUCGCCCU AGAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACCAGGCAUUAGUC AGCCGAAGUCUUUUGCCUGGGAUUUUCGAA
NO: 347
GGUCGGCUGCUUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACAUGGCAAUUGUU CGCUGAAGUCAUUUGCUACGUAUUUUUAAU NO: 348 GUCAUCGGCUGAUACUUGAUCGCCCUAGAA
GC
SEQ ID RNA GGGAAGAGCUAGCGCUACAAGGCAAUUGUC AGCCGAAGUCAUUUGCCACGUUCCUUUCAU NO: 349
GGUCGGCUGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACAUGGCAAUAGUU AGCUGGAGUCUAUUCGCCCUGUUAUGUACA
NO: 350
GUUCGGCUGCUGUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGCGCUACCAAGCUAUUAUU CAGCCAAAGUCUAUUAGCUCCGUUCAUUCA NO: 351
AGGUCGGCUGCUUUGAUACUUGAUCGCCCU AGAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAC GC AC GAGUC AGCCGAAGUCUCCUGUGUCGGUCCUUACAU NO: 352
GGUCGGCUGCAUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGACAACAUUA GCCGAAUUCUUUUGUCACGGUUUUUUCAUG
NO: 353
GUCGGCUGCAUUGAUACUUGAUCGCCCUAG AAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGCCAUUAGUC GGGGGAAGUUUUUUGGCUGGAUUAUUUCAC
NO: 354
GGUCCCCCGCGUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGC C ACUAGUCGA CCGAAGUCUUUUGGCUUGGUUAUUUCACGG NO: 355
UCGGUCGCGUUGAUACUUGAUCGCCCUAGA AGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAUGCAUUAGUC GGCCGAAGUCUGUUGUCUCGGUGUUUUCAC
NO: 356
GGUCGGCCGCGUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUACGAGGCAUUAGUC AGCCGAAGUCUGGUGUCUCAGUUUGUUUAC
NO: 357
GGUCGGCUGCGUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGGAAGAGCUAGC GCUAC AC GGC AUUAAGU CAGCCGAAGUCUUAUGGGUCCUUUGCUCAC NO: 358
GGUCGGCUGCGUUGAUACUUGAUCGCCCUA GAAGC
SEQ ID RNA GGAGCUAUUCGGAUGCGUGUCAUUAGGCCA CCGGAGUCUAAUGGCACUGGUGUCUGCAGU
NO: 359
UCGGUGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGUUAUUAGGCCA CCGGAGUCUAAUGGCACUGUUGUCUGCGUU
NO: 360
CGGUGGCUGUGAUACUUGAUCGCCCUAGAA GCAA SEQ ID RNA GGAGCUAUUCGGAUGCCGGGUCAUAAGGCC
ACCGGAGUCUUAUGGCCCUGGAAGUCUAUG
NO: 361
UUCGGUGGCAUUGAUACUUGAUCGCCCUAG AAGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCAAUAGGCCA CCGGAGUCUAUUGGCUGGGUUCUCUUCAGU NO: 362
UCGGUGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCAAUAGGCUA CCGGAGUCUAUUGGCUGGGUUGUCUCAGUU
NO: 363
CGGUGGCUGUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCUAGCCAAUAGGCCA CCGGAGUCUAUUGGCUGGGUUGUCCUCAGU
NO: 364
UCGGUGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCUAGCCAAUAGGCCA UCGGAGUCUAUUGGCUGGGUUGUCCUCAGU
NO: 365
UCGGUGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCAAUAGGCCA CCGGAGUCUAUUGGCUGGGUUGUCCUCAGU
NO: 366
UCGGUGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCAAUAGGUCA CCGGAGUCUAUUGUCUGGGUUGUCCUCAGU NO: 367
UCGGUGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGACAUUAGGCCA UCGGAGUCUAAUGCCUCGGACGUACUCAGU
NO: 368
GCGGUGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGAUUAGGCCACC GGAGUCUAAUGCCUCGGACGUAUUCAGUUC
NO: 369
GGUGGCUGUGAUACUUGAUCGCCCUAGAAG CAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGUCAUUGGCCAC CGGAGUCUAAUGUCUCGGACGUACUCAGUU NO: 370
CGGUGGCUGUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGAGAUUGGGCCG CCGGAGUCCAAUGCCUCGGAAGUCCAAUGU NO: 371
UCGGCGACAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCU AUUC GGAUGC GAGGC AUUC GUC C G CCGGAGUCGAAUCGCCUGGGUAUACUCUGU
NO: 372
UCGGCGGCAGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCAUAAGGCCA CCGAAGUCUAAUGGCUCGGGUACUCUCAGU
NO: 373
UCGGCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGUCAUUAGGCCG GCGAAGUCUAAUGGCUCGGGUAUUCUCAGU
NO: 374
UCGGCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUACCAUUAGGCCA CCGAAGUCUAAUGGUUCGAGUGUUAUCAGU NO: 375
UCGGUGGCUUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUACCAUUAGGCCA CCGAAGUCUAAUGGUUCGAGUGUUAUCAGU
NO: 376
UCGGUGCUGUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGGGCCAUUAGGGCU GCGAAGUCUAAUGGCUCGAGUGUUGUCAGU NO: 377
UCGCCGCCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGACAUUAGGCCG ACGAAGUCUAAUGGCUCUUAUGGUCUCAGU NO: 378
UCGUCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCU CUGAAGUCUAAUGGCUCGUAAUUUCUCAGU
NO: 379
UCGGUGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAUGCAUUAGUCCG CCGAAGUCUAAUGCGUCGGGUCUUCUCAGU NO: 380
UCGGCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGGUGCAUUAGUCCG CCGGAGUCUAAUGUAUCGGGUCGUCUCAGU NO: 381
UCGGCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGACGUUAGCCCG CCGAAGUCUAAUGUCUCGGGUCUUGUCAGU NO: 382
UCGGCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGC GAGC AUUAGGCC GC CGAAGUCUAAUGUCUCGGGAGUUCUCAGUU NO: 383
CGGUGGCUGUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGUAUUAGGCCG UCGAAGUCUAAUGCCUCGGUUGUUCGCAGU NO: 384
UCGGGCUGUGAUACUUGAUCGCCCUAGAAG CAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCCG UCGAAGUCUAAUGUCUCGGCGGUUUCCAGU
NO: 385
UCGGCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCUG CCGAAGUCUAAUGUCACGGUUUAUCUCAGU NO: 386 UCGGCAGCUGUGAUACUUGAUCGCCCUAGA
AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCUG CCGAAGUCUAAUGUCACGGGUGAUUUCAGU NO: 387
UCGGCAGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCACUAGGCCG CCGAAGUCUAUUGGCUCGGGUGUUCUCAUU
NO: 388
UCGGCGGAUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG ACGGAGUCUAUUGCCUCGGGUGUUCUCAGU NO: 389
UCGUCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCCAGGCAUUAGGCUG CAGAGUCUAAUGGCUUGUGUGUUUCCAGUU
NO: 390
CUGCAGCUGUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCCAGUCAUUAGGGCG UAGAAGUCUAAUGUCUAGAGUGUUCUCCGU NO: 391
UCUGCGCCGGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCCAGUCAUUAGGGCG UAGAAGUCUAAUGUCUAGAGUGUUCUCCGU
NO: 392
UCUGCGCCGGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGUCAUUAGGCCG ACGGAGUCUAAUGCCUCGGAACUUUACAGU
NO: 393
UCGUCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG ACGGAGUCUAAUGUCCCGGACUUCCCAGUU
NO: 394
CGGCAGCUGUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCCCAGCAGUAGGCCG ACGGGUCUAGUGCUUGGGCUGUUUUCAGUU
NO: 395
CGUCGGCUGUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCCAAGCAGUAGGCCG ACGAAGUCUACUGUCUUGGAUGUUGUCAGU
NO: 396
UCGGCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCCG ACGAAGUCUAAUGUCUGGGGGGUUGUCUGU
NO: 397
UCGUCGGCAGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGGGUCAUUAGGCCG GCGGAGUCUAAUGACGCGGUUGUACUCAUU
NO: 398
UCGCCGGAUGUGAUACUUGAUCGCCCUAGA AGCAA SEQ ID RNA GGAGCUAUUCGGAUGCGUGUCAUUAGGCCG
GCGGAGUCUAAUGGCUCGGGUGUUAUCAGU
NO: 399
UCGCCGGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCCGGGCAUUAGGCUG GCGGAGUCUAAUCCCUCGGUUGUUAUCUGU NO: 400
UCGCCAGCAGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGGGGCAUUGGUCAG ACGAAGUCCAUUGCCUCGGGUAACCUCAGG NO: 401
UCGUCUGCUGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCA CCUAAGUCUAAUGUUUCGCUUGAUGUAUGU NO: 402
UCGGCGGUAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAUGCAUUAGGCCG GCGAAGUCUAAUGCUUCGGUUGUUCUAUGU
NO: 403
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG UCGAAGUCUAAUGUCUCGGGUGUGUUAUGU
NO: 404
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG UCGAAGUCUAAUGUCUCGAGUGUGAUAUGU NO: 405
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG CUGAAGUCUAAUACCUCGACAGUUCUAUGU
NO: 406
UCGGUGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAUGCAUCAGGCCG GCGAAGUCUAAUGCAUCGAGUGUUCUAUGU NO: 407
UCGACGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGGGGUAUUAGGCCG UCGAAGUGUAAUGCCUUGAGUUACCAUGUC NO: 408
GGCUGCAUUGAUACUUGAUCGCCCUAGAAG CAA
SEQ ID RNA GGAGCUAUUCGGAUGCGGGGCAUUAGGCCG CUGAAGUCUAAUCGCCCUGAUGUUCAAUGU NO: 409
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCCG UCGAAGUCUAAUGUCCCAGGUGUCCUAUGU NO: 410
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCCG UCGAAGUCUAAUGUCCCAGGUGUCCUAUGU NO: 411
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCCG UCGAAGUCUAAUGUCCCAGGUGUCCUAUGU NO: 412
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCCG UUGGAGUCUAAUACCAGGAUUGUCCGAUGC NO: 413
UCGGCUGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGCUAUUAGGCCG CCGGAGUCUAAUAGCUAGGUUUUACCAUGU NO: 414
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAUGCAUUAGGCCG CCGGAGUCUAAUGCAUCGGUUGUCCCAUGU NO: 415
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCAGUGCAUUAGGCCG UCGAAGUCUAAUGCAUUGGUUGUCCUAUGU NO: 416
UCGGUGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGUCAUUAGGCCA CCGGAGUCUAAUGGCUCGGGUAAUCUAUGU NO: 417
UCGGUGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGUCAUUAGGCUA CCGAAGUCUAAUGGCUCGGAUAUUCUAUGC NO: 418
UCGGUGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCCA CCGAAGUCUAAUGGCUCGGAUUUUCAAUGU NO: 419
UCGGUGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCCG UCGAAGUCUAAUGGCUAGGAUCUUCUAUGU NO: 420
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGGCACAUAAGGUCC UCGAAGUCUUAUGUGUCGGCUGUUCUAUGU NO: 421
UCGGGGACAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGUCAAAAGGCCG UCGAAGUCUUUUGGCUCUGGUUUUGUAUGU NO: 422
UCGGCGGCAUUGAUACUUGAUCGCCCAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGCCAUUGGGCCG GCGAAGUCUAAUGCCUCGGGUGUUCUAUGU
NO: 423
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG ACGGAGUCUAAUGGCUCGGGUUUCCCAUGU NO: 424 UCGUCGGCAUUGAUACUUGAUCGCCCUAGA
AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGC GAGGC AUAGGCC GA CGGAGUCUAAUGGCUCGGGUUUCCCAUGUU NO: 425
CGUCGUCAUUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGUCG ACGGAGUCUAAUGGCUCGGGUUUCCCAUGU
NO: 426
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGUCAUUAGGCCG AUGGAGUCUAAUGGCUCGGGUUUCCCAUGU NO: 427
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGUCAUUAGGCCG AUGGAGUCUAAUGGCUCGGGUUUCCCAUGU NO: 428
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCGUUAGGCCG ACGGAGUCUAAUGGCUCGGGUGUCCCAUGU NO: 429
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCGUUAGGCCG ACGGAGUCUAAUGGCUCGGGUUUCCCAUGU NO: 430
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCAUUAGGCCG ACGGAGUCUAAUGGCUCGUGUUUCCCAUGU NO: 431
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG UCGGAGUCUAAUGGUUCGGGUUUCCCAUGU NO: 432
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG ACGGAGUCCAAUGGUUCGGGUUUCCCAUGU
NO: 433
UCGUAGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG ACGGAGUCUAAUGGAUCGGGUUUCCCAUGU NO: 434
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCCG ACGGAGUCUAAUGGCUCGGUUUUCCCAUGU NO: 435
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG ACGGGGUCUAAUGGCUAGGGUUUCACAUGU NO: 436
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA SEQ ID RNA GGAGCUAUUCGGAUGCUAGGCAUUAGGCCG
ACGGAGUCUAAUGGCUGGGUUACUGUAUGU
NO: 437
UCGUCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAUGCGUUAGGCCG CCGGAGUCUAACGAAUCGGGUCUUGUAUGU NO: 438
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG CCGGAGUCUAAUGGCUCGGAUGUCUGAUGU NO: 439
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG CCGGAGUUAAUGGCUCGGAUGUUGAUGUUC NO: 440
GGCGGCAUUGAUACUUGAUCGCCCUAGAAG CAA
SEQ ID RNA GGAGUAUUCGGAUGCGAGGCAUUAGGCCGC CGGAGUCUAAUGGCUCGUGUGUCCUAUGUU NO: 441
CGGCGGCAUUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG CCGGAGUCUAAUGGUUCGUGUGUACUAUGU NO: 442
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG CCGGAGUCUAAUGGCCGUGUUUCCUAUGUU NO: 443
CGGCGGCAUUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGUCAUUAGGCCG CCGGAGUCUAAUGGCUCGUGUGGUCUACGU NO: 444
UCGGCGGCGUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGUCCG CCGGAGUCUAAUACCUCGUGUGUCUUACGU NO: 445
UCGGCGGCGUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGUCCG CCGGAGUCUAAUACCUCGUGUGUCUUACGU NO: 446
UCGGCGGCGUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGGAUUAGGCCG CCGGAGUCUAACCCCUAGAGUGUCUUAUGU NO: 447
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG UCGAAGUCUAUUGGCUCGGGAAUUCUAUGU NO: 448
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG GCGAAGUUUAAUGGCUCAGGAAUCCUAUGU NO: 449
UCGGGGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG UCGAAGUCUAAUGGCUCGUUAGUUCUAUGU NO: 450
UCUGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCAUUAGGCCG CCGGAGUCUAGUGGUUCGCGUAUUCAAUGU NO: 451
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGCCAGUAGGUCG CCGAUGUCUUCUGGCUGGGGAUUCAUACGU NO: 452
UCGGCGGCGUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG UCGAAGUCUAAUGCUUACAGGGAUCUAUGU NO: 453
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG UCGAAGUCUAAUGCUUACAGGGAUCUAUGU NO: 454
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCCAGACAGUAGGCCG CUGAAGUCUACUUGACUGGGAGAUCUAUGU NO: 455
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCCAGACAGUAGGCCG CUGAAGUCUACUUGACUGGGAGAUCUAUGU NO: 456
UCGGCGGCAUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGC GAGGC AUUAGGUC G CAGAAGUCUAAUGCGUGGGGGAUUCUUUGU NO: 457
UCGGCGGCAGUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGACAUUAGGCCG UCGAAGUCUAAUGUCUACGGUGUUCUAAGU NO: 458
UCGGCGGCUUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGUCCG CCGAAGUCUAAUGGCUCGUGUUUUCUAAGU NO: 459
UCGGCGGCUUUGAUACUUGAUCGCCCUAGA AGCAA
SEQ ID RNA GGAGUAUUCGGAUGC GAGAC AUUAGGC AGC CGAAGUCUAAUGGCUCGGGUAUACUACGUU NO: 460
CGGCUGCGUUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGUGGCAUUAGGCAG UCUAAGUCUAAUGCUUCGGUAGUUUACGUU NO: 461
CGGCUGCGUUGAUACUUGAUCGCCCUAGAA GCAA
SEQ ID RNA GGAGCUAUUCGGAUGCGAGGCAUUAGGCCG ACGGAGUCUAAUGGCUCGUGUUCACUGAGU NO: 462 UCGUCGGCUCUGAUACUUGAUCGCCCUAGA
AGCAA
SEQ ID RNA GAGUCAUGAGUCCGCCGAAGUCUCAUGGCU CGGUUUUCUGCAGGUCGGCGGCUGU
NO: 463
SEQ ID RNA GAUGCAUUGGUCCGCCGAAGUCCAAUGUAU CCGUUUCCUCAUGUUCGGCGGCAU NO: 464
SEQ ID RNA GAGCCUUUAGUCCGUCGAAGUCUUUUAGCU CGGAUUUAUCAUGGUCGGCGGCAUU NO: 465
SEQ ID RNA UAGGCAUUAGUCAGCCGAAGUCUUUUGCCU GGAUUUAUUUCGUGGGUCGGCUGCAC
NO: 466
SEQ ID RNA CAGGCAGUAGUCCACCGAAGUCUACUGGCU CGGUUAUAUCAGUCGGUGGCUGU NO: 467
SEQ ID RNA GAGGCAUUAGGCCGCCGAAGUCUAAUGGCU CGGGUGUUCUAAGUUCGGCGGCUU NO: 468
SEQ ID RNA GAGGCAUUAGGCCGACGGAGUCUAAUGGCU CGGGUUUCCCAUGUUCGUCGGCAU NO: 469
SEQ ID RNA GUGUCAUUAGGCCACCGGAGUCUAAUGGCA CUGGUGUCUGCAGUUCGGUGGCUGU NO: 470
SEQ ID RNA GAGACGUUAGCCCGCCGAAGUCUAAUGUCU CGGGUCUUGUCAGUUCGGCGGCUG NO: 471
SEQ ID RNA GGCACAUAAGGUCCUCGAAGUCUUAUGUGU CGGCUGUUCUAUGUUCGGGGACAU NO: 472
SEQ ID RNA GAGGC AUU AGGC CGC C G AAGUCU A AUGUC C UCGGCGCUGAAAGUUCGGCGGCUUU
NO: 473
SEQ ID RNA GAGGCAUUAGUCCGCCGAAGUCUUUUGGCU CGGUUUUUUCAAGGUCGGCGGCUUU NO: 474
Table 2. fD Aptamer Sequences
SEQ ID NO. Aptamer Backbone Sequence 5' to 3'
Number
SEQ ID NO: 1 CI RNA GGGAGUGUGUACGAGGC
AUUAGGCCGCCACCCAA
with
ACUGCAGUCCUCGUAAG
modifications UCUGCCUGGCGGCUUUG
AUACUUGAUCGCCCUAG AAGC;
where G is 2'F and A,C and U are 2'OMe modified RNA.
SEQ ID NO: 2 C2 RNA GGGAGUGUGUACGAGGC
AUUAGUCCGCCGAAGUC
with
UUUUGGCUCGGUUUUUU
modifications C AAGGUC GGCGGCUUUG
AUACUUGAUCGCCCUAG AAGC;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 3 C3 RNA GGGAGUGUGUACGAGGC
AUUAGGCCGCCACCUCG
with
UUUGAUUGCGGUUGUUC
modifications GGCCGCGGGCGGCUUUG
AUACUUGAUCGCCCUAG AAGC;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 10 C4 RNA GGGAGUGUGUACGAGGC
AUU AGGC C GCC GA AGUC
with
UAAUGUCCUCGGCGCUG
modifications AAAGUUCGGCGGCUUUG
AUACUUGAUCGCCCUAG AAGC;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 11 C6 RNA GGGAGUGUGUACGAGGC
AUU AGGC C GCC GA AGUC
with
UAAUGGCUCGGGUGUUC
modifications UAAGUUCGGCGGCUUUG
AUACUUGAUCGCCCUAG AAGC;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 12 C2ml RNA C6SH-
GAGGC AUUAGUCC GCC G
with
AAGUCUUUUGGCUCGGU
modifications UUUUUCAAGGUCGGCGG
CUUU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6SH represents a six-carbon thiol containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 13 C6ml RNA C6SH-
GAGGC AUUAGGCC GCC G
with
AAGUCUAAUGGCUC GGG
modifications UGUUCUAAGUUCGGCGG
CUU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6SH represents a six-carbon thiol containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 14 C4ml RNA C6SH-
GAGGC AUUAGGCC GCC G
with
AAGUCUAAUGUCCUCGG
modifications CGCUGAAAGUUCGGCGG
CUUU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6SH represents a six-carbon thiol containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 15 RNA GAGGUAUAAGUCCGCGG
AAGUCUUUUGUCUCGGG
with
rd4-43 UAUAUGCAGGUCCGCGG modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 16 RNA GGUAGUAGUC C AC CGAA
GUCUACUGGCUCGGUUA
with
UAUACAGGUCGGUGCUG
rd4-14
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 17 RNA GAGC AAUUAGUACUCC G
AAGUCUAUUUGCCCGGU
with
UCACACAUGGUCGGAGC
rd4-16
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 18 RNA GAGGCAUAAGUCCGCCG
AAGUCUUAUGGCUCGGU
with
UUAUUUAUGGUCGGCGG
rd4-02
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 19 RNA GGGCAUGACUACGCCGA
AGUCUCUUGUCUCGGCU
with
UUCGUAUGUUCGGCGUC
rd4-08
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 20 RNA GAGGCAUCAGUCCGCCG
AAGUCUUGUGCCUUGGU
with
UUGUUAUGGUCGGCGGC
rd4-19
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA. SEQ ID NO: 21 RNA CAUAAGUCCACCGAAGU
CUUAUGGAUCGGAUUUU
with
rd4-03 UCAUGAUCGGUGGCAU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 22 RNA GCAUCAGUCCUCCGAAG
UCUUUUGGUUCGGAUUU
with
UGUCUGGUC GGGGGC AG
rd4-31
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 23 RNA GAGGC AUUAGUCUGCC G
AAGUCUUUUGGCUCGGU
with
UUAUUCAUGUUCGGCAG
rd4-36
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 24 RNA GAGCCAUUGGUCCACCG
AAGUCCUAUGACUCGUU
with
UAAUUAAUGUUCGGCGG
rd4-23
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 25 RNA GAGCCAUUGGUCCACCG
AAGUCCUAUGACUCGUU
with
UAAUUAAUGUUCGGCGG
rd4-47
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 26 RNA GAUGCAUUGGUCCGCCG
AAGUCCAAUGUAUCCGU
with
UUCCUCAUGUUCGGCGG
rd4-37
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 27 RNA UUAGUCCGCUGAAGUCU
UUUGGCUCGGGUUUUUG
with
rd4-04 AUGUUCGGCGGCAUU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 28 RNA CCAUUAGUCGGGGGAAG
UUUUUUGGCUGGAUUAU
with
rd4-21 UUCACGGUCCCCCGCGU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 29 RNA AUUAGUCCGCCGAAGUC
UUUUGGUUACGUUUAUA
with
rd4-29 CACGGUCGGCGGCGU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 30 RNA GUGCCAUUAGUCCGCCG
GAGUCUAUUGGGUACGU
with
CAUUUCAUGGUCGGCGG
rd4-15
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 31 RNA GCCAAUAGUCCGUCGAA
GUCUUUUGGCCCUGUUA
with
UUUUAUGGUCGGCGGCA
rd4-35
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 32 RNA GCCAUUAGUCCGCCGAA
GUCUAUUGGCCGGUUGC
with
UUAAUGGUCGGCGGCAU
rd4-26
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 33 RNA GUGCCAUUAGUCCGCGG
AAGUCUACUGUCACGGU
with
AUCUUGAUGGUCCGCGG
rd4-01
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 34 RNA UUAGUCCGCCGAAGUCU
UUUAGCUCGUUUUCUUC
with
rd4-05 AUGGUCGGC GGC AU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 35 RNA GAUGUCAACGUCCGUCG
AAGUCUUUGGCAUCGGU
with
UUUUUCAUGUUCGGCGG
rd4-10
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 36 RNA UAAGUCCGCCGAAGUCU
UUUGCUUAAGCUCCCGC
with
rd4-28 AUGGUCGGC GGC AU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 37 RNA GACGCAUUAGUCCGCCG
AAGUCUCUUGCGUCAGU
with
UUUUUUCAUGGUCGGCG
rd4-32
modifications GCAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 38 RNA UUAGUCCGCAUAAGUCU
UUUGGAUCGCUUUGUUC
with
rd4-22 AUGUUGUGCGGCAU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 39 RNA GAGGAAGUUGUUCGCAG
AAGUCAACUGUCUCGGA
with
AUUUUCAAGGUCUGCGG
rd4-34
modifications CUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 40 RNA AAGGCAUGUGUCCGCAG
AAGUCAUAUGGACUUGA
with
UUUUUUCAUGGUCUGCG
rd4-06
modifications GCAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 41 RNA GAUACAUUAGUGCGCUG
AAGUCUAAUGAAUCAGU
with
UUUUUCACCGUCGGCGG
rd4-39
modifications GUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 42 RNA AUGCAUUAGUCGGCCGA
AGUCUGUUGUCUCGGUG
with
UUUUCACGGUCGGCCGC
rd4-40
modifications GU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 43 RNA GAGGC AUUAGUC AGCC G
AAGUCUGGUGUCUCAGU
with
UUGUUUACGGUCGGCUG
rd4-20
modifications CGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 44 RNA GAGACAUUUGUCCGCCG
AAGUCAUCUGUCUCGGU
with
UUGUUCACGGUCGGCGG
rd4-17
modifications CGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 45 RNA GUGGCAUCAGUCCGACG
AAGUCUUUUGCCAUUUU
with
AUGUUCAAGGUCGUCGG
rd4-13
modifications CUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 46 RNA CAGGCAUUAGUCAGCCG
AAGUCUUUUGCCUGGGA
with
UUUUCGAAGGUCGGCUG
rd4-18
modifications CUUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 47 RNA GGGC AAUGGUCC GCC GA rd4-l l AGUCCAUUGUCCGGGAA with
UGUUGAUGAUCGGCGGC modifications UUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 48 RNA GGGGCAAUGGUCCACCG
AAGUCCGUUGGCUCCGU
with
AUUUUCAAGGUCGGUGG
rd4-44
modifications CUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 49 RNA GGCAAUAGUCCGCCGAA
GUCUUUUGCCACGUAUU
with
CUUCAAGGUCGGCGGCU
rd4-27
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 50 RNA GGGCACUGGUCCGCCGA
AGUCCUUUGACUCGGUU
with
UAUUCAUGGUCGGCGGC
rd4-45
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 51 RNA GAGGCAAUGGUCCGCCC
AAGUCCUUUGCCUCAGU
with
UUAUUCAAGGUGGGCGG
rd4-42
modifications CUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 52 RNA GAGUCAUGAGUCCGCCG
AAGUCUCAUGGCUCGGU
with
UUUCUGCAGGUCGGCGG
rd4-30
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 53 RNA UUAGUACGCCGAAGUCU
UAUGGCUCUAUUCCAGG
with
rd4-25 UCGGCGUCUG;
modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 54 RNA GAGUCAUAAGUCCACCG
AAGUCUUUUGGCUCUGU
with
UUUCUCCAGGUCGGUGG
rd4-24
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 55 RNA AUAAGUCUGCCGAAGUC
UUUUGUCAGUGUUUAUU
with
rd4-12 CCGGGUCGGCAGCCG; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 56 RNA UUAGUCCGGCGGAGUCU rd4-38
AUUGUUUCGGUUUUUUC with CAGGUCGCCGGCUG;
where G is 2'F and A, C and modifications
U are 2'OMe modified RNA.
SEQ ID NO: 57 RNA GAGGAAUUAGUCCGCAG
AAGUCUCUUUCCUCGGU
with
UGGUUCCAGGUCUGCGG
rd4-07
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 58 RNA GAGGCAUGAGUCCGCCG
AAGUCUCAUGUUUCGGU
with
UUCCUCAAGGUCUGCGG
rd4-46
modifications CUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 59 RNA GAGUGACU AGUC C GGC C
AAGUCUAUUCGCUCGGU
with
UUCUUUACAGUGGCCGG
rd4-41
modifications GGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 60 RNA GAGCCUUUAGUCCGUCG
AAGUCUUUUAGCUCGGA
with
UUUAUCAUGGUCGGCGG
Rd5-09
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 61 RNA UAGGCAUUAGUCAGCCG
AAGUCUUUUGCCUGGAU
with
UUAUUUCGUGGUCGGCU
Rd6-58
modifications GCAC;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 62 RNA GGGACAUCAGUCCGACG
AAGUCUGAUGGCUC GGC
with
UUACUCAUGUUCGUCGG
Rd6-35
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 63 RNA CAGGCAGUAGUCCACCG
AAGUCUACUGGCUCGGU
with
UAUAUACAGGUCGGUGG
Rd6-59
modifications UUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 64 RNA AGGCAGUAGUCCACCGA
AGUCUACUGGCUCGGUU
with
AUAUCAGGUCGGUGGCU
Rd6-05
modifications G;
where G is 2'F and A, C and U are 2'OMe modified RNA. SEQ ID NO: 65 RNA GAGCCAUAAGUCCACCG
AAGUCUUUUGGCACGUU
with
UGGUUAAUGGUCGGUGG
Rd6-30
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 66 RNA GAGAC AUAAGUC AGCC G
AAGUCUUCUGGCACGUU
with
UGGUUAAUGUUCGGUGG
Rd6-43
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 67 RNA CCAUUAGUCCGCCGAAG
UCUAUUGGGUACGUCAU
with
UUUAUGGUCGGCGGCAU
Rd6-61
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 68 RNA GCCAUUAGUACGGCGAA
GUCUCUUGGUGCGUCCU
with
UUUUAUGGUCGGCGUCA
Rd6-32
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 69 RNA GAAGCAAGAGUUCGCCG
AAGUCUCUUGCCUCGGU
with
AUAUCACUGUCGGCGUG
Rd6-06
modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 70 RNA GACGCAUACGUACGCCG
AAGUCAUAUGGUUCGGU
with
AUUUUCACUGUCGGCGG
Rd6-29
modifications GUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 71 RNA GAGGC AUUAGUC AGCC G
AAGUCUAUUGGCUCGGU
with
UUGUACAAGGUCGGCUG
Rd5-10
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 72 RNA GAGGCAUGAGUCCACCG
AAGUCUCAUGUCUCGGU
with
AUGAUCAUGGUCGGUGG
Rd6-26
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 73 RNA GAGGCAUGAGUCCACCG
Rd6-21 AAGUCUCAUGUCUCGGU with
AUGAUCAUGUCGGUGGC modifications AUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 74 RNA GAGGCUUAGUCCACCGA
AGUCUUUUGCCUCGGUU
with
UGUUGAUGGUCGGUGGC
Rd6-02
modifications AUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 75 RNA AGGC AUUAGUCC GC AGA
AGUCUUUUGCCUCGGUU
with
UUUUUGAUGGUGUGCGU
Rd6-53
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 76 RNA GAGGCAUUAGUCCGCCG
AAGUCUUUUGCCUCGGU
with
AUUUUCAUGGUCGGCGG
Rd6-39
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 77 RNA GAGACAUCAGUCCACCG
AAGUCUUCUGCCUCGGU
with
UUGUUCAUGGUGGGUGG
Rd6-42
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 78 RNA GGC AAUUGUC AGC CGAA
GUCAUUUGCCACGUUCC
with
UUUCAUGGUCGGCUGCA
Rd6-12
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 79 RNA GAGGCAUUAGUCCGCCG
AAGUCUUUUGGCUCGGU
with
UUUUUUGAUGGUCGGCG
Rd6-10
modifications GCAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 80 RNA GAGGCAGCUGUCCGCCG
AAGUCAUUUGGCUCGGU
with
UUUAUGAUGGUGGCGGC
Rd6-01
modifications AUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 81 RNA GAGGCAUCGGGCAGCCG
AAGUCCUUUGGCUCGGU
with
AUUUUUGCUGAUCGGCU
Rd5-16
modifications GCAGU;
where G is 2'F and A, C and U are 2'OMe modified RNA. SEQ ID NO: 82 RNA AGCCAUCAGUCCUCCGA
AGUCCUUUUGCUCGGCA
with
UUUUGACGGUAGGAGGC
Rd5-03
modifications AUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 83 RNA GAGUCGAUAGUCCACCG
AAGUCUCUCGGCUCGGU
with
UUGGUUCUGGUCGGUUG
Rd6-38
modifications CAG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 84 RNA GAGGCAAAAGUCCACCG
AAGUCUUUUGGUUCGAU
with
UCUUUCAUGGUCGGUGG
Rd5-17
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 85 RNA GCCACUAGUCGACCGAA
GUCUUUUGGCUUGGUUA
with
UUUCACGGUCGGUCGCG
Rd6-27
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 86 RNA GAGACAACAUUAGCCGA
AUUCUUUUGUCACGGUU
with
UUUUCAUGGUCGGCUGC
Rd6-04
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 87 RNA GGCAAUAGUUAGCUGGA
GUCUAUUCGCCCUGUUA
with
UGUACAGUUCGGCUGCU
Rd6-14
modifications GU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 88 RNA GAGUCAUAAGUUAGCCG
AAGUCUUUUGGCUCGAG
with
GACGUAUAGGUCGGCUG
Rd6-50
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 89 RNA GACGCACGAGUCAGCCG
AAGUCUCCUGUGUCGGU
with
CCUUACAUGGUCGGCUG
Rd6-62
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 90 RNA AGCUAUUAUUCAGCCAA
Rd6-31 AGUCUAUUAGCUCCGUU with
CAUUCAAGGUCGGCUGC modifications UU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 91 RNA GGCAUUAAGUCAGCCGA
AGUCUUAUGGGUCCUUU
with
GCUCACGGUCGGCUGCG
Rd5-20
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 92 RNA GAGAC AUAAGUCC GUC C
AAGUCUUGUGUCUUCGU
with
UUGUUCACGGUGGGCGG
Rd6-40
modifications CGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 93 RNA GGCAUCAGUCCACGGAA
GUCUGUUGGUUCGAUUC
with
CUUUAUGGUCCGUUCAU
Rd6-47
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 94 RNA GUGACAUAUGUCCGCCG
AUGUCAUAUGUCUCGAG
with
UCCUUGAAGGUCGGCGU
Rd6-08
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 95 RNA GAGACAUUUGUACGCGG
AAGUCAUCUGUCUCGGU
with
UACUUAAUGGUCUGCGG
Rd6-64
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 96 RNA AGCCUUAGGUACACCGA
AGUCUUAUGGCUGGGUU
with
UUAUCAUGGUCGGUGUC
Rd5-18
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 97 RNA CAGCCAUUGGCACACCG
AAGUCCUUUGGAUGGUU
with
CUAUUCACGGUCGGUGU
Rd6-16
modifications CGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 98 RNA GUGGCAUUGGUACGCCG
AAGUCUAAUGUCACGCC
with
UUAUUCACGGUCGGCGU
Rd6-63
modifications CGU;
where G is 2'F and A, C and U are 2'OMe modified RNA. SEQ ID NO: 99 RNA GAGACAUUGGUACGCCG
AAGUCCUCUGGCUCGGU
with
UUGUUCACGGUCGGCGU
Rd6-17
modifications CGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 100 RNA GUGGCAUAAGUUCGCCG
AAGUCUUAUGGCUCGGU
with
UUUGUCAUGGUCGGUGG
Rd6-44
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 101 RNA GUGGCAUUAGUGCGCCG
AAGUCUAAUGGCUC GGU
with
GUUUUCAUGGUCCGCGG
Rd6-09
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 102 RNA GAUGCAUUAGUCGGCCG
AAGUCUCUGCUUCGUCU
with
GUUUAUGGUCGGCCGCA
Rd6-54
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 103 RNA GCAUCAGUCCGCAGAAG
UCUGUUGCUUCGGUUUU
with
UUCAUGGUCUGCGGCAU
Rd6-13
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 104 RNA GAAGCAUUAGUCCGCCG
AAGUUAUUGGUUCGGAU
with
GUUGAAUGGUCGGGGGC
Rd6-45
modifications AUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 105 RNA GAACCAUGAGUCCGCCG
AAGUCUUAUGGCUCGUU
with
UGUUGGUUGGUCGGCGG
Rd5-26
modifications CAAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 106 RNA GAGCCAUAAGUCUGCAG
AAGUCUUAUGGGUUGGU
with
GUUUUGAUGGUCUGCGG
Rd5-25
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 107 RNA GAGACAUUAGUCCGCCG
Rd6-60 AAGUCUUUUGUCUCGGU with
UUUUUACAUGUUCGGCG modifications GCAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 108 RNA GAGCCAUUAGUCCGUCG
AAGUCUAUUGGCUCGGU
with
UUGUACAUGUUCGGCGG
Rd6-07
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 109 RNA GAGGAAAUAGUCCGACG
AAGUCUAUUGCCUCGUU
with
UCCCUCAUGUUCGUCGG
Rd6-37
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 110 RNA GAGGCAUUAGUCCGCGG
AAGUGUAUUGUCUCGUU
with
UCCUUCAAGUUCAGUGG
Rd6-34
modifications CUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 111 RNA AGUUACUACUCCGCCGA
AGUCUUUUGGCUGGGAU
with
CAUUCAUGGUCGGCGGC
Rd6-28
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 112 RNA GCCACUAGUCUGCCGAA
GUCUUUUGGCGCGGUAU
with
AUUCAUGGUCGGCAGCA
Rd6-49
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 113 RNA C A AU AGUUC GC C GA AGU
CUUUUCGCGCUGUUAUU
with
Rd5-l l UCAUGGUCGGCGGCAU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 114 RNA GAGCCUUAAGUCCGCGU
AAGUCUUUUGCCUCUGU
with
CUAUUCAUGGUCGGCGG
Rd5-04
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 115 RNA GAGUCAUAGGUCCGCCG
AAGUCCUUUGCUCUGUU
with
CCUUCAUGGUGGCGGCA
Rd5-06
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA. SEQ ID NO: 1 16 RNA GAGGCAUAAGUCCGCCG
AAGUCUUUUGGCUCGGU
with
UCAUUCAUGGUCGGCGG
Rd6-48
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 1 17 RNA GAGUCAUAAGUCCGCCG
AAGUCUUUUGACUCGUG
with
UUUUUCAUGGUCGGCGG
Rd6-24
modifications CUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 1 18 RNA GAGCCAUUAGUCCGCCG
AAGUCUAUUCGCUCGGU
with
UUUUCAAGGUCGGCGGC
Rd5-07
modifications UU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 1 19 RNA GAGCCAUUAGUCCACCG
AAGUCUUAUGGCCCGGU
with
UUUAUCCAGGUCGGUGG
Rd6-55
modifications CUG,
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 120 RNA GGGGCAUAAGUCCACCG
AAGUCUUUUGGCCCGGG
with
AUUUUGCAGGUCGGUGG
Rd5-22
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 121 RNA GACGCAUUGGUCCACCG
AAGUCCUCUGCCUCGGU
with
CCUGUAUAGUUCGGUGG
Rd6-36
modifications UUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 122 RNA GGGGC AUUGGUAC ACC G
AAGUCCACUGGUACCGU
with
CUUUUACAGGUCGGUGU
Rd5-02
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 123 RNA GAGGCAUAAGUCCACCG
AGUCUUAUGGCUCGGUA
with
CUUUCAUGGUCGGUGGC
Rd6-57
modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 124 RNA GUGGCAUUGGUCCGAGG
Rd5-19 AAGUCCAUUGUCACGGU with
UUAUACCAGGUCCUCGG modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 125 RNA AGUCAUUUGUCCGCGGA
AGUCAUUUGGCUACGUU
with
GUUAUCAGGUCCGUGGC
Rd5-01
modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 126 RNA UUGCCAUAAGUCCGUCG
AAGUCUUCUGGCUAGUU
with
AAUAUGUAGGUCGGCGG
Rd5-21
modifications UUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 127 RNA UAGCCAUUAGUCCGGCG
AAGUCUUCUGGCUAGGU
with
UAUUAACGGGUCGUCGG
Rd6-51
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 128 RNA GAGGCAUUAGUCCGUAG
AAGUCUAAUGGC AC GAA
with
UAUUUCCAGGUCUACGG
Rd5-23
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 129 RNA GAGGCAUAAGUCGCAGA
AGUCUUAUGUCACGGUG
with
UCAUCCAGGUCUGCGGC
Rd6-22
modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 130 RNA AGUC AUUAGUCC GC AGA
AGUCUAUUGUCUUGGAU
with
UUUUCAGGUCUGCGGCU
Rd6-20
modifications G;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 131 RNA GAGUCAUUAGUCCGCAG
AAGUCUGAUGGUUCGGU
with
UUUUGGCGGGUCUGCGG
Rd6-56
modifications CCGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 132 RNA AGGCAGUAGUCCGACCA
AGUCUCCUGUCUGUUUG
with
UUUUCAGGUGGUCGGCU
Rd5-29
modifications G;
where G is 2'F and A, C and U are 2'OMe modified RNA. SEQ ID NO: 133 RNA GGGCAAUAGUCCGACGA
AGUCUUUUGUCCCGGUU
with
UUAUCCAGGUCGUCGGG
Rd6-52
modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 134 RNA GGGGCAUUAGUCCGCCG
AAGUCUAAUGGCCCAGU
with
UUGUUCCAGUUCGGCGG
Rd5-08
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 135 RNA GAGCCAUUAGUCCGCCG
AAGUCUUUUGGCUCGGU
with
UGAUUGCAGUUCGGCGG
Rd5-13
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 136 RNA GGCAUUAGUCCGCCGAA
GUCUUUUGCCUUGGUAU
with
UCUACAGGUCGGCGGCU
Rd5-12
modifications GU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 137 RNA GGGGCAUUAGGCCGCUG
AAGUCUAAUCGCCCUGA
with
UGUUCAAUGUUCGGCGG
rd4-l l
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 138 RNA GGCAUUAGGCCGUCGAA
GUCUAAUGCUUACAGGG
with
AUCUAUGUUCGGCGGCA
rd4-16
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 139 RNA AGGCAUUAGGCCGUCGA
AGUCUAAUGCUUACAGG
with
GAUCUAUGUUCGGCGGC
rd4-21
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 140 RNA AGUAGGCCGCUGAAGUC
UACUUGACUGGGAGAUC
with
rd4-15 UAUGUUCGGCGGCAU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 141 RNA AGUAGGCCGCUGAAGUC
UACUUGACUGGGAGAUC
with rd4-26
UAUGUUCGGCGGCAU; where G is 2'F and A, C and modifications U are 2'OMe modified RNA.
SEQ ID NO: 142 RNA GAGCCAGUAGGUCGCCG
AUGUCUUCUGGCUGGGG
with
AUUCAUACGUUCGGCGG
rd4-27
modifications CGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 143 RNA CAUUAGGCAGCCGAAGU
CUAAUGGCUCGGGUAUA
with
rd4-37 CUACGUUCGGCUGCGU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 144 RNA AGUAGGCCGACGGGUCU
AGUGCUUGGGCUGUUUU
with
rd4-06 CAGUUCGUCGGCUG; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 145 RNA GGGGCAUUGGUCAGACG
AAGUCCAUUGCCUCGGG
with
UAACCUCAGGUCGUCUG
rd4-01
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 146 RNA GAGCCAUAAGGCCACCG
AAGUCUAAUGGCUCGGG
with
UACUCUCAGUUCGGCGG
rd4-24
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 147 RNA GAGUC AUUAGGCC GGC G
AAGUCUAAUGGCUCGGG
with
UAUUCUCAGUUCGGCGG
rd4-35
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 148 RNA GGGCCAUUAGGGCUGCG
AAGUCUAAUGGCUCGAG
with
UGUUGUCAGUUCGCCGC
rd4-39
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 149 RNA GAGAC AUUAGGCC GAC G
AAGUCUAAUGGCUCUUA
with
UGGUCUCAGUUCGUCGG
rd4-31
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 150 RNA GAGC AUUAGGCC GCC GA rd4-09 AGUCU A AUGUCUC GGGA with
GUUCUCAGUUCGGUGGC modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 151 RNA GUGGC AUUAGGCC GUC G
AAGUCUAAUGUCUCGGC
with
GGUUUCCAGUUCGGCGG
rd4-33
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 152 RNA AUUAGGCUGCAGAGUCU
AAUGGCUUGUGUGUUUC
with
rd4-05 CAGUUCUGCAGCUG; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 153 RNA AGACAUUAGGCCGUCGA
AGUCUAAUGUCUACGGU
with
GUUCUAAGUUCGGCGGC
rd4-17
modifications UU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 154 RNA GAGACGUUAGCCCGCCG
AAGUCUAAUGUCUCGGG
with
UCUUGUCAGUUCGGCGG
rd4-30
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 155 RNA GAGGCAUUAGUCCGCCG
AAGUCUAAUGGCUCGUG
with
UUUUCUAAGUUCGGCGG
rd4-04
modifications CUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 156 RNA AGGCAUUCGUCCGCCGG
AGUCGAAUCGCCUGGGU
with
AUACUCUGUUCGGCGGC
rd4-19
modifications AG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 157 RNA AUUGGGC C GCC GGAGUC
CAAUGCCUCGGAAGUCC
with
rd4-14 AAUGUUCGGCGACAUU; modifications where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 158 RNA GAUGC AUUAGGCC GGC G
AAGUCUAAUGCUUCGGU
with
UGUUCUAUGUUCGUCGG
rd4-40
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 159 RNA GGC AC AUAAGGUC CUC G rd4-28
AAGUCUUAUGUGUCGGC with UGUUCUAUGUUCGGGGA
CAU;
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 160 RNA GUGUC AAAAGGCC GUC G
AAGUCUUUUGGCUCUGG
with
UUUUGUAUGUUCGGCGG
rd4-10
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 161 RNA GUGUCAUUAGGCUACCG
AAGUCUAAUGGCUC GGA
with
UAUUCUAUGCUCGGUGG
rd4-18
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 162 RNA GUGGCAUUAGGCCACCG
AAGUCUAAUGGCUC GGA
with
UUUUCAAUGUUCGGUGG
rd4-08
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 163 RNA UGGCAUUAGGCCGUCGA
AGUCUAAUGGCUAGGAU
with
CUUCUAUGUUCGGCGGC
rd4-38
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 164 RNA GCUAUUAGGCCGCCGGA
GUCUAAUAGCUAGGUUU
with
UACCAUGUUCGGCGGCA
rd4-13
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 165 RNA GUGCCAUUGGGCCGGCG
AAGUCUAAUGCCUCGGG
with
UGUUCUAUGUUCGGCGG
rd4-12
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 166 RNA GUGGC AUUAGGCC GUC G
AAGUCUAAUGUCCCAGG
with
UGUCCUAUGUUCGGCGG
rd4-36
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 167 RNA GUGGC AUUAGGCC GUC G
AAGUCUAAUGUCCCAGG
with
rd4-29 UGUCCUAUGUUCGGCGG modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 168 RNA GUGGC AUUAGGCC GUC G
AAGUCUAAUGUCCCAGG
with
UGUCCUAUGUUCGGCGG
rd4-23
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 169 RNA GAGGC AUUAGGCC GUC G
AAGUCUAAUGUCUCGAG
with
UGUGAUAUGUUCGGCGG
rd4-02
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 170 RNA GAGGC AUUAGGCC GAC G
GAGUCUAAUGGCUC GGG
with
UUUCCCAUGUUCGUCGG
Rd6-04
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 171 RNA GAGGCAUAGGCCGACGG
AGUCU A AUGGCUC GGGU
with
UUCCCAUGUUCGUCGUC
Rd6-09
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 172 RNA GAGGC AUUAGGCC GAC G
GAGUCUAAUGGCUC GGG
with
UUUCCCAUGUUCGUCGG
Rd5-34
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 173 RNA GAGGCAUUAGGUCGACG
GAGUCUAAUGGCUC GGG
with
UUUCCCAUGUUCGUCGG
Rd6-03
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 174 RNA AGGCAUUAGGCCGACGG
GGUCUAAUGGCUAGGGU
with
UUCACAUGUUCGUCGGC
Rd6-26
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 175 RNA GUGGC AUUAGGCC GAC G
GAGUCUAAUGGCUC GGU
with
UUUCCCAUGUUCGUCGG
Rd5-15
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 176 RNA GGCAUUAGGCCGACGGA
Rd6-13
GUCCAAUGGUUCGGGUU with UCCCAUGUUCGUAGGCA
U;
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 177 RNA GAGGC AUUAGGCC GUC G
GAGUCUAAUGGUUCGGG
with
UUUCCCAUGUUCGUCGG
Rd6-02
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 178 RNA GAGCCGUUAGGCCGACG
GAGUCUAAUGGCUC GGG
with
UGUCCCAUGUUCGUCGG
Rd6-05
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 179 RNA GAGCCAUUAGGCCGACG
GAGUCUAAUGGCUC GUG
with
UUUCCCAUGUUCGUCGG
Rd6-52
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 180 RNA GAGUCAUUAGGCCGAUG
GAGUCUAAUGGCUC GGG
with
UUUCCCAUGUUCGUCGG
Rd6-53
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 181 RNA GUGUCAUUAGGCCACCG
GAGUCUAAUGGCACUGG
with
UGUCUGCAGUUCGGUGG
Rd5-10
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 182 RNA GGGUCAUAAGGCCACCG
GAGUCUUAUGGCCCUGG
with
AAGUCUAUGUUCGGUGG
Rd5-12
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 183 RNA GUGUCAUUAGGCCACCG
GAGUCUAAUGGCUC GGG
with
UAAUCUAUGUUCGGUGG
Rd6-12
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 184 RNA GUGUUAUUAGGCCACCG
GAGUCUAAUGGCACUGU
with
Rd6-54 UGUCUGCGUUCGGUGGC modifications UGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 185 RNA GAGUCAUUAGGCCGCCG
GAGUCUAAUGGCUC GUG
with
UGGUCUACGUUCGGCGG
Rd5-23
modifications CGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 186 RNA GAGCCAUUAGGCCGCCG
GAGUCUAGUGGUUCGCG
with
UAUUCAAUGUUCGGCGG
Rd5-13
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 187 RNA GAGGCAUUAGGCCGCCG
GAGUCUAAUGGUUCGUG
with
UGUACUAUGUUCGGCGG
Rd6-45
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 188 RNA GAGGCAUUAGGCCGCCG
GAGUCUAAUGGCUC GUG
with
UGUCCUAUGUUCGGCGG
Rd5-37
modifications CAU,
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 189 RNA GGCAUUAGGCCGCCGGA
GUCUAAUGGCCGUGUUU
with
CCUAUGUUCGGCGGCAU
Rd6-46
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 190 RNA GGCAUUAGGCCGACGAA
GUCUAAUGUCUGGGGGG
with
UUGUCUGUUCGUCGGCA
Rd6-25
modifications G;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 191 RNA GAGGCAUUAGGUCGCAG
AAGUCUAAUGCGUGGGG
with
GAUUCUUUGUUCGGCGG
Rd5-30
modifications CAG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 192 RNA UAGGC AUUAGGCC GAC G
GAGUCUAAUGGCUGGGU
with
UACUGUAUGUUCGUCGG
Rd6-43
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 193 RNA GAGGCAUUAGUCCGCCG
Rd5-27
GAGUCUAAUACCUCGUG with UGUCUUACGUUCGGCGG
CGU;
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 194 RNA AGGGAUUAGGCCGCCGG
AGUCUAACCCCUAGAGU
with
GUCUUAUGUUCGGCGGC
Rd6-16
modifications AU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 195 RNA GUGGCAUUAGGCAGUCU
AAGUCUAAUGCUUCGGU
with
AGUUUACGUUCGGCUGC
Rd5-07
modifications GU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 196 RNA GAGGCAUUAGGCCACCU
AAGUCUAAUGUUUCGCU
with
UGAUGUAUGUUCGGCGG
Rd6-17
modifications UAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 197 RNA GAGGC AUUAGGCC GUC G
AAGUCUAAUGUCUC GGG
with
UGUGUUAUGUUCGGCGG
Rd5-20
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 198 RNA GAUGCGUUAGGCCGCCG
GAGUCU A AC GAAUC GGG
with
UCUUGUAUGUUCGGCGG
Rd5-16
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 199 RNA GAUGCAUCAGGCCGGCG
AAGUCUAAUGC AUC GAG
with
UGUUCUAUGUUCGACGG
Rd5-19
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 200 RNA GAUGCAUUAGGCCGCCG
GAGUCU AAUGC AUC GGU
with
UGUCCCAUGUUCGGCGG
Rd6-36
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 201 RNA AGUGC AUUAGGCC GUC G
AAGUCUAAUGCAUUGGU
with
Rd6-34 UGUCCUAUGUUCGGUGG modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 202 RNA GGGGUAUUAGGCCGUCG
AAGUGUAAUGCCUUGAG
with
UUACCAUGUCGGCUGCA
Rd6-48
modifications U;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 203 RNA GUGGCAUUAGGCCGUUG
GAGUCUAAUACCAGGAU
with
UGUCCGAUGCUCGGCUG
Rd6-19
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 204 RNA GAGGCAUUAGGCCGCUG
AAGUCUAAUACCUCGAC
with
AGUUCUAUGUUCGGUGG
Rd6-08
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 205 RNA GAGGC AUUAGGCC GUC G
AAGUCUAAUGGCUCGUU
with
AGUUCUAUGUUCUGCGG
Rd5-08
modifications CAU,
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 206 RNA GAGGC AUUAGGCC GUC G
AAGUCUAUUGGCUC GGG
with
AAUUCUAUGUUCGGCGG
Rd5-l l
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 207 RNA GAGGC AUUAGGCC GGC G
AAGUUUAAUGGCUCAGG
with
AAUCCUAUGUUCGGGGG
Rd5-17
modifications CAU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 208 RNA GAGGCAUUAGGCCGCCG
GAGUUAAUGGCUCGGAU
with
GUUGAUGUUCGGCGGCA
Rd6-06
modifications UU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 209 RNA GAGGCAUUAGGCCGCCG
GAGUCUAAUGGCUC GGA
with
UGUCUGAUGUUCGGCGG
Rd5-29
modifications CAUU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 210 RNA GAGGC AUUAGGCC GAC G
Rd6-07
GAGUCUAAUGGCUC GUG with UUCACUGAGUUCGUCGG
CUC;
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 211 RNA GGGUC AUUAGGCC GGC G
GAGUCUAAUGACGCGGU
with
UGUACUCAUUUCGCCGG
Rd5-40
modifications AUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 212 RNA GUGUC AUUAGGCC GGC G
GAGUCUAAUGGCUC GGG
with
UGUUAUCAGUUCGCCGG
Rd5-28
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 213 RNA GGGCAUUAGGCUGGCGG
AGUCUAAUCCCUCGGUU
with
GUUAUCUGUUCGCCAGC
Rd5-35
modifications AG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 214 RNA GAGACAUUAGGCCAUCG
GAGUCUAAUGCCUCGGA
with
CGUACUC AGUGC GGUGG
Rd6-29
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 215 RNA GAGAUUAGGC C AC CGGA
GUCUAAUGCCUCGGACG
with
UAUUCAGUUCGGUGGCU
Rd6-l l
modifications G;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 216 RNA GAGUCAUUGGCCACCGG
AGUCUAAUGUCUCGGAC
with
GUACUCAGUUCGGUGGC
Rd6-10
modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 217 RNA GUACCAUUAGGCCACCG
AAGUCUAAUGGUUCGAG
with
UGUUAUCAGUUCGGUGG
Rd5-32
modifications CU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 218 RNA GUACCAUUAGGCCACCG
AAGUCUAAUGCUUCGAG
with
Rd5-03 UGUUAUCAGUUCGGUGC modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 219 RNA GAGCCAAUAGGCCACCG
GAGUCUAUUGGCUGGGU
with
UGUCCUCAGUUCGGUGG
Rd5-18
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 220 RNA GAGCCAAUAGGCUACCG
GAGUCUAUUGGCUGGGU
with
UGUCUCAGUUCGGUGGC
Rd6-18
modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 221 RNA UAGCCAAUAGGCCAUCG
GAGUCUAUUGGCUGGGU
with
UGUCCUCAGUUCGGUGG
Rd6-30
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 222 RNA GAGCCAAUAGGCCACCG
GAGUCUAUUGGCUGGGU
with
UCUCUUCAGUUCGGUGG
Rd6-37
modifications CUG,
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 223 RNA GAGCCAAUAGGUCACCG
GAGUCUAUUGUCUGGGU
with
UGUCCUCAGUUCGGUGG
Rd6-50
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 224 RNA GAGGCAUUAGGCCUCUG
AAGUCUAAUGGCUC GUA
with
AUUUCUCAGUUCGGUGG
Rd6-24
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 225 RNA GAUGCAUUAGUCCGCCG
AAGUCUAAUGCGUC GGG
with
UCUUCUCAGUUCGGCGG
Rd6-32
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 226 RNA GGUGCAUUAGUCCGCCG
GAGUCUAAUGUAUCGGG
with
UCGUCUCAGUUCGGCGG
Rd5-31
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 227 RNA GUGGC AUUAGGCUGCC G
Rd6-51
AAGUCUAAUGUC AC GGU with UUAUCUCAGUUCGGCAG
CUG;
modifications
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 228 RNA GUGGC AUUAGGCUGCC G
AAGUCUAAUGUC AC GGG
with
UGAUUUCAGUUCGGCAG
Rd6-15
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 229 RNA GUGGUAUUAGGCCGUCG
AAGUCUAAUGCCUCGGU
with
UGUUCGCAGUUCGGGCU
Rd6-23
modifications GU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 230 RNA C AGUC AUUAGGGC GUAG
AAGUCUAAUGUCUAGAG
with
UGUUCUCCGUUCUGCGC
Rd6-21
modifications CGG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 231 RNA C A AGC AGU AGGC C GAC G
AAGUCUACUGUCUUGGA
with
UGUUGUCAGUUCGGCGG
Rd5-24
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 232 RNA GAGGC AUUAGGCC GAC G
GAGUCUAUUGCCUCGGG
with
UGUUCUCAGUUCGUCGG
Rd5-22
modifications CUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 233 RNA GAGGC AUUAGGCC GAC G
GAGUCUAAUGUCCCGGA
with
CUUCCCAGUUCGGCAGC
Rd5-06
modifications UG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 234 RNA GAGUC AUUAGGCC GAC G
GAGUCUAAUGCCUCGGA
with
ACUUUACAGUUCGUCGG
Rd5-36
modifications CUGU;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 235 RNA GAGGCACUAGGCCGCCG
AAGUCUAUUGGCUCGGG
with
Rd5-02 UGUUCUCAUUUCGGCGG modifications AUG;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 236 RNA GGGAAGAGCUAGCGCUA
CAAGGCAUGUGUCCGCA
with
GAAGUCAUAUGGACUUG
modifications Rd4-06 full AUUUUUUCAUGGUCUGC length GGCAUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 237 RNA GGGAAGAGCUAGCGCUA
CGAGGAAGUUGUUC GC A
with
GAAGUCAACUGUCUCGG
modifications Rd4-34 full AAUUUUCAAGGUCUGCG length GCUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 238 RNA GGGAAGAGCUAGCGCUA
CGAGUGACUAGUC CGGC
with
CAAGUCUAUUCGCUCGG
modifications Rd4-41 full UUUCUUUACAGUGGCCG length GGGUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 239 RNA GGGAAGAGCUAGCGCUA
CGAGGC AGUAGUC CGAC
with
CAAGUCUCCUGUCUGUU
modifications Rd5-29 full UGUUUUCAGGUGGUCGG length CUGUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 240 RNA GGGAAGAGCUAGCGCUA
CGAGAC AUAAGUC CGUC
with
CAAGUCUUGUGUCUUCG
modifications Rd6-40 full UUUGUUACGGUGGGCGG length CGUUGAUACUUGAUCGC
CCUAGAAGCC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 241 RNA GGGAAGAGCUAGCGCUA
CGAUACAUUAGUGCGCU
with
GAAGUCUAAUGAAUCAG
modifications Rd4-39 full UUUUUUCCCGUCGGCGG length GUGUGAUACUUGAUCGC
CCUAGAAGC A;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 242 Rd6-54 full RNA GGGAAGAGCUAGCGCUA length CGAUGCAUUAGUCGGCC with GAAGUCUCUGCUUCGUC
UGUUUAUGGUCGGCCGC
modifications
AUUGAUACUUGAUCGCC
CUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 243 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUAGUCCGCC
with
GAAGUCUAUUCGCUCGG
modifications Rd5-07 full UUUUUCAAGGUCGGCGG length CUUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 244 RNA GGGAAGAGCUAGCGCUA
CGAGUCAUGAGUCCGCC
with
GAAGUCUCAUGGCUCGG
modifications Rd4-30 full UUUUCUGCAGGUCGGCG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 245 RNA GGGAAGAGCUAGCGCUA
CGAGUCAUUAGUCCGCA
with
GAAGUCUGAUGGUUCGG
modifications Rd6-56 full UUUUUGCGGGUCUGCGG length CCGUGAUACUUGAUCGC
CCUAGAAGCG;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 246 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUAGUCCGCC
with
GAAGUCUUUUGGCUCGG
modifications Rd5-13 full UUGAUUCAGUUCGGCGG length CUGUGAUACUUGAUCGC
CCUAGAAGCG;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 247 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUUAGUC CGUA
with
GAAGUCUAAUGGCACGA
modifications Rd5-23 full AUAUUUCCAGGUCUACG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 248 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUAAGUC GC AG
with Rd6-22 full
AAGUCUUAUGUCACGGU
length
modifications GUCAUCAGGUCUGCGGC
UGUGAUACUUGAUCGCC CUAGAAGCC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 249 RNA GGGAAGAGCUAGCGCUA
CGAGGAAUUAGUCCGCA
with
GAAGUCUCUUUCCUCGG
modifications Rd4-07 full UUGGUUCCAGGUCUGCG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 250 RNA GGGAAGAGCUAGCGCUA
CCAGUCAUUAGUCCGCA
with
GAAGUCUAUUGUCUUGG
modifications Rd6-20 full AUUUUUCAGGUCUGCGG length CUGUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 251 RNA GGGAAGAGCUAGCGCUA
CGAGGU AU A AGUC C GC G
with
GAAGUCUUUUGUCUCGG
modifications Rd4-43 full GUAUAUCAGGUCCGCGG length CUGUGAUACUUGAUCGC
CCUAGAAGCG;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 252 RNA GGGAAGAGCUAGCGCUA
CGAGUCAUAAGUUAGCC
with
GAAGUCUUUUGGCUCGA
modifications Rd6-50 full GGACGUAUAGGUCGGCU length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 253 RNA GGGAAGAGCUAGCGCUA
CGAGUCAUUUGUCCGCG
with
GAAGUCAUUUGGCUACG
modifications Rd5-01 full UUGUUAUCAGGUCCGUG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 254 RNA GGGAAGAGCUAGCGCUA
CUUGCCAUAAGUCCGUC
with
GAAGUCUUCUGGCUAGU
modifications Rd5-21 full UAAUAUGUAGGUCGGCG length GUUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 255 RNA GGGAAGAGCUAGCGCUA
CUAGCCAUUAGUCCGGC
with
GAAGUCUUCUGGCUAGG
modifications Rd6-51 full UUAUUAACGGGUCGUCG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 256 RNA GGGAAGAGCUAGCGCUA
CC GGGC AAUAGUC CGAC
with
GAAGUCUUUUGUCCCGG
modifications Rd6-52 full UUUUAUCCAGGUCGUCG length GGUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 257 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUUAGUC CGGC
with
GGAGUCUAUUGUUUCGG
modifications Rd4-38 full UUUUUUCCAGGUCGCCG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 258 RNA GGGAAGAGCUAGCGCUA
CGUGGC AUUGGUC CGAG
with
GAAGUCCAUUGUCACGG
modifications Rd5-19 full UUUAUACCAGGUCCUCG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 259 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUUAGU AC GC C
with
GAAGUCUUAUGGCUCUA
modifications Rd4-25 full UUCCAGGUCGGCGUCUG length UGAUACUUGAUCGCCCU
AGAAGC;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 260 RNA GGGAAGAGCUAGCGCUA
CGGGGCAUUAGUCCGCC
with
GAAGUCUAAUGGCCCAG
modifications Rd5-08 full UUUGUUCCAGUUCGGCG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 261 RNA GGGAAGAGCUAGCGCUA
Rd4-12 full
CGAGGC AUAAGUCUGC C
with length
GAAGUCUUUUGUCAGUG modifications UUUAUUCGGGUCGGCAG
CCGUGAUACUUGAUCGC
CCUAGAAGCC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 262 RNA GGGAAGAGCUAGCGCUA
CGAGUCAUAAGUCCACC
with
GAAGUCUUUUGGCUCUG
modifications Rd4-24 full UUUUCUCCAGGUCGGUG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 263 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUAGUCCACC
with
GAAGUCUUAUGGCCCGG
modifications Rd6-55 full UUUUAUCCAGGUCGGUG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 264 RNA GGGAAGAGCUAGCGCUA
CGGGGC AUAAGUC C AC C
with
GAAGUCUUUUGGCCCGG
modifications Rd5-22 full GAUUUUGCAGGUCGGUG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 265 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUAAGUC C AC C
with
GAGUCUUAUGGCUCGGU
modifications Rd6-57 full ACUUUAUGGUCGGUGGC length UGUGAUACUUGAUCGCC
CUAGAAGCC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 266 RNA GGGAAGAGCUAGCGCUA
CCAGGUAGUAGUCCACC
with
GAAGUCUACUGGCUCGG
modifications Rd4-14 full UUAUAUACAGGUCGGUG length CUGUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 267 RNA GGGAAGAGCUAGCGCUA
CCAGGCAGUAGUCCACC
with
Rd6-59 full GAAGUCUACUGGCUCGG modifications length UUAUAUACAGGUCGGUG
GUUGUGAUACUUGAUCG
CCCUAGAAGC; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 268 RNA GGGAAGAGCUAGCGCUA
CCAGGCAGUAGUCCACC
with
GAAGUCUACUGGCUCGG
modifications Rd6-05 full UUAUAUCAGGUCGGUGG length CUGUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 269 RNA GGGAAGAGCUAGCGCUA
CGGGGCAUUGGUACACC
with
GAAGUCCACUGGUACCG
modifications Rd5-02 full UCUUUUACAGGUCGGUG length UCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 270 RNA GGGAAGAGCUAGCGCUA
CGACGCAUUGGUCCACC
with
GAAGUCCUCUGCCUCGG
modifications Rd6-36 full UCCUGUAUAGUUCGGUG length GUUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 271 RNA GGGAAGAGCUAGCGCUA
CGUGGCAUCAGUCCGAC
with
GAAGUCUUUUGCCAUUU
modifications Rd4-13 full UAUGUUCAAGGUCGUCG length GCUUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 272 RNA GGGAAGAGCUAGCGCUA
CGUGGCAUUAGUCCGCC
with
GAAGUCUUUUGCCUUGG
modifications Rd5-12 full UAUUCUACAGGUCGGCG length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 273 RNA GGGAAGAGCUAGCGCUA
CGGGGC AAUGGUC CGC C
with
GAAGUCCAUUGUCCGGG
modifications Rd4-l l full AAUGUUGAUGAUCGGCG length GCUUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 274 RNA GGGAAGAGCUAGCGCUA
CGGGGC AAUGGUC C AC C
with
GAAGUCCGUUGGCUCCG
modifications Rd4-44 full UAUUUUCAAGGUCGGUG length GCUUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 275 RNA GGGAAGAGCUAGCGCUA
CGGGGC AAUAGUCCGCC
with
GAAGUCUUUUGCCACGU
modifications Rd4-27 full AUUCUUCAAGGUCGGCG length GCUUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 276 RNA GGGAAGAGCUAGCGCUA
CGAGGC AAUGGUC CGC C
with
CAAGUCCUUUGCCUCAG
modifications Rd4-42 full UUUAUUCAAGGUGGGCG length GCUUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 277 RNA GGGAAGAGCUAGCGCUA
CGGGGCACUGGUCCGCC
with
GAAGUCCUUUGACUCGG
modifications Rd4-45 full UUUAUUCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 278 RNA GGGAAGAGCUAGCGCUA
CGAAGC AAGAGUUC GC C
with
GAAGUCUCUUGCCUCGG
modifications Rd6-06 full UAUAUCACUGUCGGCGU length GUGUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 279 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUAAGUC CGC C
with
GAAGUCUUAUGGCUCGG
modifications Rd4-02 full UUUAUUUAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 280 RNA GGGAAGAGCUAGCGCUA
Rd6-48 full
CGAGGC AUAAGUC CGC C
with length
GAAGUCUUUUGGCUCGG modifications UUCAUUCAUGGUCGGCG
GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 281 RNA GGGAAGAGCUAGCGCUA
CGAGUUACUACUCCGCC
with
GAAGUCUUUUGGCUGGG
modifications Rd6-28 full AUCAUUCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 282 RNA GGGAAGAGCUAGCGCUA
CGAUGUCAACGUCCGUC
with
GAAGUCUUUGGCAUCGG
modifications Rd4-10 full UUUUUUCAUGUUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 283 RNA GGGAAGAGCUAGCGCUA
CGAGUCAUAGGUCCGCC
with
GAAGUCCUUUGCUCUGU
modifications Rd5-06 full UCCUUCAUGGUGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 284 RNA GGGAAGAGCUAGCGCUA
CGAUGCAUAAGUCCGCC
with
GAAGUCUUUUGCUUAAG
modifications Rd4-28 full CUCCCGCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 285 RNA GGGAAGAGCUAGCGCUA
CGAGCCUUAAGUCCGCG
with
UAAGUCUUUUGCCUCUG
modifications Rd5-04 full UCUAUUCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 286 RNA GGGAAGAGCUAGCGCUA
CGAGCCAAUAGUCCGUC
with
Rd4-35 full GAAGUCUUUUGGCCCUG modifications length UUAUUUUAUGGUCGGCG
GCAUUGAUACUUGAUCG
CCCUAGAAGC; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 287 RNA GGGAAGAGCUAGCGCUA
CGAGAC AAUAGUUC GC C
with
GAAGUCUUUUCGCGCUG
modifications Rd5-l l full UUAUUUCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 288 RNA GGGAAGAGCUAGCGCUA
CGAGGCAUUAGUCUGCC
with
GAAGUCUUUUGGCUCGG
modifications Rd4-36 full UUUAUUCAUGUUCGGCA length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 289 RNA GGGAAGAGCUAGCGCUA
CGAGCCACUAGUCUGCC
with
GAAGUCUUUUGGCGCGG
modifications Rd6-49 full UAUAUUCAUGGUCGGCA length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 290 RNA GGGAAGAGCUAGCGCUA
CGAGUCGAUAGUCCACC
with
GAAGUCUCUCGGCUCGG
modifications Rd6-38 full UUUGGUUCUGGUCGGUU length GCAGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 291 RNA GGGAAGAGCUAGCGCUA
CGAGCCUUUAGUCCGUC
with
GAAGUCUUUUAGCUCGG
modifications Rd5-09 full AUUUAUCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 292 RNA GGGAAGAGCUAGCGCUA
CGAGCCUUUAGUCCGUC
with
GAAGUCUUUUAGCUCGG
modifications Rd5-05 full AUUUAUCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 293 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUAGUCCGCC
with
GAAGUCUUUUAGCUCGU
modifications Rd4-05 full UUUCUUCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 294 RNA GGGAAGAGCUAGCGCUA
CGAGACAUUAGUCCGCC
with
GAAGUCUUUUGUCUCGG
modifications Rd6-60 full UUUUUUACAUGUUCGGC length GGCAUUGAUACUUGAUC
GCCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 295 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUAGUCCGUC
with
GAAGUCUAUUGGCUCGG
modifications Rd6-07 full UUUGUACAUGUUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 296 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUAAGUCCACC
with
GAAGUCUUUUGGCACGU
modifications Rd6-30 full UUGGUUAAUGGUCGGUG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 297 RNA GGGAAGAGCUAGCGCUA
CGAGACAUAAGUCAGCC
with
GAAGUCUUCUGGCACGU
modifications Rd6-43 full UUGGUUAAUGUUCGGUG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 298 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUAGUCCGCC
with
GAAGUCUAUUGGCCGGU
modifications Rd4-26 full UGCUUAAUGGUCGGCGG length CAUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 299 RNA GGGAAGAGCUAGCGCUA
Rd6-47 full
CGAGGCAUCAGUCCACG
with length
GAAGUCUGUUGGUUCGA modifications UUCCUUUAUGGUCCGUU
CAUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 300 RNA GGGAAGAGCUAGCGCUA
CGAGGC AAAAGUC C AC C
with
GAAGUCUUUUGGUUCGA
modifications Rd5-17 full UUCUUUCAUGGUCGGUG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 301 RNA GGGAAGAGCUAGCGCUA
CAAGCCUUAGGUACACC
with
GAAGUCUUAUGGCUGGG
modifications Rd5-18 full UUUUAUCAUGGUCGGUG length UCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 302 RNA GGGAAGAGCUAGCGCUA
CCAGCCAUUGGCACACC
with
GAAGUCCUUUGGAUGGU
modifications Rd6-16 full UCUAUUCACGGUCGGUG length UCGUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 303 RNA GGGAAGAGCUAGCGCUA
CGAGCAAUUAGUACUCC
with
GAAGUCUAUUUGCCCGG
modifications Rd4-16 full UUC AC AC AUGGUC GGAG length CAUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 304 RNA GGGAAGAGCUAGCGCUA
CGAGACAUCAGUCCACC
with
GAAGUCUUCUGCCUCGG
modifications Rd6-42 full UUUGUUCAUGGUGGGUG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 305 RNA GGGAAGAGCUAGCGCUA
CGAGGCUUAGUCCACCG
with
Rd6-02 full AAGUCUUUUGCCUCGGU modifications length UUGUUGAUGGUCGGUGG
CAUUGAUACUUGAUCGC
CCUAGAAGC; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 306 RNA GGGAAGAGCUAGCGCUA
CGAGGCAUCAGUCCGCC
with
GAAGUCUUGUGCCUUGG
modifications Rd4-19 full UUUGUUAUGGUCGGCGG length CAUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 307 RNA GGGAAGAGCUAGCGCUA
CGAGGCAUUAGUCCGCU
with
GAAGUCUUUUGGCUCGG
modifications Rd4-04 full GUUUUUGAUGUUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 308 RNA GGGAAGAGCUAGCGCUA
CGAGGCAUUAGUCCGCC
with
GAAGUCUUUUGGCUCGG
modifications Rd6-10 full UUUUUUUGAUGGUCGGC length GGCAUUGAUACUUGAUC
GCCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 309 RNA GGGAAGAGCUAGCGCUA
CGAUGCAUUAGUCCGCA
with
UAAGUCUUUUGGAUCGC
modifications Rd4-22 full UUUGUUCAUGUUGUGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 310 RNA GGGAAGAGCUAGCGCUA
CGUUGCAUCAGUCCGCA
with
GAAGUCUGUUGCUUCGG
modifications Rd6-13 full UUUUUUCAUGGUCUGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 311 RNA GGGAAGAGCUAGCGCUA
CAAGGCAUUAGUCCGCA
with
GAAGUCUUUUGCCUCGG
modifications Rd6-53 full UUUUUUUGAUGGUGUGC length GUCAUUGAUACUUGAUC
GCCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 312 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUAAGUCUGCA
with
GAAGUCUUAUGGGUUGG
modifications Rd5-25 full UGUUUUGAUGGUCUGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 313 RNA GGGAAGAGCUAGCGCUA
CGACGCAUUAGUCCGCC
with
GAAGUCUCUUGCGUCAG
modifications Rd4-32 full UUUUUUUCAUGGUCGGC length GGCAUUGAUACUUGAUC
GCCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 314 RNA GGGAAGAGCUAGCGCUA
CGAGGCAUUAGUCCGCC
with
GAAGUCUUUUGCCUCGG
modifications Rd6-39 full UAUUUUCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 315 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUAAGUC C AC C
with
GAAGUCUUAUGGAUCGG
modifications Rd4-03 full AUUUUUCAUGAUCGGUG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 316 RNA GGGAAGAGCUAGCGCUA
CGUGGCAUAAGUUCGCC
with
GAAGUCUUAUGGCUCGG
modifications Rd6-44 full UUUUGUCAUGGUCGGUG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 317 RNA GGGAAGAGCUAGCGCUA
CGAGUCAUAAGUCCGCC
with
GAAGUCUUUUGACUCGU
modifications Rd6-24 full GUUUUUCAUGGUCGGCG length GCUUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 318 RNA GGGAAGAGCUAGCGCUA
Rd6-01 full
CGAGGCAGCUGUCCGCC
with length
GAAGUCAUUUGGCUCGG modifications UUUUAUGAUGGUGGCGG
CAUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 319 RNA GGGAAGAGCUAGCGCUA
CGUGACAUAUGUCCGCC
with
GAUGUCAUAUGUCUCGA
modifications Rd6-08 full GUC CUUGA AGGUC GGC G length UCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 320 RNA GGGAAGAGCUAGCGCUA
CUAGCCAUCAGUCCUCC
with
GAAGUCCUUUUGCUCGG
modifications Rd5-03 full C AUUUUGAC GGU AGGAG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 321 RNA GGGAAGAGCUAGCGCUA
CGAGGCAUCGGGCAGCC
with
GAAGUCCUUUGGCUCGG
modifications Rd5-16 full UAUUUUUGCUGAUCGGC length UGCAGUGAUACUUGAUC
GCCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 322 RNA GGGAAGAGCUAGCGCUA
CGAAGCAUCAGUCCUCC
with
GAAGUCUUUUGGUUCGG
modifications Rd4-31 full AUUUUGUCUGGUCGGGG length GCAGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 323 RNA GGGAAGAGCUAGCGCUA
CGAAGCAUUAGUCCGCC
with
GAAGUUAUUGGUUCGGA
modifications Rd6-45 full UGUUGAAUGGUCGGGGG length CAUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 324 RNA GGGAAGAGCUAGCGCUA
CGAACCAUGAGUCCGCC
with
Rd5-26 full GAAGUCUUAUGGCUCGU modifications length UUGUUGGUUGGUCGGCG
GCAAUGAUACUUGAUCG
CCCUAGAAGC; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 325 RNA GGGAAGAGCUAGCGCUA
CGGGGCAUGACUACGCC
with
GAAGUCUCUUGUCUCGG
modifications Rd4-08 full CUUUCGUAUGUUCGGCG length UCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 326 RNA GGGAAGAGCUAGCGCUA
CGGGACAUCAGUCCGAC
with
GAAGUCUGAUGGCUCGG
modifications Rd6-35 full CUUACUCAUGUUCGUCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 327 RNA GGGAAGAGCUAGCGCUA
CGGUACAUCAGUCCGAC
with
GAAGUCUGAUGGCUCGG
modifications Rd6-23 full CUUACUCAUGUUCGUCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 328 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUGAGUC C AC C
with
GAAGUCUCAUGUCUCGG
modifications Rd6-26 full UAUGAUCAUGGUCGGUG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 329 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUGAGUC C AC C
with
GAAGUCUCAUGUCUCGG
modifications Rd6-21 full UAUGAUCAUGUCGGUGG length CAUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 330 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUGAGUC CGC C
with
GAAGUCUCAUGUUUCGG
modifications Rd4-46 full UUUCCUCAAGGUCUGCG length GCUUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 331 RNA GGGAAGAGCUAGCGCUA
CGAGGAAAUAGUCCGAC
with
GAAGUCUAUUGCCUCGU
modifications Rd6-37 full UUCCCUCAUGUUCGUCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 332 RNA GGGAAGAGCUAGCGCUA
CGAGGC AUUAGUC CGC G
with
GAAGUGUAUUGUCUCGU
modifications Rd6-34 full UUCCUUCAAGUUCAGUG length GCUUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 333 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUGGUCCACC
with
GAAGUCCUAUGACUCGU
modifications Rd4-23 full UUAAUUAAUGUUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 334 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUGGUCCACC
with
GAAGUCCUAUGACUCGU
modifications Rd4-47 full UUAAUUAAUGUUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 335 RNA GGGAAGAGCUAGCGCUA
CGAUGCAUUGGUCCGCC
with
GAAGUCCAAUGUAUCCG
modifications Rd4-37 full UUUCCUCAUGUUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 336 RNA GGGAAGAGCUAGCGCUA
CGAGACAUUUGUCCGCC
with
GAAGUCAUCUGUCUCGG
modifications Rd4-17 full UUUGUUCACGGUCGGCG length GCGUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 337 RNA GGGAAGAGCUAGCGCUA
Rd6-17 full
CGAGAC AUUGGUAC GC C
with length
GAAGUCCUCUGGCUCGG modifications UUUGUUCACGGUCGGCG
UCGUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 338 RNA GGGAAGAGCUAGCGCUA
CGAGAC AUUUGUAC GC G
with
GAAGUCAUCUGUCUCGG
modifications Rd6-64 full UUACUUAAUGGUCUGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 339 RNA GGGAAGAGCUAGCGCUA
CGUGCCAUUAGUCCGCC
with
GGAGUCUAUUGGGUACG
modifications Rd4-15 full UCAUUUCAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 340 RNA GGGAAGAGCUAGCGCUA
CGUGCCAUUAGUCCGCC
with
GAAGUCUAUUGGGUACG
modifications Rd6-61 full UCAUUUUAUGGUCGGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 341 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUAGUCCGCC
with
GAAGUCUUUUGGUUACG
modifications Rd4-29 full UUUAUACACGGUCGGCG length GCGUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 342 RNA GGGAAGAGCUAGCGCUA
CGUGGCAUUGGUACGCC
with
GAAGUCUAAUGUCACGC
modifications Rd6-63 full CUUAUUCACGGUCGGCG length UCGUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 343 RNA GGGAAGAGCUAGCGCUA
CGUGCCAUUAGUCCGCG
with
Rd4-01 full GAAGUCUACUGUCACGG modifications length UAUCUUGAUGGUCCGCG
GCAUUGAUACUUGAUCG CCCUAGAAGC; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 344 RNA GGGAAGAGCUAGCGCUA
CGUGGCAUUAGUGCGCC
with
GAAGUCUAAUGGCUCGG
modifications Rd6-09 full UGUUUUCAUGGUCCGCG length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 345 RNA GGGAAGAGCUAGCGCUA
CUAGGCAUUAGUCAGCC
with
GAAGUCUUUUGCCUGGA
modifications Rd6-58 full UUUAUUUCGUGGUCGGC length UGCACUGAUACUUGAUC
GCCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 346 RNA GGGAAGAGCUAGCGCUA
CUAGGCAUUAGUCAGCC
with
GAAGUCUUUUGCCUGGA
modifications Rd6-15 full UUUAUUUCGUGGUCGGC length UGCACUGAUACUUGAUC
GCCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 347 RNA GGGAAGAGCUAGCGCUA
CCAGGCAUUAGUCAGCC
with
GAAGUCUUUUGCCUGGG
modifications Rd4-18 full AUUUUCGAAGGUCGGCU length GCUUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 348 RNA GGGAAGAGCUAGCGCUA
CAUGGCAAUUGUUCGCU
with
GAAGUCAUUUGCUACGU
modifications Rd6-19 full AUUUUUAAUGUCAUCGG length CUGAUACUUGAUCGCCC
UAGAAGC;
where G is 2'F and A, C and U are 2'OMe modified RNA.
SEQ ID NO: 349 RNA GGGAAGAGCUAGCGCUA
CAAGGCAAUUGUCAGCC
with
GAAGUCAUUUGCCACGU
modifications Rd6-12 full UCCUUUCAUGGUCGGCU length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 350 RNA GGGAAGAGCUAGCGCUA
CAUGGCAAUAGUUAGCU
with
GGAGUCUAUUCGCCCUG
modifications Rd6-14 full UUAUGUACAGUUCGGCU length GCUGUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 351 RNA GGGAAGAGCUAGCGCUA
CCAAGCUAUUAUUCAGC
with
CAAAGUCUAUUAGCUCC
modifications Rd6-31 full GUUCAUUCAAGGUCGGC length UGCUUUGAUACUUGAUC
GCCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 352 RNA GGGAAGAGCUAGCGCUA
CGAC GC AC GAGUC AGCC
with
GAAGUCUCCUGUGUCGG
modifications Rd6-62 full UCCUUACAUGGUCGGCU length GCAUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 353 RNA GGGAAGAGCUAGCGCUA
CGAGACAACAUUAGCCG
with
AAUUCUUUUGUCACGGU
modifications Rd6-04 full UUUUUCAUGGUCGGCUG length CAUUGAUACUUGAUCGC
CCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 354 RNA GGGAAGAGCUAGCGCUA
CGAGCCAUUAGUCGGGG
with
GAAGUUUUUUGGCUGGA
modifications Rd4-21 full UUAUUUCACGGUCCCCC length GCGUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 355 RNA GGGAAGAGCUAGCGCUA
CGCCACUAGUCGACCGA
with
AGUCUUUUGGCUUGGUU
modifications Rd6-27 full AUUUCACGGUCGGUCGC length GUUGAUACUUGAUCGCC
CUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 356 RNA GGGAAGAGCUAGCGCUA
Rd4-40 full
CGAUGCAUUAGUCGGCC
with length
GAAGUCUGUUGUCUCGG modifications UGUUUUCACGGUCGGCC
GCGUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 357 RNA GGGAAGAGCUAGCGCUA
CGAGGCAUUAGUCAGCC
with
GAAGUCUGGUGUCUCAG
modifications Rd4-20 full UUUGUUUACGGUCGGCU length GCGUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 358 RNA GGGAAGAGCUAGCGCUA
C AC GGC AUUAAGUC AGC
with
CGAAGUCUUAUGGGUCC
modifications Rd5-20 full UUUGCUCACGGUCGGCU length GCGUUGAUACUUGAUCG
CCCUAGAAGC;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 359 RNA GGAGCUAUUCGGAUGCG
UGUCAUUAGGCCACCGG
with
AGUCUAAUGGCACUGGU
modifications Rd5-10 full GUCUGCAGUUCGGUGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 360 RNA GGAGCUAUUCGGAUGCG
UGUUAUUAGGCCACCGG
with
AGUCUAAUGGCACUGUU
modifications Rd6-54 full GUCUGCGUUCGGUGGCU length GUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 361 RNA GGAGCUAUUCGGAUGCC
GGGUCAUAAGGCCACCG
with
GAGUCUUAUGGCCCUGG
modifications Rd5-12 full AAGUCUAUGUUCGGUGG length CAUUGAUACUUGAUCGC
CCUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 362 RNA GGAGCUAUUCGGAUGCG
AGCCAAUAGGCCACCGG
with
Rd6-37 full AGUCUAUUGGCUGGGUU modifications length CUCUUCAGUUCGGUGGC
UGUGAUACUUGAUCGCC CUAGAAGCAA; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 363 RNA GGAGCUAUUCGGAUGCG
AGCCAAUAGGCUACCGG
with
AGUCUAUUGGCUGGGUU
modifications Rd6-18 full GUCUCAGUUCGGUGGCU length GUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 364 RNA GGAGCUAUUCGGAUGCU
AGCCAAUAGGCCACCGG
with
AGUCUAUUGGCUGGGUU
modifications Rd6-40 full GUCCUCAGUUCGGUGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 365 RNA GGAGCUAUUCGGAUGCU
AGCCAAUAGGCCAUCGG
with
AGUCUAUUGGCUGGGUU
modifications Rd6-30 full GUCCUCAGUUCGGUGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 366 RNA GGAGCUAUUCGGAUGCG
AGCCAAUAGGCCACCGG
with
AGUCUAUUGGCUGGGUU
modifications Rd6-20 full GUCCUCAGUUCGGUGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 367 RNA GGAGCUAUUCGGAUGCG
AGCCAAUAGGUCACCGG
with
AGUCUAUUGUCUGGGUU
modifications Rd6-50 full GUCCUCAGUUCGGUGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 368 RNA GGAGCUAUUCGGAUGCG
AGACAUUAGGCCAUCGG
with
AGUCUAAUGCCUCGGAC
modifications Rd6-29 full GUACUCAGUGCGGUGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 369 RNA GGAGCUAUUCGGAUGCG
AGAUU AGGC C AC C GGAG
with
UCUAAUGCCUCGGACGU
modifications Rd6-l l full AUUCAGUUCGGUGGCUG length UGAUACUUGAUCGCCCU
AGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 370 RNA GGAGCUAUUCGGAUGCG
AGUCAUUGGCCACCGGA
with
GUCUAAUGUCUCGGACG
modifications Rd6- 10 ful- UACUCAGUUCGGUGGCU length GUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 371 RNA GGAGCUAUUCGGAUGCG
AGAGAUUGGGCCGCCGG
with
AGUCCAAUGCCUCGGAA
modifications Rd4-14 full GUCCAAUGUUCGGCGAC length AUUGAUACUUGAUCGCC
CU AGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 372 RNA GGAGCUAUUCGGAUGCG
AGGCAUUCGUCCGCCGG
with
AGUCGAAUCGCCUGGGU
modifications Rd4-19 full AUACUCUGUUCGGCGGC length AGUGAUACUUGAUCGCC
CU AGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 373 RNA GGAGCUAUUCGGAUGCG
AGCCAUAAGGCCACCGA
with
AGUCU A AUGGCUC GGGU
modifications Rd4-24 full ACUCUCAGUUCGGCGGC length UGUGAUACUUGAUCGCC
CU AGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 374 RNA GGAGCUAUUCGGAUGCG
AGUCAUUAGGCCGGCGA
with
AGUCU A AUGGCUC GGGU
modifications Rd4-35 full AUUCUCAGUUCGGCGGC length UGUGAUACUUGAUCGCC
CU AGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 375 RNA GGAGCUAUUCGGAUGCG
Rd5-32 full
UACCAUUAGGCCACCGA
with length
AGUCUAAUGGUUCGAGU modifications GUUAUCAGUUCGGUGGC
UUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 376 RNA GGAGCUAUUCGGAUGCG
UACCAUUAGGCCACCGA
with
AGUCUAAUGCUUCGAGU
modifications Rd5-03 full GUUAUCAGUUCGGUGCU length GUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 377 RNA GGAGCUAUUCGGAUGCG
GGCCAUUAGGGCUGCGA
with
AGUCUAAUGGCUCGAGU
modifications Rd4-39 full GUUGUCAGUUCGCCGCC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 378 RNA GGAGCUAUUCGGAUGCG
AGACAUUAGGCCGACGA
with
AGUCUAAUGGCUCUUAU
modifications Rd4-31 full GGUCUCAGUUCGUCGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 379 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCUCUGA
with
AGUCU A AUGGCUC GU A A
modifications Rd6-22 full UUUCUCAGUUCGGUGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 380 RNA GGAGCUAUUCGGAUGCG
AUGCAUUAGUCCGCCGA
with
AGUCU AAUGCGUCGGGU
modifications Rd6-32 full CUUCUCAGUUCGGCGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 381 RNA GGAGCUAUUCGGAUGCG
GUGCAUUAGUCCGCCGG
with
Rd5-31 full AGUCUAAUGUAUCGGGU modifications length CGUCUCAGUUCGGCGGC
UGUGAUACUUGAUCGCC
CUAGAAGCAA; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 382 RNA GGAGCUAUUCGGAUGCG
AGACGUUAGCCCGCCGA
with
AGUCUAAUGUCUCGGGU
modifications Rd4-30 full CUUGUCAGUUCGGCGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 383 RNA GGAGCUAUUCGGAUGCG
AGCAUUAGGCCGCCGAA
with
GUCUAAUGUCUCGGGAG
modifications Rd4-09 full UUCUCAGUUCGGUGGCU length GUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 384 RNA GGAGCUAUUCGGAUGCG
UGGUAUUAGGCCGUCGA
with
AGUCUAAUGCCUCGGUU
modifications Rd6-23 full GUUCGCAGUUCGGGCUG length UGAUACUUGAUCGCCCU
AGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 385 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCCGUCGA
with
AGUCUAAUGUCUCGGCG
modifications Rd4-33 full GUUUCCAGUUCGGCGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 386 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCUGCCGA
with
AGUCUAAUGUCACGGUU
modifications Rd6-51 full UAUCUCAGUUCGGCAGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 387 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCUGCCGA
with
AGUCUAAUGUCACGGGU
modifications Rd6-15 full GAUUUCAGUUCGGCAGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 388 RNA GGAGCUAUUCGGAUGCG
AGGCACUAGGCCGCCGA
with
AGUCUAUUGGCUCGGGU
modifications Rd5-14 full GUUCUCAUUUCGGCGGA length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 389 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGACGG
with
AGUCUAUUGCCUCGGGU
modifications Rd5-26 full GUUCUCAGUUCGUCGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 390 RNA GGAGCUAUUCGGAUGCC
AGGCAUUAGGCUGCAGA
with
GUCUAAUGGCUUGUGUG
modifications Rd4-05 full UUUCCAGUUCUGCAGCU length GUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 391 RNA GGAGCUAUUCGGAUGCC
AGUCAUUAGGGCGUAGA
with
AGUCUAAUGUCUAGAGU
modifications Rd6-42 full GUUCUCCGUUCUGCGCC length GGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 392 RNA GGAGCUAUUCGGAUGCC
AGUCAUUAGGGCGUAGA
with
AGUCUAAUGUCUAGAGU
modifications Rd6-21 full GUUCUCCGUUCUGCGCC length GGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 393 RNA GGAGCUAUUCGGAUGCG
AGUCAUUAGGCCGACGG
with
AGUCUAAUGCCUCGGAA
modifications Rd5-36 full CUUUACAGUUCGUCGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 394 RNA GGAGCUAUUCGGAUGCG
Rd5-06 full
AGGCAUUAGGCCGACGG
with length
AGUCUAAUGUCCCGGAC modifications UUCCCAGUUCGGCAGCU
GUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 395 RNA GGAGCUAUUCGGAUGCC
CAGCAGUAGGCCGACGG
with
GUCUAGUGCUUGGGCUG
modifications Rd4-06 full UUUUCAGUUCGUCGGCU length GUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 396 RNA GGAGCUAUUCGGAUGCC
AAGCAGUAGGCCGACGA
with
AGUCUACUGUCUUGGAU
modifications Rd5-21 full GUUGUCAGUUCGGCGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 397 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCCGACGA
with
AGUCUAAUGUCUGGGGG
modifications Rd6-25 full GUUGUCUGUUCGUCGGC length AGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 398 RNA GGAGCUAUUCGGAUGCG
GGUCAUUAGGCCGGCGG
with
AGUCUAAUGACGCGGUU
modifications Rd5-40 full GUACUCAUUUCGCCGGA length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 399 RNA GGAGCUAUUCGGAUGCG
UGUCAUUAGGCCGGCGG
with
AGUCU A AUGGCUC GGGU
modifications Rd5-28 full GUUAUCAGUUCGCCGGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 400 RNA GGAGCUAUUCGGAUGCC
GGGCAUUAGGCUGGCGG
with
Rd5-35 full AGUCUAAUCCCUCGGUU modifications length GUUAUCUGUUCGCCAGC
AGUGAUACUUGAUCGCC CUAGAAGCAA; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 401 RNA GGAGCUAUUCGGAUGCG
GGGCAUUGGUCAGACGA
with
AGUCCAUUGCCUCGGGU
modifications Rd4-01 full AACCUCAGGUCGUCUGC length UGUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 402 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCACCUA
with
AGUCUAAUGUUUCGCUU
modifications Rd6-17 full GAUGUAUGUUCGGCGGU length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 403 RNA GGAGCUAUUCGGAUGCG
AUGCAUUAGGCCGGCGA
with
AGUCUAAUGCUUCGGUU
modifications Rd4-40 full GUUCUAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 404 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGUCGA
with
AGUCUAAUGUCUCGGGU
modifications Rd5-20 full GUGUUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 405 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGUCGA
with
AGUCUAAUGUCUCGAGU
modifications Rd4-02 full GUGAUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 406 RNA GGAGCUAUUCGGAUGCG
AGGC AUUAGGCC GCUGA
with
AGUCUAAUACCUCGACA
modifications Rd6-08 full GUUCUAUGUUCGGUGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 407 RNA GGAGCUAUUCGGAUGCG
AUGCAUCAGGCCGGCGA
with
AGUCUAAUGCAUCGAGU
modifications Rd5-19 full GUUCUAUGUUCGACGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 408 RNA GGAGCUAUUCGGAUGCG
GGGUAUUAGGCCGUCGA
with
AGUGUAAUGCCUUGAGU
modifications Rd6-48 full UACCAUGUCGGCUGCAU length UGAUACUUGAUCGCCCU
AGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 409 RNA GGAGCUAUUCGGAUGCG
GGGC AUUAGGCC GCUGA
with
AGUCUAAUCGCCCUGAU
modifications Rd4-l l full GUUCAAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 410 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCCGUCGA
with
AGUCUAAUGUCCCAGGU
modifications Rd4-23 full GUCCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 411 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCCGUCGA
with
AGUCUAAUGUCCCAGGU
modifications Rd4-36 full GUCCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 412 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCCGUCGA
with
AGUCUAAUGUCCCAGGU
modifications Rd4-29 full GUCCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 413 RNA GGAGCUAUUCGGAUGCG
Rd6-19 full
UGGCAUUAGGCCGUUGG
with length
AGUCUAAUACCAGGAUU modifications GUCCGAUGCUCGGCUGC
AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 414 RNA GGAGCUAUUCGGAUGCG
UGCUAUUAGGCCGCCGG
with
AGUCUAAUAGCUAGGUU
modifications Rd4-13 full UUACCAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 415 RNA GGAGCUAUUCGGAUGCG
AUGC AUUAGGCC GCC GG
with
AGUCUAAUGCAUCGGUU
modifications Rd6-36 full GUCCCAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 416 RNA GGAGCUAUUCGGAUGCA
GUGCAUUAGGCCGUCGA
with
AGUCUAAUGCAUUGGUU
modifications Rd6-34 full GUCCUAUGUUCGGUGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 417 RNA GGAGCUAUUCGGAUGCG
UGUCAUUAGGCCACCGG
with
AGUCU A AUGGCUC GGGU
modifications Rd6-12 full AAUCUAUGUUCGGUGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 418 RNA GGAGCUAUUCGGAUGCG
UGUCAUUAGGCUACCGA
with
AGUCU A AUGGCUC GGAU
modifications Rd4-18 full AUUCUAUGCUCGGUGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 419 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCCACCGA
with
Rd4-08 full AGUCU A AUGGCUC GGAU modifications length UUUCAAUGUUCGGUGGC
AUUGAUACUUGAUCGCC CUAGAAGCAA; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 420 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCCGUCGA
with
AGUCUAAUGGCUAGGAU
modifications Rd4-38 full CUUCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 421 RNA GGAGCUAUUCGGAUGCG
GCACAUAAGGUCCUCGA
with
AGUCUUAUGUGUCGGCU
modifications Rd4-28 full GUUCUAUGUUCGGGGAC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 422 RNA GGAGCUAUUCGGAUGCG
UGUCAAAAGGCCGUCGA
with
AGUCUUUUGGCUCUGGU
modifications Rd4-10 full UUUGUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 423 RNA GGAGCUAUUCGGAUGCG
UGCCAUUGGGCCGGCGA
with
AGUCUAAUGCCUCGGGU
modifications Rd4-12 full GUUCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 424 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGACGG
with
AGUCUAAUGGCUCGGGU
modifications Rd6-04 full UUCCCAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 425 RNA GGAGCUAUUCGGAUGCG
AGGCAUAGGCCGACGGA
with
GUCUAAUGGCUCGGGUU
modifications Rd6-09 full UCCCAUGUUCGUCGUCA length UUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 426 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGUCGACGG
with
AGUCU A AUGGCUC GGGU
modifications Rd6-03 full UUCCCAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 427 RNA GGAGCUAUUCGGAUGCG
AGUCAUUAGGCCGAUGG
with
AGUCU A AUGGCUC GGGU
modifications Rd6-31 full UUCCCAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 428 RNA GGAGCUAUUCGGAUGCG
AGUCAUUAGGCCGAUGG
with
AGUCU A AUGGCUC GGGU
modifications Rd6-53 full UUCCCAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 429 RNA GGAGCUAUUCGGAUGCG
AGCCGUUAGGCCGACGG
with
AGUCU A AUGGCUC GGGU
modifications Rd6-39 full GUCCCAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 430 RNA GGAGCUAUUCGGAUGCG
AGCCGUUAGGCCGACGG
with
AGUCU A AUGGCUC GGGU
modifications Rd6-33 full UUCCCAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 431 RNA GGAGCUAUUCGGAUGCG
AGCCAUUAGGCCGACGG
with
AGUCUAAUGGCUCGUGU
modifications Rd6-52 full UUCCCAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 432 RNA GGAGCUAUUCGGAUGCG
Rd6-02 full
AGGCAUUAGGCCGUCGG
with length
AGUCUAAUGGUUCGGGU modifications UUCCCAUGUUCGUCGGC
AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 433 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGACGG
with
AGUCCAAUGGUUCGGGU
modifications Rd6-13 full UUCCCAUGUUCGUAGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 434 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGACGG
with
AGUCUAAUGGAUCGGGU
modifications Rd6-41 full UUCCCAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 435 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCCGACGG
with
AGUCUAAUGGCUCGGUU
modifications Rd5-15 full UUCCCAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 436 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGACGG
with
GGUCUAAUGGCUAGGGU
modifications Rd6-26 full UUCACAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 437 RNA GGAGCUAUUCGGAUGCU
AGGCAUUAGGCCGACGG
with
AGUCUAAUGGCUGGGUU
modifications Rd6-43 full ACUGUAUGUUCGUCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 438 RNA GGAGCUAUUCGGAUGCG
AUGCGUUAGGCC GCC GG
with
Rd5-16 full AGUCU A AC GA AUC GGGU modifications length CUUGUAUGUUCGGCGGC
AUUGAUACUUGAUCGCC CUAGAAGCAA; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 439 RNA GGAGCUAUUCGGAUGCG
AGGC AUUAGGCC GCC GG
with
AGUCU A AUGGCUC GGAU
modifications Rd5-29 full GUCUGAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 440 RNA GGAGCUAUUCGGAUGCG
AGGC AUUAGGCC GCC GG
with
AGUUAAUGGCUCGGAUG
modifications Rd6-06 full UUGAUGUUCGGCGGCAU length UGAUACUUGAUCGCCCU
AGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 441 RNA GGAGUAUUCGGAUGCGA
GGCAUUAGGCCGCCGGA
with
GUCUAAUGGCUCGUGUG
modifications Rd5-37 full UCCUAUGUUCGGCGGCA length UUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 442 RNA GGAGCUAUUCGGAUGCG
AGGC AUUAGGCC GCC GG
with
AGUCUAAUGGUUCGUGU
modifications Rd6-45 full GUACUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 443 RNA GGAGCUAUUCGGAUGCG
AGGC AUUAGGCC GCC GG
with
AGUCUAAUGGCCGUGUU
modifications Rd6-46 full UCCUAUGUUCGGCGGCA length UUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 444 RNA GGAGCUAUUCGGAUGCG
AGUC AUUAGGCC GCC GG
with
AGUCUAAUGGCUCGUGU
modifications Rd5-23 full GGUCUACGUUCGGCGGC length GUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA. SEQ ID NO: 445 RNA GGAGCUAUUCGGAUGCG
AGGC AUUAGUCC GCC GG
with
AGUCUAAUACCUCGUGU
modifications Rd5-27 full GUCUUACGUUCGGCGGC length GUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 446 RNA GGAGCUAUUCGGAUGCG
AGGC AUUAGUCC GCC GG
with
AGUCUAAUACCUCGUGU
modifications Rd5-05 full GUCUUACGUUCGGCGGC length GUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 447 RNA GGAGCUAUUCGGAUGCG
AGGGAUUAGGCCGCCGG
with
AGUCUAACCCCUAGAGU
modifications Rd6-16 fUll GUCUUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 448 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGUCGA
with
AGUCU AUUGGCUC GGGA
modifications Rd5-l l full AUUCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 449 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGGCGA
with
AGUUUAAUGGCUCAGGA
modifications Rd5-17 full AUCCUAUGUUCGGGGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 450 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGUCGA
with
AGUCUAAUGGCUCGUUA
modifications Rd5-04 full GUUCUAUGUUCUGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 451 RNA GGAGCUAUUCGGAUGCG
Rd5-13 full
AGCCAUUAGGCCGCCGG
with length
AGUCUAGUGGUUCGCGU modifications AUUCAAUGUUCGGCGGC
AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 452 RNA GGAGCUAUUCGGAUGCG
AGCCAGUAGGUCGCCGA
with
UGUCUUCUGGCUGGGGA
modifications Rd4-27 full UUCAUACGUUCGGCGGC length GUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 453 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGUCGA
with
AGUCUAAUGCUUACAGG
modifications Rd4-16 full GAUCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 454 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGUCGA
with
AGUCUAAUGCUUACAGG
modifications Rd4-21 full GAUCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 455 RNA GGAGCUAUUCGGAUGCC
AGAC AGUAGGCC GCUGA
with
AGUCUACUUGACUGGGA
modifications Rd4-26 full GAUCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 456 RNA GGAGCUAUUCGGAUGCC
AGAC AGUAGGCC GCUGA
with
AGUCUACUUGACUGGGA
modifications Rd4-15 full GAUCUAUGUUCGGCGGC length AUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 457 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGUCGCAGA
with
Rd5-30 full AGUCUAAUGCGUGGGGG modifications length AUUCUUUGUUCGGCGGC
AGUGAUACUUGAUCGCC CUAGAAGCAA; where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 458 RNA GGAGCUAUUCGGAUGCG
AGACAUUAGGCCGUCGA
with
AGUCUAAUGUCUACGGU
modifications Rd4-17 full GUUCUAAGUUCGGCGGC length UUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 459 RNA GGAGCUAUUCGGAUGCG
AGGC AUUAGUCC GCC GA
with
AGUCUAAUGGCUCGUGU
modifications Rd4-04 full UUUCUAAGUUCGGCGGC length UUUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 460 RNA GGAGUAUUCGGAUGCGA
GAC AUUAGGC AGC CGAA
with
GUCUAAUGGCUCGGGUA
modifications Rd4-37 full UACUACGUUCGGCUGCG length UUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 461 RNA GGAGCUAUUCGGAUGCG
UGGCAUUAGGCAGUCUA
with
AGUCUAAUGCUUCGGUA
modifications Rd5-07 full GUUUACGUUCGGCUGCG length UUGAUACUUGAUCGCCC
UAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 462 RNA GGAGCUAUUCGGAUGCG
AGGCAUUAGGCCGACGG
with
AGUCUAAUGGCUCGUGU
modifications Rd6-07 full UCACUGAGUUCGUCGGC length UCUGAUACUUGAUCGCC
CUAGAAGCAA;
where G is 2'F and A, C and
U are 2'OMe modified RNA.
SEQ ID NO: 463 Aptamer 41 RNA C6NH2-
GAGUC AUGAGUCC GCC G
with
AAGUCUCAUGGCUCGGU
modifications UUUCUGCAGGUCGGCGG
CUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 464 Aptamer 42 RNA C6NH2-
GAUGCAUUGGUCCGCCG
with
AAGUCCAAUGUAUCCGU
modifications UUCCUCAUGUUCGGCGG
CAU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 465 Aptamer 43 RNA C6NH2-
GAGCCUUUAGUCCGUCG
with
AAGUCUUUUAGCUCGGA
modifications UUUAUCAUGGUCGGCGG
CAUU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 466 Aptamer 44 RNA C6NH2-
UAGGCAUUAGUCAGCCG
with
AAGUCUUUUGCCUGGAU
modifications UUAUUUCGUGGGUCGGC
UGCAC-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 467 Aptamer 45 RNA C6NH2-
CAGGCAGUAGUCCACCG
with
AAGUCUACUGGCU
modifications CGGUUAUAUCAGUCGGU
GGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3 ' inverted deoxythymidine residue. SEQ ID NO: 468 Aptamer 46 RNA C6NH2-
GAGGC AUUAGGCC GCC G
with
AAGUCUAAUGGCUCGGG
modifications UGUUCUAAGUUCGGCGG
CUU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 469 Aptamer 47 RNA C6NH2-
GAGGCAUUAGGCCGACG
with
GAGUCUAAUGGCU
modifications CGGGUUUCCCAUGUUCG
UCGGCAU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 470 Aptamer 48 RNA C6NH2-
GUGUCAUUAGGCCACCG
with
GAGUCUAAUGGCACUGG
modifications UGUCUGCAGUUCGGUGG
CUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 471 Aptamer 49 RNA C6NH2-
GAGACGUUAGCCCGCCG
with
AAGUCUAAUGUCUCGGG
modifications UCUUGUCAGUUCGGCGG
CUG-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 472 Aptamer 50 RNA C6NH2-
GGCACAUAAGGUCCUCG
with
AAGUCUUAUGUGUCGGC modifications UGUUCUAUGUUCGGGGA
CAU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3' inverted deoxythymidine residue.
SEQ ID NO: 473 Aptamer 51 RNA C6NH2-
GAGGC AUUAGGCC GCC G
with
AAGUCUAAUGUCCUCGG
modifications CGCUGAAAGUUCGGCGG
CUUU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3' inverted deoxythymidine residue.
SEQ ID NO: 474 Aptamer 52 RNA C6NH2-
GAGGC AUUAGUCC GCC G
with
AAGUCUUUUGGCUCGGU
modifications UUUUUCAAGGUCGGCGG
CUUU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; and idT represents a 3' inverted deoxythymidine residue.
SEQ ID NO:543 Aptamer 122 RNA C6NH2-
GAGUC AUGAGUCC GCC G
with
AAGUCUC AUGGCUC(Sp 1 modifications 8)GCAGGUCGGCGGCUGU
-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue; and Spl8 represents a Spacer 18 linker.
SEQ ID NO:544 Aptamer 123 RNA C6NH2-
GAGUC AUGAGUCC GCC G
with
AAGUCUCAUGGCUC(Sp9) modifications GC AGGUC GGCGGCUGU- idT; where G is 2'F and A, C and
U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3' inverted deoxythymidine residue, and Sp9 represents a Spacer 9 linker.
SEQ ID NO:545 Aptamer 124 RNA C6NH2-
GAGUC AUGAGUCC GCC G
with
AAGUCUCAUGGCUC(L6) modifications GC AGGUC GGCGGCUGU- idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue; and L6 represents a 6-carbon spacer.
SEQ ID NO:546 Aptamer 125 RNA C6NH2-
GAGUC AUGAGUCC GCC G
with
AAGUCUCAUGGCUC(Sp3) modifications GC AGGUC GGCGGCUGU- idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue; and Sp3 represents a Spacer 3 linker.
SEQ ID NO:547 Aptamer 126 RNA C6NH2-
CAUGAGUCCGCCGAAGU
with
CUCAUG(Sp 18Sp 18)GCAG modifications GUCGGCGGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3' inverted deoxythymidine residue; and Spl8 represents a Spacer 18 linker.
SEQ ID NO:548 Aptamer 127 RNA C6NH2-
CAUGAGUCCGCCGAAGU
with
CUC AUG(Sp 18)GC AGGUC modifications GGCGGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA;
C6NH2 represents a six- carbon amino containing linker; idT represents a 3' inverted deoxythymidine residue; and Spl8 represents a Spacer 18 linker.
SEQ ID NO:549 Aptamer 128 RNA C6NH2-
CAUGAGUCCGCCGAAGU
with
CUCAUG(Sp9)GCAGGUCG modifications GCGGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3' inverted deoxythymidine residue; and Sp9 represents a Spacer 9 linker.
SEQ ID NO:550 Aptamer 129 RNA C6NH2-
CAUGAGUCCGCCGAAGU
with
CUCAUG(L6)GCAGGUCG modifications GCGGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue; and L6 represents a 6-carbon spacer.
SEQ ID NO:551 Aptamer 130 RNA C6NH2-
CAUGAGUCCGCCGAAGU
with
CUCAUG(Sp3)GCAGGUCG modifications GCGGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue; and Sp3 represents a Spacer 3 linker.
SEQ ID NO:552 Aptamer 131 RNA C6NH2-
GUCAUGAGUCCGCCGAA
with
GUCUCAUGGC(Sp 18)GCA modifications GGUCGGCGGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue; and Spl8 represents a Spacer 18 linker.
SEQ ID NO:553 Aptamer 132 RNA C6NH2-
GUCAUGAGUCCGCCGAA
with
GUCUCAUGGC(Sp9)GCAG modifications GUCGGCGGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue; and Sp9 represents a Spacer 9 linker.
SEQ ID NO:554 Aptamer 133 RNA C6NH2-
GUCAUGAGUCCGCCGAA
with
GUCUCAUGGC(L6)GCAG modifications GUCGGCGGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue and L6 represents a 6- carbon spacer.
SEQ ID NO:555 Aptamer 134 RNA C6NH2-
GUCAUGAGUCCGCCGAA
with
GUCUCAUGGC(Sp3)GCAG modifications GUCGGCGGCUGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue; and Sp3 represents a Spacer 3 linker.
SEQ ID NO:556 Aptamer 1736 RNA C6NH2-
GAGUC AUGAGUCC GCC G
with
AAGUCUC AUGGCUC(Sp 1 modifications 8 Sp 18)GCAGGUCGGCGGC
UGU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6NH2 represents a six- carbon amino containing linker; idT represents a 3 ' inverted deoxythymidine residue; and Spl8 represents a
Spacer 18 linker.
[0092] In some cases, an aptamer of the disclosure does not comprise any one of SEQ ID NOs: 1-3, 475-542 as described in Table 3. In some cases, an aptamer of the disclosure does not comprise any one of SEQ ID NOs: 475-542 as described in Table 3. In some cases, an aptamer of the disclosure does not comprise any one of SEQ ID NOs: 535-542 as described in Table 3. Table 3. fD Aptamer Sequences
SEQ ID Backbone Sequence 5' to 3'
NO.
SEQ ID RNA GGGAGUGUGU AC GAGGC AUU AGGCC G CCACCCAAACUGCAGUCCUCGUAAGUC NO: 1
UGCCUGGCGGCUUUGAUACUUGAUCG CCCUAGAAGC
SEQ ID RNA GGGAGUGUGU AC GAGGC AUU AGUCC G CCGAAGUCUUUUGGCUCGGUUUUUUC
NO: 2
AAGGUCGGCGGCUUUGAUACUUGAUC GCCCUAGAAGC
SEQ ID RNA GGGAGUGUGU AC GAGGC AUU AGGCC G CCACCUCGUUUGAUUGCGGUUGUUCG
NO: 3
GCCGCGGGCGGCUUUGAUACUUGAUC GCCCUAGAAGC
SEQ ID DNA GTGACGACTGACATATCTGCTCCGAGG TTATTGGGGTTGGGGCCTGGGCGATTG
NO: 475
GGGCCTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGTTTGGG
GTTGGGGCCTGGGAGTTTGGGGAGCAG
NO: 476
AAAGGACGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTGGGT GTTGTGGGGGTGGGTGGTGGGCCCTTC
NO: 477
GCCATGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGGCGGTT GGGGTCGAAGGGCGAGGGGTGGGAGG
NO: 478
TCGCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTATTTTGG
GGCCTGGGTGTTGGGGATTGGGGACTA
NO: 479
TGTGTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTGGAT
GGTGGGGGGTGGTGTGGGAGGGCTGGT
NO: 480
CGGTCGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCCCTATAG
GGGTGTGGGCGAGGGGTGGGTGGTAGG NO: 481
GCGGCTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGGAGGTG GGTGGGTGGGTGCGTGCGAGGGCGGTG
NO: 482
TAGGTCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCAAAAGTT AGATTGACATGGTATGCACCGTCTGAG NO: 483 GTTGGTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCACCACGC
TAGGGGTGAGGGCGAGGGGTGGGTAGC NO: 484
GCGTGGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTGGGT GTTGTGGGGGCGGGTGGTGGGTGCGTC NO: 485
GGTGGTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGCTTCCA
GCGGTCATGATATGCACTGTCTGAAGC NO: 486
TCGGTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTGTTAT
GATATGCACCGTCTGAGGGTAGTCGCG NO: 487
GGGTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGCTTGTT
TAGTGGGTGGGTGGGTGGTGTGGTGGT NO: 488
GATGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCCTTGGGG TTGGGGCCTGGGTGTTTGGGGTGGCCT NO: 489
AGAAGTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGCTAGGG GTGGGTTGGGGTTGGTGGTGTGCGTGT
NO: 490
GGGTTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTTGAG
GTTGGTGGGGGGTGGGCGGTGGGATGG NO: 491
TTGTGCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTTGACAG TCTGCTTTGCAGGGGCCGAGAGCGCCA NO: 492
TTGCGTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTGGTTG GTGGGGGGTGGAGGGTGGGAGGCCGTG
NO: 493
TGTCCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTGGTG
GTGGGGGAGGGTGGTGGGGTGGCCGGC
NO: 494
GCTCGTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGGGTTA
CGTGGTTCGGGGCTAGGGGGGTGGGGT NO: 495
GTGTTTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGGTGGT GTGCGGTGGGTTCTTGGGTGGGATGGG
NO: 496
TGGTACCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTATTAGAT
CCTCGGTGGGTGGGTGGGTGTGTGGTG
NO: 497
GTGTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGGGCGTC
TGAGCGCATGGATGACCCACCGACAGA NO: 498
TTGCGGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGCTTTGG GTGGGCTCGGTGTGCGGTGTGCGGGTG
NO: 499
GGTTTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGTTTGGG NO: 500 GTTGGGGCCTGGGAGTTTGGGGAGCAG
AAAGGGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGGGTGGG TTGGGTTGGGTTTGGTGGTGGTGCCTGT NO: 501
TAGTTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCAGGTGGG TGGGTGGGTGTGTGTGCGGTGGTGTGA
NO: 502
TTTGGCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTGGTTG GTGGGGGGCGGCGGGTGGGGAGCCTGG NO: 503
TGTTCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTCCCGTTT GAGGGCTTGTCGGACAGATTGCTGGCA
NO: 504
CGTCACGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTCTTGGTG
GTGGTGGTGGGTTGGGATGGGTCTTGG NO: 505
GCTGCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCCTGTGAG
GGGAGGGAGGGTGGGTTTGGCGGTGGC
NO: 506
GCAGGCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGTGGTGG
TGCGTGGGTGGTGGGGGGGGGAGCTGG NO: 507
GTGCCCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTGGGT GTTGTGGGGGTGGGTGGTGGGCCCTTC NO: 508
GCCGTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTTCCGGTA
TGTGTGGGTGGGTGGGTGGTGTGGTGG NO: 509
TGTTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTCTCTTCT
GTTGTGGGTGGGTGGGTGGTGTGGTGC NO: 510
GTGTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGGCTGGG TGGGTTGGGTTAGGGTGGTGTGCGGTG NO: 511
GGTTGCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGTTTAGGT GGGCGGGTGGGTGTGCGGTGGGCGGTG NO: 512
TTGAACGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGGTGATT
GGGGTTGGGGCCTGGGCGTTTGGGGAC NO: 513
CGCATGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGTTTGGG
GTTGGGGCCTGGGAGTTTGGGGAGCAG NO: 514
AGAGGACGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTAACTTGT
TGGGGTTTGGGGCCTGGGTGTTGGGGT NO: 515
TGTTTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGGGGTT
GGTGGGGGGAGGTGGGTGGGTTATGTG NO: 516
CGCTGGCGTAGTTGAGTCTGAGTGCT SEQ ID DNA GTGACGACTGACATATCTGCTGTGGGT
GTTGTGGGGGTGGGTTGGTGGGCATTG
NO: 517
CGTGTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGAGTGGG
TTCGGTGGTGGTGTGTGGGAGGGTTGG
NO: 518
GTACGTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGGACAT
GATTGCACCGTATGAGGTTTAGTCGTTA
NO: 519
ATGTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCAGTGGGG
CCTGGGCGTTGGGGTTTGGGGTGCCTC
NO: 520
GTCAGTCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCATGGATTT
TCGGTGGGTGGGTGGGTTGGTGTGGTG
NO: 521
GTGTGCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCTGTGGTTG
GTGGGGGGTGGGTGGTGGGAAGGTTCC
NO: 522
GGTGCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGGTTGGG
GTTGGGGCCTGGGTGTTGGGGAGCAGG
NO: 523
TAGCACCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA GTGACGACTGACATATCTGCGGCCTGG
GAGGGTTCGGTGGTGGTGCGAGGGTGG
NO: 524
GCAAGCCGTAGTTGAGTCTGAGTGCT
SEQ ID DNA ACCTAGTTTGGCTTGCAXAAGTAACYA
GC AC GTGGGC TAG,
NO: 525
where X= 5-(amino-l-propenyl)-2'- deoxyuridine; and Y= 5-(4- pivaloylbenzamido- 1 -propenyl)-2'- deoxyuridine.
SEQ ID DNA ACGATCGCCCCYGTCTWTAAGAXCGAA
TACTATGGGCTAG,
NO: 526
where W= 5-(indole-3-acetamido-l-propenyl)- 2'-deoxyuridine; X= 5-(amino-l-propenyl)-2'- deoxyuridine; and Y= 5-(4- pivaloylbenzamido- 1 -propenyl)-2'- deoxyuridine.
SEQ ID DNA ACCTAGAAAGGCTTAGTGAAGTAAWG
ATCAGGGCGGGATC,
NO: 527
where W= 5-(indole-3-acetamido-l-propenyl)- 2'-deoxyuridine.
SEQ ID DNA ACCTAGTTCCCYGTCTAXYAGAXCCGA
GXGTATGCCGATC,
NO: 528
where X= 5-(amino-l-propenyl)-2'- deoxyuridine; and Y= 5-(4- pivaloylbenzamido- 1 -propenyl)-2'- deoxyuridine.
SEQ ID DNA ACCTAGGCAGTCTTGCCGAATTTACGA
GXGGGGAGGGATC,
NO: 529
where X= 5-(amino-l-propenyl)-2'- deoxyuridine. SEQ ID DNA ACGATCACTGCYCAGCWTYATTAACYA
GCYTCGACCCTAG,
NO: 530
where W= 5-(indole-3-acetamido-l-propenyl)- 2'-deoxyuridine; and Y= 5-(4- pivaloylbenzamido- 1 -propenyl)-2'- deoxyuridine.
SEQ ID DNA ACGATCTTCCGCCAGCTGYATTXCGAA
GXGCGTGAGGATC,
NO: 531
where X= 5-(amino-l-propenyl)-2'- deoxyuridine; and Y= 5-(4- pivaloylbenzamido- 1 -propenyl)-2'- deoxyuridine
SEQ ID DNA ACCTAGGCGGTCTTXCCGTCGTTACGTC
CYCGGCCCCTAG,
NO: 532
where X= 5-(amino-l-propenyl)-2'- deoxyuridine; and Y= 5-(4- pivaloylbenzamido- 1 -propenyl)-2'- deoxyuridine.
SEQ ID DNA ACCTAGTTTGGCGTAGCGYATTAAWGG GXGCGGCAGCTAG,
NO: 533
where W= 5-(indole-3-acetamido-l-propenyl)- 2'-deoxyuridine; X= 5-(amino-l-propenyl)-2'- deoxyuridine; and Y= 5-(4- pivaloylbenzamido- 1 -propenyl)-2'- deoxyuridine.
SEQ ID DNA ACGATCGCTGACGTXCAXYAGTATGAG
GC AC GTGGGC TAG,
NO: 534
where X= 5-(amino-l-propenyl)-2'- deoxyuridine; and Y= 5-(4- pivaloylbenzamido- 1 -propenyl)-2'- deoxyuridine.
SEQ ID DNA ACGGAGAAAGAGAGAGTGTAATTGCTA GCATAACCGCTGC NO: 535
SEQ ID DNA GTAACCACGTTGCCAGACCGAGTCTAC CAGCGATCCTCAG NO: 536
SEQ ID DNA TATGCCCAAATCCCTCAAGTCGGCCAG GATACACCACCGT NO: 537
SEQ ID DNA AATCAAAAGGCTCACGCGCGGATTGGT CAACCTTACAACC NO: 538
SEQ ID DNA TCGGCCTTCCCAGACCACCGCAATCCCC AGGGAACAGGCA NO: 539
SEQ ID DNA CATCACACTGTCAACATACCCAGCCTG GGGA A AGAC GAAC NO: 540
SEQ ID DNA AACCCGCATGCCGATCGATGTCGTGCC TCGCTCCACGCTC NO: 541
SEQ ID DNA ACCAGGCACCCGACGGACTAACTCATC ACTCAGGCGAGGG NO: 542
[0093] In some cases, an aptamer of the disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any aptamer described herein. For example, an anti-fD aptamer of the disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any aptamer described in Table 1 or Table 2. In some cases, an aptamer of the disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%), 97%, 98%, or 99% sequence homology with any aptamer described herein. For example, an anti-fD aptamer of the disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with any aptamer described in Table 1 or Table 2.
[0094] In such cases where specific nucleotide modifications have been recited, it should be understood that any number and type of nucleotide modifications may be substituted. For example, 2'OMeG may be substituted for 2'FG. Non-limiting examples of nucleotide modifications have been provided herein. In some instances, all of the nucleotides of an aptamer are modified. In some instances, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotides of an aptamer of the disclosure may be modified.
[0095] Aptamers of the disclosure may have a hairpin-loop-hairpin-loop (H-H; Brierly, Pennell, and Gilbert (2007) Viral RNA Pseudoknots: versatile motifs in gene expression and replication. Nat. Rev. Microbiology 5, 598-610.) (also referred to as a "kissing loop") pseudoknot secondary structure comprising at least one stem and at least one loop. FIGs. 4A-C, FIGs. 5A-G, and FIGs. 6A-G depict non-limiting examples of various anti-fD aptamer secondary structures in accordance with embodiments of the disclosure. Although particular sequences and structural elements are described in FIGs. 4A-C, FIGs. 5A-G, and FIGs. 6A-G, it should be understood that FIGs. 4A-C, FIGs. 5A-G, and FIGs. 6A-G are meant as examples of various anti-fD aptamers. Other structurally-related families of anti-fD aptamers are disclosed herein. In some cases, aptamers of the disclosure may be structurally related to any aptamer described in FIGs. 4A-C, FIGs. 5A-G, and FIGs. 6A-G, but may tolerate some variation in size or nucleic acid sequence while still retaining anti-fD activity. [0096] In various aspects, aptamers of the disclosure may have a stem-loop secondary structure that includes, in a 5' to 3 ' direction, a first stem, a first loop, a second stem, a second loop, a third loop joining a first stem to a third stem, a third stem, and a fourth loop. As demonstrated in FIGs. 4A-C, the first loop may join the 5' side of the first stem to the 5' end of the second stem. The second loop may join the 5' side of the second stem to the 3 ' side of the first stem. The third loop may join the 3 ' side of the first stem to the 5' side of the third stem. The fourth loop may join the 5' side of the third stem to the 3' side of the second stem
[0097] In various aspects, an aptamer of the disclosure has a nucleic acid sequence. The aptamer may form a pseudoknot secondary structure which specifically binds to fD. In some cases, the nucleic acid sequence does not comprise any one of SEQ ID NOs: 1-3, 475-534. In some cases, the nucleic acid sequence does not comprise any one of SEQ ID NOs: 475-534. In some cases, the nucleic acid sequence does not comprise any one of SEQ ID NOs: 535-542. In some cases, the anti-fD aptamer specifically binds to an exosite of fD. In some cases, the anti-fD aptamer has a nucleic acid sequence comprising from 30 to 90 nucleotides. For example, the anti-fD aptamer may have a nucleic acid sequence comprising 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 nucleotides.
[0098] In some cases, a secondary structure of the anti-fD aptamer as defined by comparative sequence analysis and multiple sequence alignment contains less than a total of 15 unpaired residues at the 5' terminus. For example, a secondary structure of the anti-fD aptamer as defined by comparative sequence analysis and multiple sequence alignment may contain 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 unpaired nucleotide residues at the 5' terminus. In some cases, a secondary structure of the anti-fD aptamer as defined by comparative sequence analysis and multiple sequence alignment contains less than 15 unpaired residues at the 3' terminus. For example, a secondary structure of the anti-fD aptamer as defined by comparative sequence analysis and multiple sequence alignment may contain 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 unpaired nucleotide residues at the 3 ' terminus. In some cases, a secondary structure of the anti-fD aptamer as defined by comparative sequence analysis and multiple sequence alignment contains less than 30 total unpaired nucleotide residues at the 5 ' and 3 ' termini. For example, a secondary structure of the anti-fD aptamer as defined by comparative sequence analysis and multiple sequence alignment may contain 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 total unpaired nucleotide residues at the 5' and 3' termini. In some cases, a secondary structure of the anti-fD aptamer as defined by comparative sequence analysis and multiple sequence alignment has less 4 loops.
[0099] In various aspects, the pseudoknot secondary structure comprises up to four loops. For example, the pseudoknot secondary structure may comprise one loop, two loops, three loops, or four loops. Each of the up to four loops may have up to 12 nucleotides. For example, each of the up to four loops may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides.
[00100] In various aspects, the pseudoknot secondary structure comprises up to three stems. For example, the pseudoknot secondary structure may comprise one stem, two stems, or three stems. Each of the up to three stems may have up to 15 base pairs. For example, each of the up to three stems may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs.
[00101] In a particular aspect, the pseudoknot secondary structure may comprise, in a 5' to 3 ' direction, a first stem, a first loop, a second stem, a second loop, a third loop joining a first stem to a third stem, a third stem, and a fourth loop.
[00102] In various aspects, the first stem may have from 2 to 12 base pairs. For example, the first stem may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs. In more particular aspects, the first stem may have from 3 to 9 base pairs. For example, the first stem may have 3, 4, 5, 6, 7, 8, or 9 base pairs. In some cases, the first stem may have one or more mismatched base pairs.
[00103] In various aspects, the second stem may have from 2 to 9 base pairs. For example, the second stem may have 2, 3, 4, 5, 6, 7, 8, or 9 base pairs. In more particular aspects, the second stem may have 6 or 7 base pairs.
[00104] In various aspects, the third stem may have from 2 to 6 base pairs. For example, the third stem may have 2, 3, 4, 5, or 6 base pairs. In more particular aspects, the third stem may have 3 or 4 base pairs. In some cases, the third stem may comprise a nucleic acid sequence of 5'-NMHG-3 ', where N is any nucleotide; M is A or C; and H is A, C, or U.
[00105] In various aspects, the first loop may have from 1 to 5 nucleotides. For example, the first loop may have 1, 2, 3, 4, or 5 nucleotides. In various aspects, the first loop may have from 2 to 5 nucleotides. For example, the first loop may have 2, 3, 4, or 5 nucleotides. In more particular aspects, the first loop may have 2 nucleotides. In some cases, the first loop may have a nucleic acid sequence comprising, in a 5' to 3 ' direction, GU. In some cases, the first loop may have a nucleic acid sequence comprising, in a 5' to 3 ' direction, GG.
[00106] In various aspects, the second loop may have from 2 to 9 nucleotides. For example, the second loop may have 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In more particular aspects, the second loop may have from 4 to 6 nucleotides. For example, the second loop may have 4, 5, or 6 nucleotides. In some cases, the second loop may have a nucleic acid sequence comprising, in a 5' to 3 ' direction, AGUC.
[00107] In various aspects, the third loop may have from 2 to 12 nucleotides. For example, the third loop may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 nucleotides. In more particular aspects, the third loop may have from 3 to 14 nucleotides. For example, the third loop may have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides. In some cases, the third loop may comprise a nucleic acid sequence having at least 50% uridines In some cases, the third loop may include a non- nucleotidyl linker.
[00108] In various aspects, the fourth loop may have from 0 to 2 nucleotides. For example, the fourth loop may have 0, 1, or 2 nucleotides. In more particular aspects, the fourth loop may have a single nucleotide. In some cases, the single nucleotide is G or U.
[00109] In various aspects, an aptamer of the disclosure includes a polyethylene glycol (PEG) molecule covalently attached to the 5' end of the aptamer. Non-limiting examples of PEG molecules suitable for use with an aptamer of the disclosure are described throughout. In some cases, the PEG molecule has a molecular weight of 80kDa or less (e.g., 40kDa).
Anti-fD Compositions
[00110] fD is a component of the alternative complement pathway and is believed to be involved in the pathogenesis of AMD and other ocular disorders. fD is unique among serine proteases in that it does not require cleavage of a zymogen for expression of proteolytic activity. Rather, fD requires a conformational change that is believed to be induced by the complex C3bB resulting in a reversible reorientation of the catalytic center and substrate binding site of fD. fD is primarily produced by adipocytes and is systemically available in serum at low levels. fD contains a self-inhibitory loop that prevents catalytic activity of fD. Binding of the C3bB complex to fD displaces the self-inhibitory loop and fD cleaves C3bB to form the C3 convertase C3bBb. The catalytic activity of fD only occurs in the context of complexed fB; fD does not cleave uncomplexed fB. The complex of fD, fB, and C3b forms an amplification loop of the alternative complement pathway of which fD is the rate-limited enzyme.
[00111] In some aspects, the methods and compositions described herein involve inhibition of fD, resulting in inhibition of the amplification step of the alternative complement pathway. The anti-fD compositions herein may involve the use of one or more anti-fD aptamers for the treatment of ocular diseases. In some cases, the ocular disease is macular degeneration. In some cases, macular degeneration is age-related macular degeneration. In some cases, age-related macular degeneration is dry age-related macular degeneration. In some cases, dry age-related macular degeneration is advanced dry age-related macular degeneration (i.e., geographic atrophy). In some cases, age-related macular degeneration is wet age-related macular degeneration. In some cases, macular degeneration is Stargardt disease or Stargardt-like disease.
Anti-fD Inhibitors
[00112] The anti-fD compositions disclosed herein may be designed to bind to specific regions of fD with high specificity and affinity. The compositions may bind to fD in such a way as to inhibit, either directly or indirectly, the catalytic activity of the enzyme. In some cases, the anti- fD aptamers can bind to the active site (e.g., the catalytic cleft) of fD and directly inhibit the catalytic activity of fD. In this example, the aptamer may be designed to target the active site (e.g., the catalytic cleft) of fD. When the aptamer is bound to the active site of fD, it can prevent the substrate (e.g., C3bB) from accessing the active site. In some cases, the anti-fD aptamer can bind to an exosite of fD and indirectly inhibit the catalytic activity of fD by e.g., preventing the binding of C3bB. In some cases, the exosite may be remote from the catalytic site. In other cases, there may be some overlap with the catalytic site. In some cases the anti-fD aptamer can bind to the self-inhibitory loop of fD to prevent displacement of the self-inhibitory loop and thus, prevent activation of fD.
[00113] Amino acid residues of fD may be referenced according to the chymotrypsin numbering scheme and this numbering system is used throughout the disclosure to refer to specific amino acid residues of fD. Chymotrypsin numbering scheme for fD may be as depicted in FIG. 7 (SEQ ID NO: 9)(chymotrypsin numbering displayed above amino acid sequence and fD numbering scheme below amino acid sequence).
[00114] Anti-fD aptamers as described herein can modulate or inhibit the activity of fD or a fD variant thereof. A fD variant as used herein encompasses variants that perform essentially the same function as fD. A fD variant includes essentially the same structure as fD and in some cases includes at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to the amino acid sequence (shown above) of the fD protein.
[00115] In certain embodiments of the disclosure, methods are provided for the identification of fD aptamers that specifically bind to epitopes of fD. These methods may be utilized, for example, to determine the binding site and/or the mechanism of action of the aptamer.
[00116] In one instance, methods are provided for testing a fD aptamer in alternative
complement dependent hemolysis of red blood cells. Human serum that is rendered deficient in the classical complement pathway by depleting C lq may be dependent on alternative complement activity to lyse rabbit red blood cells, an activity that may be dependent on fD (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892). In some cases, the fD aptamers disclosed herein may inhibit alternative complement dependent hemolysis of red blood cells (see Example 2).
[00117] In another instance, methods are provided for testing a fD aptamer in fD esterase activity assays (see Example 3). Cleavage of a modified peptide substrate of fD, Z-lys-S-Bzl, may be monitored by the cleaved product reducing 5,5 '-Dithiobis(2-nitrobenzoic acid). FD may have a lower catalytic rate than other complement proteases when using peptide thioester substrates, and one such substrate Z-lys-SBzl was found to be cleaved by fD and useful as a synthetic substrate (fD is called protein D in Kam, McRae et al. (1987) Human complement proteins D, C2, and B. J. Biol. Chem. 262, 3444-3451). In some cases, a molecule that binds fD may block catalytic activity by binding in the catalytic cleft to sterically prevent access of the peptide substrate to the catalytic residues of fD (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892). In other cases, a molecule that binds fD may block catalytic activity by an allosteric mechanism that induces structural changes in the enzyme. In yet other cases, a molecule that binds fD may bind to the fD exosite region to sterically inhibit binding of the physiologic substrate protein C3bB, but not of the synthetic modified peptide substrate Z-Lys-SBzl (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892). In some instances, where a molecule inhibits fD binding and proteolytic cleavage of fB but not Z-Lys-SBzl, the binding may be similar to how anti-factor D FAb antibody fragment binds to the exosite and induces a subtle conformational change that increases fD cleaving Z-Lys-S-Bzl (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892).
[00118] In another instance, methods are provided for testing a fD aptamer in a reconstituted biochemical fD activity assay which is composed of purified proteins fD, fB, and C3b (see
Example 4). When fD binds to the complex of fB and C3b (C3bB), fB is cleaved by fD into fragments Ba and Bb (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892). The activity of fD can be monitored by the rate of fB cleavage and Ba fragment production using an ELISA that uses an antibody that specifically binds Ba (Quidel, A033), or by other means known in the art to measure Ba levels. In some cases, the concentrations of fB and C3b are equal so they form a 1 : 1 complex which can then bind fD and allow enzymatically active fD to cleave fB to fragments Ba and Bb. In some cases, the fB:C3b complex is present in 4-fold excess of fD. In other cases, the concentrations of fD and/or C3bB are varied in such a manner as to measure enzymatic constants, including, but not limited to kcat, Km and kcat/Km
[00119] In yet another instance, methods are provided for the identification of fD binding to C3bB in complex (see Example 5). FD is the rate-limiting enzyme in the alternative complement pathway, and converts the proconvertases C3bB and C3b2B to form the active C3 convertase C3bBb or the active C5 convertase C3b2Bb (Katschke et al 2012). For surface plasmon resonance (SPR) to detect fD in a stable complex with fB, catalytically inactive fD (SI 95 A) may be used so that it does not cleave the fB upon binding to the fB:C3b complex (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892). When C3b is amine-coupled to a CM5 chip, SPR may detect binding of fB as increased mass, and binding of fD to the C3b:fB complex as a further increase in mass. In one aspect, the fD binding compounds are aptamers that bind fD and prevent fD binding to fB:C3b as determined by a reduced mass detected by SPR.
[00120] In some cases, a cell model of Stargardt disease may be used to detect activity of anti-fD aptamers (see Example 6). Retinal pigment epithelial (RPE) cells may undergo cell death early during the progress of Stargardt disease, and evidence points toward the involvement of the alternative complement pathway (AP) in RPE cell death (Berchuck, Yang, et al (2013) All-trans- retinal (atRal) sensitizes human RPE cells to alternative complement pathway-induced cell death. Invest Ophthalmol Vis Sci 54, 2669-2677). ARPE-19 cells are a spontaneously arising RPE cell line derived from the normal eyes of a 19-year-old male. The ARPE-19 cell line, established using the cuboidal basal cell layer cultured in specific culture media, expresses the RPE-specific markers cellular retinaldehyde binding protein and RPE-65. Stargardt disease is a hereditary juvenile macular degeneration that occurs in patients with homozygous mutations in the ABCA4 genes, which encode a protein that is responsible for removal of bisretinoid fluorophores, which can include N-retinylidene-N-retinyethanolamine (A2E), all-trans-retinal and related photo-oxidation products of vitamin A aldehyde which together constitute lipofuscin from photoreceptor cells (Molday (2007) ATP -binding cassette transporter ABCA4: molecular properties and role in vision and macular degeneration. J. Bioenerg Biomembr 39, 507-517). An ABCA4 and RDH8 mouse model of Stargardt disease presents with retinal pathology caused by accumulated atRal, and ABCA4 mutations are present in 16% of AMD patients, suggesting that elevated atRal may contribute to Stargardt disease and AMD disease progression (Berchuck et al 2013). Mechanistically, atRal decreased expression of CD46 and CD59 on RPE cells in vitro, which increased susceptibility to cell lysis mediated by alternative complement in response to anti-RPE antibody binding to the RPE cell membranes (Berchuck et al 2013). In some cases, the disclosure provides for the identification of fD inhibitors that inhibit alternative complement- mediated lysis of human retinal pigmented epithelial cells.
[00121] The anti-fD aptamers as disclosed herein, in some cases, may bind to the region of fD that includes the active site cleft. Upon activation by binding to C3bB, fD exhibits serine protease activity towards fB. Activation of fD by substrate binding is a two-step process: first, fD binds to fB in the open C3bB configuration at the Von Willebrand factor type-A (VWA)- serine protease (SP) interface of fB, interacting mainly via its exosite residues within loops 145- 149, 169-173, 185-188 and 220-224. Binding of fD to C3bB causes the self-inhibitory loop of fD to be displaced from the active site cleft. The global architecture of fD is comprised of two anti-parallel beta barrel domains, each composed of six or seven beta strands that have the same topology in both domains. The beta-strands are connected by 14 turns/loops and three short alpha helices. The active site cleft is located within the loop formed between the two beta barrels, and encompasses structural elements including helix 1, loop 7 and beta-strand 7, loop 11 and beta- strand 11, beta-strand 12, loop 13 and beta-strand 13 (Jing et. al. 1998). Aptamers which bind the active site cleft could recognize any portion of the alpha helices, loops and beta strands which comprise the portion of fD within which the active site cleft resides, and by binding to this region, may prevent access to the active site cleft. Such residues include the catalytic triad, His57, Aspl02 and Serl95, the oxyanion hole including the backbone amine of residue 193 and Serl95, the residues linking the catalytic triad to the oxyanion hole via a salt bridge including residue 16, 194 and Serl95, the S I pocket, including residues 189-192, 214-216, and 224-228, as well as other elements of the specificity pocket including those residues comprising the S2, S3, S4 and Sn pockets. In particular, such aptamers would prevent interaction of P2-Pn residues of fB with specificity pockets S2-Sn of fD. In some cases, the aptamers as described herein specifically bind to the active site cleft or a region comprising the active site cleft of fD.
Aptamers that are said to bind to the active site cleft or a region comprising the active site cleft may include any aptamers that bind to one or more of the regions including the catalytic triad (His57, Aspl02 and Serl95); the oxyanion hole including the backbone amine of residue 193 and Serl95; the residues linking the catalytic triad to the oxyanion hole via a salt bridge including residue 16, 194 and Serl95; the S I pocket, including residues 189-192, 214-216, and 224-228; as well as other elements of the specificity pocket including those residues comprising the S2, S3, S4 and Sn pockets. [00122] Such fD inhibitors may inhibit alternative complement dependent hemolysis of red blood cells, may inhibit esterase activity of fD against thioester substrates of fD such as Z-Lys-S- Bzl, and may inhibit fB cleavage in the C3bB complex by fD. In esterase assays, such inhibitors may reduce kcat and increase Km of fD, with the primary effect decreasing kcat and decreasing kcat/Km (Hedstrom). In complete biochemical assays, such inhibitors may decrease kcat and increase Km, with a primary effect decreasing kcat and decreasing kcat/Km. Such inhibitors may not prevent formation of the enzyme-substrate complex (fD-C3bB complex) as assessed in enzymatic assays or enzyme-substrate assembly assays, such as surface plasmon resonance (SPR) assays described in Forneris et. al. or Katschke et. al., or similar E-S assembly assays assessed by ELISA or similar assays.
[00123] The anti-fD aptamers as disclosed herein, in some cases, may bind to the region of fD that includes the self-inhibitory loop (residues 212-218) and regions adjacent to the self- inhibitory loop, so as to stabilize the self-inhibited state of fD. Mature fD maintains a self- inhibited state through a set of conformations in the free fD state including the conformation of residues 212-218, which may be referred to as the self-inhibitory loop of fD. These residues may comprise portions of the polypeptide binding site as well as the SI specificity pocket of fD. In the inactive state of fD, this loop is in an elevated conformation and forms specific bonds with key components of the catalytic triad and SI specificity pocket, rendering fD inactive. In some cases, the anti-fD compounds of the disclosure are designed to target the self-inhibitory loop of fD to prevent the activation of fD. For example, the anti-fD compounds may bind to the self- inhibitory loop or to regions around the self-inhibitory loop to prevent displacement of the self- inhibitory loop from the active site cleft. In some cases, the anti-fD compounds may be designed to target residues 212-218 of fD. In cases where anti-fD aptamers bind to a region comprising one or more of amino acid residues 212-218 of fD, it may be said that such anti-fD aptamers bind to the self-inhibitory loop or a portion thereof of fD.
[00124] Such fD inhibitors may inhibit alternative complement dependent hemolysis of red blood cells, may inhibit esterase activity of fD against thioester substrates of fD such as Z-Lys-S- Bzl, and may inhibit fB cleavage in the C3bB complex by fD. In esterase assays, such inhibitors may reduce kcat and increase Km of fD, with the primary effect decreasing kcat and decreasing kcat Km. In complete biochemical assays, such inhibitors may decrease kcat and increase Km, with a primary effect decreasing kcat and decreasing kcat/Km. Such inhibitors may not prevent formation of the enzyme-substrate complex (fD-C3bB complex) as assessed in enzymatic assays or enzyme-substrate assembly assays, such as surface plasmon resonance (SPR) assays described in Forneris et. al. or Katschke et. al., or similar E-S assembly assays assessed by ELISA or similar assays.
[00125] The anti-fD aptamers as disclosed herein, in some cases, may bind to the exosite of fD so as to prevent formation of the ES complex. Without wishing to be bound by theory, the high specificity of fD for fB may be due to protein-protein interactions between the exosites of fD and fB. The exosite of fD is approximately 25 A from the catalytic center and consists of 4 loops comprised by residues 145-149, 169-173, 185-188 and 220-224. In some cases, the anti-fD compounds of the disclosure may target the exosite of fD and prevent the interaction of fD with fB. Anti-fD compounds of this nature may target one or more of the 4 loops of the fD exosite, for example, the anti-fD compounds may be designed to target one or more of amino acid residues 145-149, 169-173, 185-188 and 220-224 of fD. In cases where an anti-fD aptamer binds to one or more of amino acid residues 145-149, 169-173, 185-188, and 220-224, it may be said that such aptamers bind to the exosite of fD.
[00126] Aptamer inhibitors that block binding of the C3bB substrate to fD may inhibit alternative complement dependent hemolysis of red blood cells. Such inhibitors may enhance the esterase activity of fD against thioester substrates of fD such as Z-Lys-S-Bzl, as observed for the anti-fD Fab's when bound to human fD (Katschke et. al.). Alternatively, aptamers which bind to the exosite of fD may not impact the esterase activity of fD, as for example, when the anti-fD Fab in Katschke et. al. binds fD from cynomolgus monkeys, it neither inhibits nor enhances fD esterase activity. Exosite binding aptamers would inhibit fB cleavage in the C3bB complex by fD. In esterase assays, such inhibitors may increase kcat and have no or minimal impact on Km of fD, with the primary effect increasing kcat and increasing kcat/Km, or such inhibitors would neither impact kcat or Km or kcat/Km In complete biochemical assays, such inhibitors would primarily increase Km and decrease kcat/Km. Such inhibitors may prevent formation of the enzyme-substrate complex (fD-C3bB complex) as assessed in enzymatic assays or enzyme-substrate assembly assays, such as surface plasmon resonance (SPR) assays described in Forneris et. al. or Katschke et. al., or similar ES assembly assays assessed by ELISA or similar assays.
[00127] In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 50nM and may inhibit at least 85% of fD activity in an alternative complement dependent hemolysis assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 25nM and may inhibit at least 85% of fD activity in an alternative complement dependent hemolysis assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about ΙΟηΜ and may inhibit at least 85% of fD activity in an alternative complement dependent hemolysis assay. In some cases, an anti- fD aptamer as disclosed herein may bind to fD with a K of less than about 5nM and may inhibit at least 85% of fD activity in an alternative complement dependent hemolysis assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a K of less than about lnM and may inhibit at least 85% of fD activity in an alternative complement dependent hemolysis assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 500pM and may inhibit at least 85% of fD activity in an alternative complement dependent hemolysis assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 50pM and may inhibit at least 85% of fD activity in an alternative complement dependent hemolysis assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 5pM and may inhibit at least 85% of fD activity in an alternative complement dependent hemolysis assay.
[00128] In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 50nM and may inhibit at least 85% of fD activity in a fD convertase assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 25nM and may inhibit at least 85% of fD activity in a fD convertase assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about lOnM and may inhibit at least 85%) of fD activity in a fD convertase assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 5nM and may inhibit at least 85% of fD activity in a fD convertase assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about lnM and may inhibit at least 85% of fD activity in a fD convertase assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 500pM and may inhibit at least 85% of fD activity in a fD convertase assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 50pM and may inhibit at least 85% of fD activity in a fD convertase assay. In some cases, an anti-fD aptamer as disclosed herein may bind to fD with a Kd of less than about 5pM and may inhibit at least 85% of fD activity in a fD convertase assay.
[00129] Catalytic turn-over of fD activation of ffi requires dissociation of the ES complex if bound in a non-productive state or the EP (fD-C3bBb) complex upon fB cleavage. The anti-fD aptamers as disclosed herein, in some cases, may bind to fD in such a way as to prevent dissociation of fD from C3bB or C3bBb. As envisioned, such aptamers may bind near the exosite of fD and bind to fD in such a manner as to increase the affinity of fD for C3bB or C3bBb by decreasing the off-rate of this interaction. Such aptamers could be generated by selection against the fD-C3bB complex, by for example using a catalytically inactivated form of fD such as a mutant form in which Serl95 is mutated to Alal95 (Forneris et. al.), to provide a stable, non-reactive ES complex as a target for selection. Aptamers possessing such a mechanism of action would inhibit alternative complement dependent hemolysis of red blood cells. Such inhibitors may inhibit the esterase activity of fD against thioester substrates of fD such as Z-Lys-S-Bzl, or may not impact the esterase activity of fD. Such binding aptamers would inhibit the turn-over of fB cleavage in the C3bB complex by fD. In esterase assays, such inhibitors may decrease the kcat and have no or minimal impact on Km of fD, with the primary effect decreasing kcat and decreasing kcat Km, or such inhibitors would neither impact kcat or Km or kcat m In complete biochemical assays, such inhibitors would primarily decrease Kcat and decrease kcat/Km. Such inhibitors would enhance formation of the enzyme-substrate complex (fD-C3bB complex) as assessed in enzymatic assays or enzyme-substrate assembly assays, such as surface plasmon resonance (SPR) assays described in Forneris et. al., and may increase the apparent affinity of fD for C3bB or C3bBb
[00130] In some cases, an aptamer as described herein may bind the same epitope as an anti-fD antibody or antibody fragment thereof. In some cases, an aptamer as described herein may bind to the same epitope as an anti-fD therapeutic antibody. For example, the anti-fD aptamer may bind to the same or similar region of fD to that which an anti-fD therapeutic antibody such as an anti-fD Fab with an amino acid sequence of heavy chain variable region according to SEQ ID NO: 7 and an amino acid sequence of light chain variable region according to SEQ ID NO:8; Mab 166-3 or LS-C135735 bind. For example, an anti-fD Fab with an amino acid sequence of heavy chain variable region according to SEQ ID NO: 7 and light chain variable region according to SEQ ID NO: 8 may bind residues 129-132, residues 164-178, Arg223 and Lys224, with the bulk of the interaction involving the loop encompassing amino acid 170 (the "170 loop"). In some cases, an aptamer that binds to the same or similar region of fD to that which an anti-fD Fab with an amino acid sequence of heavy chain variable region according to SEQ ID NO: 7 and light chain variable region according to SEQ ID NO: 8 binds (e.g., a region comprising one or more of amino acid residues 129-132, 164-178, Arg223 and Lys224) may be said to be binding to the exosite of fD.
[00131] In some cases, an anti-fD aptamer for the modulation of fD is provided. In some cases, an anti-fD aptamer for the inhibition of a function associated with fD is provided. In some cases, the anti-fD aptamer inhibits the catalytic activity of fD. In some cases, an anti-fD aptamer for the treatment of dry AMD or geographic atrophy is provided. In some cases, an anti-fD aptamer for the treatment of wet AMD is provided. In some cases, an anti-fD aptamer for the treatment of Stargardt disease is provided. [00132] The dissociation constant (¾) can be used to describe the affinity of an aptamer for a target (or to describe how tightly the aptamer binds to the target) or to describe the affinity of an aptamer for a specific epitope of a target (e.g., exosite, catalytic cleft, etc.). The dissociation constant is defined as the molar concentration at which half of the binding sites of a target are occupied by the aptamer. Thus, the smaller the ¾, the tighter the binding of the aptamer to its target. In some cases, an anti-fD aptamer has a dissociation constant (Kd) for fD protein of less than ImM, less than 100 μΜ, less than 10μΜ, less than ΙμΜ, less than ΙΟΟηΜ, less than lOnM, less than lnM, less than 500pM, or less than ΙΟΟρΜ. In some cases, an anti-fD aptamer has a dissociation constant (¾) for fD protein of less than 50nM. In some cases, an anti-fD aptamer has a dissociation constant (Kd) for fD protein of less than 25nM. In some cases, an anti-fD aptamer has a dissociation constant (Kd) for fD protein of less than lOnM. In some cases, an anti-fD aptamer has a dissociation constant (Kd) for fD protein of less than 5nM. In some cases, an anti-fD aptamer has a dissociation constant (Kd) for fD protein of less than 500pM. In some cases, an anti-fD aptamer has a dissociation constant (Kd) for fD protein of less than 50pM. In some cases, an anti-fD aptamer has a dissociation constant (Kd) for fD protein of less than 5pM. In some cases, the aptamer binds to the catalytic cleft, the active site, the exosite, and/or the self- inhibitory loop of fD with a Kd of less than about ImM, ΙΟΟμΜ, 10μΜ, Ι μΜ, ΙΟΟηΜ, 50nM, 25nM, lOnM, 5nM, 500pM, 50pM, or 5pM. In some cases, the Kd is determined by a flow cytometry assay as described herein.
[00133] The aptamers disclosed herein may bind to the catalytic cleft of fD with a Kd of less than about 50nM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the catalytic cleft of fD with a Kd of less than about 50nM and have an IC50 of less than about lOnM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the catalytic cleft of fD with a Kd of less than about 50nM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the catalytic cleft of fD with a Kd of less than about lOnM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the catalytic cleft of fD with a Kd of less than about lOnM and have an IC50 of less than about lOnM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the catalytic cleft of fD with a Kd of less than about lOnM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the catalytic cleft of fD with a Kd of less than about 5nM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the catalytic cleft of fD with a Kd of less than about 5nM and have an IC50 of less than about ΙΟηΜ as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the catalytic cleft of fD with a Kd of less than about 5nM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay.
[00134] The aptamers disclosed herein may bind to the active site of fD with a K of less than about 50nM and have an IC50 of less than about 50nM as measured by an alternative
complement dependent hemolysis assay. The aptamers disclosed herein may bind to the active site of fD with a Kd of less than about 50nM and have an IC50 of less than about lOnM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the active site of fD with a Kd of less than about 50nM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the active site of fD with a Kd of less than about lOnM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the active site of fD with a Kd of less than about lOnM and have an IC50 of less than about lOnM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the active site of fD with a Kd of less than about lOnM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the active site of fD with a Kd of less than about 5nM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the active site of fD with a Kd of less than about 5nM and have an IC50 of less than about lOnM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the active site of fD with a Kd of less than about 5nM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay.
[00135] The aptamers disclosed herein may bind to the exosite of fD with a Kd of less than about 50nM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the exosite of fD with a Kd of less than about 50nM and have an IC50 of less than about lOnM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the exosite of fD with a Kd of less than about 50nM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the exosite of fD with a ¾ of less than about ΙΟηΜ and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the exosite of fD with a Kd of less than about ΙΟηΜ and have an IC50 of less than about ΙΟηΜ as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the exosite of fD with a K of less than about ΙΟηΜ and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the exosite of fD with a Kd of less than about 5nM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the exosite of fD with a ¾ of less than about 5nM and have an IC50 of less than about ΙΟηΜ as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the exosite of fD with a Kd of less than about 5nM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay.
[00136] The aptamers disclosed herein may bind to the self-inhibitory loop of fD with a Kd of less than about 50nM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the self- inhibitory loop of fD with a Kd of less than about 50nM and have an IC50 of less than about lOnM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the self-inhibitory loop of fD with a ¾ of less than about 50nM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the self-inhibitory loop of fD with a Kd of less than about lOnM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the self-inhibitory loop of fD with a Kd of less than about lOnM and have an IC50 of less than about lOnM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the self-inhibitory loop of fD with a Kd of less than about lOnM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the self-inhibitory loop of fD with a Kd of less than about 5nM and have an IC50 of less than about 50nM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the self-inhibitory loop of fD with a Kd of less than about 5nM and have an IC50 of less than about lOnM as measured by an alternative complement dependent hemolysis assay. The aptamers disclosed herein may bind to the self-inhibitory loop of fD with a Kd of less than about 5nM and have an IC50 of less than about 5nM as measured by an alternative complement dependent hemolysis assay.
[00137] In some aspects, the aptamers disclosed herein have an improved half-life as compared to other therapeutics, including antibodies. In some cases, the aptamers have an improved half- life in a biological fluid or solution as compared to an antibody. In some cases, the aptamers have an improved half-life in vivo as compared to an antibody. In one example, the aptamers have an improved half-life when injected into the eye (intraocular half-life) as compared to an antibody. In some cases, the aptamers may have an improved intraocular half-life when injected into the eye of a human. In some cases, the aptamers may demonstrate improved stability over antibodies under physiological conditions.
[00138] In some cases, the aptamers described herein have an intraocular half-life of at least 7 days in a human. In some cases, the aptamers described herein have an intraocular half-life of at least 8 days, at least 9 days, at least 10 days, at least 1 1 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 20 days or greater in a human.
[00139] In some cases, the aptamers described herein have an intraocular half-life of at least 1 day in a non-human animal (e.g., rodent/rabbit/monkey). In some cases, the aptamers described herein have an intraocular half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days or greater in a non-human animal such as a rodent, rabbit or monkey.
[00140] In some aspects, the aptamers described herein may have a shorter half-life as compared to other therapeutics. For example, an unmodified or unconjugated aptamer may have a lower half-life as compared to a modified or conjugated aptamer, however, the low molecular weight of the unmodified or unconjugated forms may allow for orders of magnitude greater initial concentrations, thereby achieving greater duration/efficacy. In some examples, the aptamer may have an intraocular half-life of less than about 7 days in a human. In some examples, the aptamers described herein have an intraocular half-life of less than about 6 days, less than about 5 days or even less than about 4 days in a human.
[00141] The aptamers disclosed herein may demonstrate high specificity for fD versus other complement pathway components. Generally, the aptamer may be selected such that the aptamer has high affinity for fD, but with little to no affinity for other complement pathway components or serine proteases. In some cases, the aptamers bind to fD with a specificity of at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, or greater than 20-fold greater than the aptamers bind to any of C3, C5, Factor B, Factor H or Factor I (or any of their related dimeric, trimeric, or multimeric complexes, units or subunits) at relative serum concentrations. For example, in some cases the aptamers bind to fD with a specificity of at least 50-fold greater than the aptamers bind to any of C3, C5, Factor B, Factor H or Factor I (or any of their related dimeric, trimeric, or multimeric complexes, units or subunits) at relative serum concentrations. For example, in some cases the aptamers bind to FD with a specificity of at least 100-fold greater than the aptamers bind to any of C3, C5, Factor B, Factor H or Factor I (or any of their related dimeric, trimeric, or multimeric complexes, units or subunits) at relative serum concentrations.
[00142] The activity of a therapeutic agent can be characterized by the half maximal inhibitory concentration (IC50). The IC50 is calculated as the concentration of therapeutic agent in nM at which half of the maximum inhibitory effect of the therapeutic agent is achieved. The IC50 is dependent upon the assay utilized to calculate the value. In some examples, the IC50 of an aptamer described herein is less than ΙΟΟηΜ, less than 50nM, less than 25nM, less than ΙΟηΜ, less than 5nM, less than InM, less than 0.5nM, less than O. lnM or less than O.OlnM as measured by an alternative complement dependent hemolysis assay (Pangburn, 1988, Methods in
Enzymology; and Katschke, 2009, Journal of Biological Chemistry).
[00143] In some examples, the aptamers described herein increase the activity of fD as measured by a fD esterase activity assay as compared to a control, and inhibit activity of fD as measured by an alternative complement dependent hemolysis assay. In other examples, the aptamers described herein inhibit activity of fD as measured by a fD esterase activity assay as compared to a control, and inhibit activity of fD as measured by an alternative complement dependent hemolysis assay. In yet other cases, the aptamer does not inhibit activity of complement fD as measured by a fD esterase activity assay as compared to a control, and does inhibit activity of fD as measured by an alternative complement dependent hemolysis assay.
[00144] Aptamers generally have high stability at ambient temperatures for extended periods of time. The aptamers described herein demonstrate greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9% activity in solution under physiological conditions at 30 days or later.
[00145] In some cases, a composition of the disclosure comprises anti-fD aptamers, wherein essentially 100% of the anti-fD aptamers comprise nucleotides having ribose in the β-D- ribofuranose configuration. In other examples, a composition of the disclosure may comprise anti-fD aptamers, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90% of the anti-fD aptamers have ribose in the β-D-ribofuranose configuration. Indications [00146] In some aspects, the methods and compositions provided herein are used for the treatment of ocular diseases or disorders. Ocular diseases or disorders can include, without limitation, any complement-mediated ocular disorders such as inflammatory conjunctivitis, including allergic and giant papillary conjunctivitis, macular edema, uveitis, endophthalmitis, scleritis, corneal ulcers, dry eye syndrome, glaucoma, ischemic retinal disease, corneal transplant rejection, complications related to intraocular surgery such intraocular lens implantation and inflammation associated with cataract surgery, Behcet's disease, Stargardt disease, immune complex vasculitis, Fuch's disease, Vogt-Koyanagi-Harada disease, subretinal fibrosis, keratitis, vitreo-retinal inflammation, ocular parasitic infestation/migration, retinitis pigmentosa, cytomeglavirus retinitis and choroidal inflammation.
[00147] Other examples of ocular diseases or disorders that may be amendable to treatment by the methods and compositions provided herein may include, without limitation, ectropion, lagophthalmos, blepharochalasis, ptosis, xanthelasma of the eyelid, parasitic infestation of the eyelid, dermatitis of the eyelid, dacryoadenitis, epiphora, dysthyroid exophthalmos,
conjunctivitis, scleritis, keratitis, corneal ulcer, corneal abrasion, snow blindness, arc eye, Thygeson's superficial punctate keratopathy, corneal neovascularization, Fuchs' dystrophy, keratoconus, keratoconjunctivitis sicca, iritis, uveitis, sympathetic ophthalmia, cataracts, chorioretinal inflammation, focal chorioretinal inflammation, focal chorioretinitis, focal choroiditis, focal retinitis, focal retinochoroiditis, disseminated chorioretinal inflammation, disseminated chorioretinitis, disseminated choroiditis, disseminated retinitis, disseminated retinochoroiditis, exudative retinopathy, posterior cyclitis, pars planitis, Harada's disease, chorioretinal scars, macula scars of posterior pole, solar retinopathy, choroidal degeneration, choroidal atrophy, choroidal sclerosis, angioid streaks, hereditary choroidal dystrophy, choroideremia, choroidal dystrophy (central arealor), gyrate atrophy (choroid), ornithinaemia, choroidal haemorrhage and rupture, choroidal haemorrhage (not otherwise specified), choroidal haemorrhage (expulsive), choroidal detachment, retinoschisis, retinal artery occlusion, retinal vein occlusion, hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathy of prematurity, macular degeneration, Bull's Eye maculopathy, epiretinal membrane, peripheral retinal degeneration, hereditary retinal dystrophy, retinitis pigmentosa, retinal haemorrhage, separation of retinal layers, central serous retinopathy, retinal detachment, macular edema, glaucoma - optic neuropathy, glaucoma suspect - ocular hypertension, primary open-angle glaucoma, primary angle-closure glaucoma, floaters, Leber's hereditary optic neuropathy, optic disc drusen, strabismus, ophthalmoparesis, progressive external ophthaloplegia, esotropia, exotropia, disorders of refraction and accommodation, hypermetropia, myopia, astigmastism, anisometropia, presbyopia, internal ophthalmoplegia, amblyopia, Leber's congenital amaurosis, scotoma, anopsia, color blindness, achromatopsia, maskun, nyctalopia, blindness, River blindness, micropthalmia, coloboma, red eye, Argyll Robertson pupil, keratomycosis, xerophthalmia, aniridia, sickle cell retinopathy, ocular neovascularization, retinal
neovascularization, subretinal neovascularization; rubeosis iritis inflammatory diseases, chronic posterior and pan uveitis, neoplasms, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting following a combined vitrectomy-2 and lensectomy, vascular diseases, retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis,
neovascularization of the optic nerve, diabetic macular edema, cystoid macular edema, proliferative vitreoretinopathy, and neovascularization due to penetration of the eye or ocular injury.
[00148] In some aspects, the methods and compositions provided herein are suitable for the treatment of macular degeneration. In some cases, macular degeneration is age-related macular degeneration. In some cases, the methods and compositions can be utilized to treat neovascular or exudative ("wet") age-related macular degeneration. In other cases, the methods and compositions can be utilized to treat non-exudative ("dry") age-related macular degeneration. In some cases, advanced forms of dry age-related macular degeneration can be treated, including geographic atrophy. In some cases, the methods and compositions herein can be utilized to prevent age-related macular degeneration and associated diseases thereof. In other cases, the methods and compositions herein can be utilized to slow or halt the progression of age-related macular degeneration and associated diseases thereof.
[00149] In some aspects, the methods and compositions provided herein are suitable for the treatment of Stargardt disease. In some cases, the methods and compositions herein can be utilized to prevent age-related Stargardt disease. In other cases, the methods and compositions herein can be utilized to slow or halt the progression of Stargardt disease.
[00150] In some aspects, the methods and compositions provided herein are suitable for the treatment of diseases causing ocular symptoms. Examples of symptoms which may be amenable to treatment with the methods disclosed herein include: increased drusen volume, reduced reading speed, reduced color vision, retinal thickening, increase in central retinal volume and/or, macular sensitivity, loss of retinal cells, increase in area of retinal atrophy, reduced best corrected visual acuity such as measured by Snellen or ETDRS scales, Best Corrected Visual Acuity under low luminance conditions, impaired night vision, impaired light sensitivity, impaired dark adaptation, contrast sensitivity, and patient reported outcomes. [00151] In some cases, the methods and compositions provided herein may alleviate or reduce a symptom of a disease. In some cases, treatment with an aptamer provided herein may result in a reduction in the severity of any of the symptoms described herein. In some cases, treatment with an aptamer described herein may slow, halt or reverse the progression of any of the symptoms described herein. In some cases, treatment with an aptamer described herein may prevent the development of any of the symptoms described herein. In some cases, treatment with an aptamer described herein may slow, halt or reverse the progression of a disease, as measured by the number and severity of symptoms experienced. Examples of symptoms and relevant endpoints where the aptamer may have a therapeutic effect include increased drusen volume, reduced reading speed, reduced color vision, retinal thickening, increase in central retinal volume and/or, macular sensitivity, loss of retinal cells, increase in area of retinal atrophy, reduced best corrected visual acuity such as measured by Snellen or ETDRS scales, Best Corrected Visual Acuity under low luminance conditions, impaired night vision, impaired light sensitivity, impaired dark adaptation, contrast sensitivity, and patient reported outcomes. In some instances, treatment with an aptamer described herein may have beneficial effects as measured by clinical endpoints including drusen volume, reading speed, retinal thickness as measured by Optical Coherence Tomography or other techniques, central retinal volume, number and density of retinal cells, area of retinal atrophy as measured by Fundus Photography or Fundus
Autofluoresence or other techniques, best corrected visual acuity such as measured by Snellen or ETDRS scales, Best Corrected Visual Acuity under low luminance conditions, light sensitivity, dark adaptation, contrast sensitivity, and patient reported outcomes as measured by such tools as the National Eye Institute Visual Function Questionnaire and Health Related Quality of Life Questionnaires.
Subjects
[00152] In some aspects, the methods and compositions provided herein are utilized to treat a subject in need thereof. In some cases, the subject suffers from an ocular disease or disorder. In some cases, the subject is a human. In some cases, the human is a patient at a hospital or a clinic. In some cases, the subject is a non-human animal, for example, a non-human primate, a livestock animal, a domestic pet, or a laboratory animal. For example, a non-human animal can be an ape (e.g., a chimpanzee, a baboon, a gorilla, or an orangutan), an old world monkey (e.g., a rhesus monkey), a new world monkey, a dog, a cat, a bison, a camel, a cow, a deer, a pig, a donkey, a horse, a mule, a lama, a sheep, a goat, a buffalo, a reindeer, a yak, a mouse, a rat, a rabbit, or any other non-human animal. In some cases, the subject is a human. In some cases, the human is a patient at a hospital or a clinic.
[00153] In cases where the subject is a human, the subject may be of any age. In some cases, the subject has an age-related ocular disease or disorder (e.g., age-related macular degeneration, Stargardt disease). In some cases, the subj ect is about 50 years or older. In some cases, the subject is about 55 years or older. In some cases, the subject is about 60 years or older. In some cases, the subject is about 65 years or older. In some cases, the subject is about 70 years or older. In some cases, the subject is about 75 years or older. In some cases, the subject is about 80 years or older. In some cases, the subject is about 85 years or older. In some cases, the subject is about 90 years or older. In some cases, the subject is about 95 years or older. In some cases, the subject is about 100 years or older. In some cases, the subject is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or greater than 100 years old. In some cases, the subject is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater than 20 years old.
[00154] In cases where the subject is a human, the subject may have any genetic profile. In some cases, the subject may have mutations in complement Factor H (CFH), complement component 3 (C3), complement component 2 (C2), complement Factor B, complement Factor I (CFI), ABC4A, ELOVL4, or any combination thereof.
[00155] In some aspects, the methods and compositions provided herein are utilized to treat a subject suffering from ocular symptoms as described herein. In some aspects, the methods and compositions provided herein are utilized to treat a subject suffering from an ocular disease as provided herein. In some cases, the methods and compositions provided herein are utilized to treat a subject suffering from wet AMD. In some cases, the methods and compositions provided herein are utilized to treat a subject suffering from dry AMD or geographic atrophy. In some cases, the methods and compositions provided herein are utilized to treat a subject suffering from Stargardt disease.
[00156] In some aspects, the methods and compositions provided herein may be utilized to treat a subject with a highly active immune system. In some cases, the methods and compositions provided herein may be used to treat a subject with an autoimmune disease. In some cases, the methods and compositions provided herein may be used to treat a subject with an inflammatory disease. In some cases, the methods and compositions provided herein may be used to treat a subject undergoing an inflammatory reaction to a disease such as an infectious disease. For example, the aptamers described herein may be used to treat a subject with a fever. In some cases, the aptamers described herein may be used to treat a subject with an allergy. In some cases, the aptamers described herein may be used to treat a subject suffering from an allergic response. In some cases, the aptamers described herein may be particularly useful for treating a subject who has experienced an allergic reaction to an antibody treatment, and/or who has developed neutralizing antibodies against an antibody treatment.
Pharmaceutical compositions or medicaments
[00157] Disclosed herein are pharmaceutical compositions or medicaments, used
interchangeably, for use in a method of therapy, or for use in a method of medical treatment. Such use may be for the treatment of ocular diseases. In some cases, the pharmaceutical compositions can be used to treat AMD. In some cases, the pharmaceutical compositions can be used to treat non-exudative (dry) AMD. In some cases, the pharmaceutical compositions can be used to treat geographic atrophy (advanced dry AMD). In some cases, the pharmaceutical compositions can be used to treat wet AMD. In some cases, the pharmaceutical compositions can be used to treat Stargardt disease. Pharmaceutical compositions described herein may include one or more aptamers for the treatment of dry AMD. Pharmaceutical compositions described herein may include one or more aptamers for the treatment of wet AMD.
Pharmaceutical compositions described herein may include one or more aptamers for the treatment of Stargardt disease. In some cases, the one or more aptamers bind to and inhibit a component of the alternative complement pathway. In some cases, the one or more aptamers bind to one or more targets of fD as described herein. In some cases, the one or more aptamers inhibit fD as described herein. In some cases, the compositions include, e.g., an effective amount of the aptamer, alone or in combination, with one or more vehicles (e.g.,
pharmaceutically acceptable compositions or e.g., pharmaceutically acceptable carriers). In some cases, the compositions described herein are administered with one or more additional pharmaceutical treatments (e.g., co-administered, sequentially administered or co-formulated). In some examples, the compositions described herein are co-administered with one or more of an anti-vascular endothelial growth factor (VEGF) therapy, an anti-Factor P therapy, an anti- complement component 5 (C5) therapy, an anti-complement component 3 (C3) therapy, an anti- platelet-derived growth factor (PDGF) therapy, an anti -hypoxia-inducible factor 1 -alpha (HIFla) therapy, an anti-FAS therapy, an anti-integrin therapy or an anti-angiopoietin-2 (Ang2) therapy.
Formulations [00158] Compositions as described herein may comprise a liquid formulation, a solid formulation or a combination thereof. Non-limiting examples of formulations may include a tablet, a capsule, a gel, a paste, a liquid solution and a cream. The compositions of the present disclosure may further comprise any number of excipients. Excipients may include any and all solvents, coatings, flavorings, colorings, lubricants, disintegrants, preservatives, sweeteners, binders, diluents, and vehicles (or carriers). Generally, the excipient is compatible with the therapeutic compositions of the present disclosure. The pharmaceutical composition may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as, for example, sodium acetate, and
triethanolamine oleate.
Dosage and Routes of Administration
[00159] Therapeutic doses of formulations of the disclosure can be administered to a subject in need thereof. In some cases, a formulation is administered to the eye of a subject to treat, for example, dry AMD, geographic atrophy, wet AMD or Stargardt disease. Administration to the eye can be a) topical; b) local ocular delivery; or c) systemic. A topical formulation can be applied directly to the eye (e.g., eye drops, contact lens loaded with the formulation) or to the eyelid (e.g., cream, lotion, gel). In some cases, topical administration can be to a site remote from the eye, for example, to the skin of an extremity. This form of administration may be suitable for targets that are not produced directly by the eye. In one non-limiting example, fD is thought to be produced primarily by adipose cells, and thus an anti-fD aptamer may be administered topically to a non-ocular region of the body. In some cases, a formulation of the disclosure is administered by local ocular delivery. Non-limiting examples of local ocular delivery include intravitreal (IVT), intracamarel, subconjunctival, subtenon, retrobulbar, posterior juxtascleral, and peribulbar. In some cases, a formulation of the disclosure is delivered by intravitreal administration (IVT). Local ocular delivery may generally involve injection of a liquid formulation. In other cases, a formulation of the disclosure is administered systemically. Systemic administration can involve oral administration. In some cases, systemic administration can be intravenous administration, subcutaneous administration, infusion, implantation, and the like.
[00160] Other formulations suitable for delivery of the pharmaceutical compositions described herein may include a sustained release gel or polymer formulations by surgical implantation of a biodegradable microsize polymer system, e.g., microdevice, microparticle, or sponge, or other slow release transscleral devices, implanted during the treatment of an ophthalmic disease, or by an ocular delivery device, e.g. polymer contact lens sustained delivery device. In some cases, the formulation is a polymer gel, a self-assembling gel, a durable implant, an eluting implant, a biodegradable matrix or biodegradable polymers. In some cases, the formulation may be administered by iontophoresis using electric current to drive the composition from the surface to the posterior of the eye. In some cases, the formulation may be administered by a surgically implanted port with an intravitreal reservoir, an extra-vitreal reservoir or a combination thereof. Examples of implantable ocular devices can include, without limitation, the Durasert™ technology developed by Bausch & Lomb, the ODTx device developed by On Demand
Therapeutics, the Port Delivery System developed by ForSight VISION4 and the Replenish MicroPump™ System developed by Replenish, Inc.
[00161] In some cases, nanotechnologies can be used to deliver the pharmaceutical compositions including nanospheres, nanoparticles, nanocapsules, liposomes, nanomicelles and dendrimers.
[00162] A composition of the disclosure can be administered once or more than once each day. In some cases, the composition is administered as a single dose (i.e., one-time use). In this example, the single dose may be curative. In other cases, the composition may be administered serially (e.g., taken every day without a break for the duration of the treatment regimen). In some cases, the treatment regime can be less than a week, a week, two weeks, three weeks, a month, or greater than a month. In some cases, the composition is administered over a period of at least 12 weeks. In other cases, the composition is administered for a day, at least two consecutive days, at least three consecutive days, at least four consecutive days, at least five consecutive days, at least six consecutive days, at least seven consecutive days, at least eight consecutive days, at least nine consecutive days, at least ten consecutive days, or at least greater than ten consecutive days. In some cases, a therapeutically effective amount can be administered one time per week, two times per week, three times per week, four times per week, five times per week, six times per week, seven times per week, eight times per week, nine times per week, 10 times per week, 1 1 times per week, 12 times per week, 13 times per week, 14 times per week, 15 times per week, 16 times per week, 17 times per week, 18 times per week, 19 times per week, 20 times per week, 25 times per week, 30 times per week, 35 times per week, 40 times per week, or greater than 40 times per week. In some cases, a therapeutically effective amount can be administered one time per day, two times per day, three times per day, four times per day, five times per day, six times per day, seven times per day, eight times per day, nine times per day, 10 times per day, or greater than 10 times per day. In some cases, the composition is administered at least twice a day. In further cases, the composition is administered at least every hour, at least every two hours, at least every three hours, at least every four hours, at least every five hours, at least every six hours, at least every seven hours, at least every eight hours, at least every nine hours, at least every 10 hours, at least every 1 1 hours, at least every 12 hours, at least every 13 hours, at least every 14 hours, at least every 15 hours, at least every 16 hours, at least every 17 hours, at least every 18 hours, at least every 19 hours, at least every 20 hours, at least every 21 hours, at least every 22 hours, at least every 23 hours, or at least every day.
[00163] Aptamers as described herein may be particularly advantageous over antibodies as they may sustain therapeutic intravitreal concentrations of drug for longer periods of time, thus requiring less frequent administration. For example, an anti-fD Fab having an amino acid sequence of heavy chain variable region according to SEQ ID NO: 7 and a light chain variable region according to SEQ ID NO: 8, may show clinical efficacy for the treatment of geographic atrophy at lOmg when dosed every 4 weeks (q4w) but not every 8 weeks (q8w). The aptamers described herein have a longer intraocular half-life, and/or sustain therapeutic intravitreal concentrations of drug for longer periods of time, than an anti-fD Fab with an amino acid sequence of heavy chain variable region according to SEQ ID NO: 7 and light chain variable region according to SEQ ID NO: 8 and other antibody therapies and thus, can be dosed less frequently. In some cases, the aptamers are dosed at least every 4 weeks (q4w), every 5 weeks (q5w), every 6 weeks (q6w), every 7 weeks (q7w), every 8 weeks (q8w), every 9 weeks (q9w), every 10 weeks (qlOw), every 12 weeks (ql2w) or greater than ql2w.
[00164] In some aspects, a therapeutically effective amount of the aptamer is administered. A "therapeutically effective amount" or "therapeutically effective dose" are used interchangeably herein and refer to an amount of a therapeutic agent (e.g., an aptamer) that provokes a therapeutic or desired response in a subject. The therapeutically effective amount of the composition may be dependent on the route of administration. In the case of systemic administration, a therapeutically effective amount may be about 10 mg/kg to about 100 mg/kg. In some cases, a therapeutically effective amount may be about 10 g/kg to about 1000 g/kg for systemic administration. For intravitreal administration, a therapeutically effective amount can be about 0.01 mg to about 150 mg in about 25 μΐ to about 100 μΐ volume per eye.
Methods
[00165] Disclosed herein are methods for the treatment of ocular diseases. In some cases, the ocular disease is dry age-related macular degeneration or geographic atrophy. In some cases, the method involves administering a therapeutically effective amount of a composition to a subject to treat the disease. In some cases, the composition includes one or more aptamers as described herein. The aptamers may inhibit a function associated with fD as described herein. The methods can be performed at a hospital or a clinic, for example, the pharmaceutical compositions can be administered by a health-care professional. In other cases, the pharmaceutical compositions can be self-administered by the subject. Treatment may commence with the diagnosis of a subject with an ocular disease (e.g., AMD). In the event that further treatments are necessary, follow-up appointments may be scheduled for the administration of subsequence doses of the composition, for example, administration every 8 weeks.
Methods of Generating Aptamers
The SELEX™ Method
[00166] The aptamers described herein can be generated by any method suitable for generating aptamers. In some cases, the aptamers described herein are generated by a process known as Systematic Evolution of Ligands by Exponential Enrichment" ("SELEX™"). The SELEX™ process is described in, e.g., U.S. patent application Ser. No 07/536,428, filed lun. 1 1, 1990, now abandoned, U.S. Pat. No. 5,475,096 entitled "Nucleic Acid Ligands", and U. S. Pat. No. 5,270, 163 (see also WO 91/19813) entitled "Nucleic Acid Ligands", each of which are herein incorporated by reference. By performing iterative cycles of selection and amplification, SELEX™ may be used to obtain aptamers with any desired level of target binding affinity.
[00167] The SELEX™ method relies as a starting point upon a large library or pool of single stranded oligonucleotides comprising randomized sequences. The oligonucleotides can be modified or unmodified DNA, RNA, or DNA/RNA hybrids. In some examples, the pool comprises 100% random or partially random oligonucleotides. In other examples, the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence incorporated within randomized sequence. In other examples, the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence at its 5' and/or 3' end which may comprise a sequence shared by all the molecules of the oligonucleotide pool. Fixed sequences are sequences common to oligonucleotides in the pool which are incorporated for a preselected purpose such as, CpG motifs, hybridization sites for PGR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, and SP6), sequences to form stems to present the randomized region of the library within a defined terminal stem structure, restriction sites, or homopolymeric sequences, such as poly A or poly T tracts, catalytic cores, sites for selective binding to affinity columns, and other sequences to facilitate cloning and/or sequencing of an oligonucleotide of interest. Conserved sequences are sequences, other than the previously described fixed sequences, shared by a number of aptamers that bind to the same target. [00168] The oligonucleotides of the pool can include a randomized sequence portion as well as fixed sequences necessary for efficient amplification. Typically the oligonucleotides of the starting pool contain fixed 5' and 3' terminal sequences which flank an internal region of 30-50 random nucleotides. The randomized nucleotides can be produced in a number of ways including chemical synthesis and size selection from randomly cleaved cellular nucleic acids. Sequence variation in test nucleic acids can also be introduced or increased by mutagenesis before or during the selection/amplification iterations.
[00169] The random sequence portion of the oligonucleotide can be of any length and can comprise ribonucleotides and/or deoxyribonucleotides and can include modified or non-natural nucleotides or nucleotide analogs. Typical syntheses carried out on automated DNA synthesis equipment yield 1014-1016 individual molecules, a number sufficient for most SELEX™ experiments. Sufficiently large regions of random sequence in the sequence design increases the likelihood that each synthesized molecule is likely to represent a unique sequence.
[00170] The starting library of oligonucleotides may be generated by automated chemical synthesis on a DNA synthesizer. To synthesize randomized sequences, mixtures of all four nucleotides are added at each nucleotide addition step during the synthesis process, allowing for random incorporation of nucleotides. As stated above, in some cases, random oligonucleotides comprise entirely random sequences; however, in other cases, random oligonucleotides can comprise stretches of nonrandom or partially random sequences. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.
[00171] The starting library of oligonucleotides may be RNA, DNA, substituted RNA or DNA or combinations thereof. In those instances where an RNA library is to be used as the starting library it is typically generated by synthesizing a DNA library, optionally PCR amplifying, then transcribing the DNA library in vitro using T7 RNA polymerase or modified T7 RNA polymerases, and purifying the transcribed library. The nucleic acid library is then mixed with the target under conditions favorable for binding and subjected to step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. More specifically, starting with a mixture containing the starting pool of nucleic acids, the SELEX™ method includes steps of: (a) contacting the mixture with the target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; (c) dissociating the nucleic acid-target complexes; (d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids; and (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule. In those instances where RNA aptamers are being selected, the SELEX™ method further comprises the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes before amplification in step (d); and (ii) transcribing the amplified nucleic acids from step (d) before restarting the process.
[00172] Within a nucleic acid mixture containing a large number of possible sequences and structures, there is a wide range of binding affinities for a given target Those which have the higher affinity (lower dissociation constants) for the target are most likely to bind to the target. After partitioning, dissociation and amplification, a second nucleic acid mixture is generated, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested as ligands or aptamers for 1) target binding affinity; and 2) ability to effect target function.
[00173] Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle. The method is typically used to sample approximately 1014 different nucleic acid species but may be used to sample as many as about 1018 different nucleic acid species. Generally, nucleic acid aptamer molecules are selected in a 5 to 20 cycle procedure.
[00174] In some cases, the aptamers of the disclosure are generated using the SELEX™ method as described above. In other cases, the aptamers of the disclosure are generated using any modification or variant of the SELEX™ method.
[00175] In some cases, the aptamers described herein have been generated using methodologies to select for specific sites related to activity or function of a target protein. In some cases, the aptamers described herein may be selected using methods that improve the chances of selecting an aptamer with a desired function or desired binding site. In some cases, the aptamers described herein are generated using methods that increase the chances of selecting an aptamer that binds to a region of fD that serves as an epitope for an anti-fD therapeutic antibody, which anti-fD therapeutic antibody inhibits a function associated with fD.
EXAMPLES
[00176] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
Example 1. Identification of modified RNA aptamers to fD.
A. Selection of anti-factor D aptamers
[00177] Anti-factor D (fD) aptamers were identified using an N30 library (N30S) comprised of a 30-nucleotide random region flanked by constant regions containing a built-in stem region as depicted in FIG. 8A. The sequence in italics represents the forward and reverse primer binding sites. The built-in stem region is underlined. FIG. 8B depicts a representation of the N30S library with the reverse oligo hybridized. For nuclease stability, the library was composed of 2'- fluoro-G (2'F GTP) and 2'-0-methyl (2'OMe) A/C/U. FIG. 8C depicts structures of modified nucleotides used to generate the N30S library for selection against target fD For simplicity, the nucleosides, and not the nucleotide triphosphates are shown.
[00178] The library sequence (underlined sequences represent the built-in stem) and the sequence of oligos used to amplify the library are described in Table 4.
Table 4. Library sequence and sequence of oligos used to amplify the library
Figure imgf000162_0001
[00179] The starting library was transcribed from a pool of -10 double-stranded DNA
(dsDNA) molecules. The dsDNA library was generated by primer extension using Klenow exo (-) DNA polymerase, the pool forward primer (N30S.F) and synthetic single-stranded DNA (ssDNA) molecule encoding the library. The dsDNA was subsequently converted to 100% backbone modified RNA via transcription using a mixture of 2'F GTP, 2'-OMe ATP/CTP/UTP and a variant of T7 RNA polymerase in buffer optimized to facilitate efficient transcription. Following transcription, RNAs were treated with DNAse to remove the template dsDNA and purified. [00180] The selection targeting fD was facilitated by the use of a His-tagged recombinant human complement Factor D protein and magnetic His capture beads. Briefly, beads (the amount varied with the amount of target protein coupled) were washed three times with immobilization buffer (50 mM sodium phosphate, pH 8.0, 300mM aCl, 0.01% Tween-20) and were resuspended in 50 μΐ^ of immobilization buffer. His-tagged recombinant fD, in immobilization buffer, was then added to the beads and incubated at room temperature for 30 minutes. The amount of target protein varied with the rounds (Table 5). The beads were washed three times with binding buffer SB1T (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KC1, 1 mM MgCl2, 1 mM CaCl2, 0.05% Tween-20) to remove any unbound protein and then re-suspended in 50 μΐ^ SB1T buffer containing 1 μ^μΐ ssDNA and 0.1% BSA.
[00181] For the first round of selection, ~3 nanomoles of the Round 0 RNA pool, ~1014 sequences, was used. Prior to each round, the library was thermally equilibrated by heating at 80°C for 5 minutes and cooled at room temperature for 15 minutes in the presence of a 1.5-fold molar excess of reverse primer (N30S.R) to allow the library to refold and simultaneously block the 3 ' end of the pool. Following renaturation, the final volume of the reaction was adjusted to 50 μΐ. in SB1T supplemented with 1 g/ml ssDNA and 0.1% BSA.
[00182] For the first round, the library was added to the fD immobilized on beads and incubated at 37°C for 1 hour with intermittent mixing. After one hour, the beads were washed using 3 x 1 ml SB IT buffer to remove unbound aptamers. For round 0, each wash step was incubated for 5 minutes. After washing, fD-bound aptamers were eluted using 200 μΙ_. elution buffer (2M Guanidine-HCl in SB IT buffer) two times (total volume 400 μΙΤ). The eluted aptamers, in 400 μΐ^ of elution buffer, were precipitated by adding 40 μΐ, 3M NaOAc, pH 5.2, 1 ml ethanol and 2 μΐ glycogen and incubating at -80°C for 15 minutes. The recovered library was converted to DNA by reverse transcription using Super Script rv reverse transcriptase, and the ssDNA was subsequently amplified by PCR. The resulting dsDNA library was subsequently converted back into modified RNA via transcription as described above. DNased, purified RNA was used for subsequent rounds.
[00183] For subsequent rounds, the washing time and number of washes was varied as the selection progressed, the input RNA was kept fixed at 25 picomole, and the protein input varied (Table 5). After the first round, a negative selection step was included in all the subsequent rounds. For the negative selection, the pool was prepared as described before and first incubated with non-labelled beads for 1 hour at 37°C in SB IT buffer. The beads were then spun down and the supernatant containing molecules that did not bind to the unlabeled beads was incubated with fD-labeled beads for an additional 1 hour at 37°C. B. Assessing the progress of selection
[00184] Flow cytometry was used to assess the progress of the selection. For these assays, RNA from each round was first hybridized with reverse complement oligonucleotide composed of 2'OMe RNA labeled with Dylight® 650 (Dy650-N30S.ROMe). Briefly, the library was combined with 1.5-fold molar excess of Dy650-N30S.R.OMe, heated at 80°C for 6 minutes and allowed to cool at room temperature for 15 min. after which it was incubated with beads labelled with fD, in SB IT buffer containing 0.1% BSA and 1 μg μl ssDNA. Following incubation for 1 hour at 37°C, the beads were washed 3 times with SB IT, re-suspended in SB IT buffer and analyzed by flow cytometry. As shown in FIG. 9, an improvement in fluorescent signal with the progressing rounds was seen as early as Round 3 After Round 6, there was little change in the binding signal through Round 8. "Beads" refers to the signal of fD-labelled beads in the absence of labeled RNA. The apparent affinity of rounds 6, 7, and 8 for fD was also measured using flow cytometry-based assays and revealed IQs in the range of 8-45nM (FIG. 10A, Table 7).
C. Selection, purification and characterization of clones
[00185] The enriched aptamer populations recovered from rounds 6, 7, and 8 of the selection were sequenced to identify individual functional clones. The sequences were grouped in families based on sequence similarity. From an analysis of Rounds 6, 7 and 8, 7 individual clones were selected for testing. Individual bacterial colonies corresponding to these clones were picked up and plasmid isolated using QIAGEN Mini Prep Kit. The sequences for each clone were PCR amplified using the F and R oligo of the library. Each full length clone was transcribed from the PCR product using the protocol described before. The clones were gel purified and used for further analysis.
[00186] A summary of the clones tested is shown in Table 6. For simplicity, the constant regions have been omitted from sequences C I though C3.
D. Assaying individual clones for binding
[00187] Individual clones were assayed by flow cytometry in a manner similar to that described above for individual rounds of selection. In the case of clones CI through C3, fluorescent labeling of each aptamer was achieved via hybridization to Dy650-N30S.R.OMe as described above.
[00188] As an initial assay, the binding of each aptamer to fD was assessed using bead- immobilized fD when incubated at 100 nM for 1 hour at 37°C. As shown in FIG. 5, clones Cl- C3 displayed significant levels of binding to fD beads. No binding was observed when similar experiments were performed using beads bearing no target or a non-specific target, human growth factor. E. Measurement of apparent Kd on beads
[00189] Flow cytometry was used to measure the binding affinity of each individual aptamer to fD. Assays were again performed as described before but using serially diluted solutions of each aptamer. Following incubation for 1 hour at 37°C, the beads were washed and fluorescence was measured using flow cytometry and a plot of median fluorescent intensity versus aptamer concentration (FIG. 12) was used to determine the apparent binding constant for each clone. Apparent IQ values were obtained using the equation Y = Bmax*X/(KD + X). The apparent binding constant for each clone is also reported in Table 7. The apparent affinity of aptamers to fD ranged from approximately 3 to 20 nM.
F. Competition assays with rounds or individual clones
[00190] Competition binding assays were performed using a clone of an anti-fD Fab with an amino acid sequence of heavy chain variable region according to SEQ ID NO: 7 and light chain variable region according to SEQ ID NO: 8 (hereinafter, "AFD") to further assess binding. For the competition assays, beads labelled with fD were first incubated with 50 nM round or individual aptamer, in 50 μΐ SB 1T (with ssDNA and BSA), for 30 minutes at 37°C. The beads were then washed with SB IT to remove unbound aptamers and incubated with or without 100 nM AFD for 30 minutes at 37°C. Following incubation, the beads were washed three times with SB1T, and assayed by flow cytometry (FIG. 7). These assays revealed that binding of AFD reduced the aptamer signal by -75% - -90%, for both the Round 7 and 8 populations as well all selected aptamers. In cases where aptamers are sufficiently outcompeted by AFD as described above, such aptamers were presumed to be binding to the exosite or the self-inhibitory loop of fD.
Table 5. Selection details
Figure imgf000165_0001
Table 6. Se uences of random re ion-derived se uences of select Π) a tamers
Figure imgf000166_0001
Table 7. Affinity constant of selected rounds and aptamers generated in selection to fD
Figure imgf000166_0002
Example 2. Identification of I I) inhibitors in hemolysis assays.
[00191] In some cases, the disclosure provides for the identification of aptamers that inhibit a function associated with fD. In some cases, the identification of aptamers that that inhibit a function associated with fD may involve performing an alternative complement-dependent hemolysis assay. Human serum that is rendered deficient in the classical complement pathway by depleting Clq may be dependent on alternative complement activity to lyse rabbit red blood cells, an activity that may be dependent on fD. (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892).
[00192] Briefly, citrated rabbit blood was centrifuged at 500xg for 5 minutes at room
temperature. The top plasma fraction was removed and the volume was replaced with IX Veronal buffer containing 0.1% gelatin (prepared from 5X Veronal buffer, Lonza #12-624E and 2% gelatin solution, Sigma-Aldrich, G1393). The red blood cells were washed two more times. The washed rabbit red blood cells were diluted in IX Veronal buffer to a concentration of 2xl09 cells/mL (RBCs).
[00193] In V-bottom 96-well plates the following reagents were added to a final volume of 250 μΐ^: appropriate volume of IX Veronal buffer with 0.1% gelatin, 100 μΐ, aptamer, 30 μΐ, of Clq- depleted human serum and 20 μΐ^ RBCs. This mixture was incubated for 25 minutes at room temperature, then the reaction was stopped by the addition of 5 of 500 mM EDTA. The plate was centrifuged for 5 minutes at 500xg at room temperature, then 100 μΐ, of supernatant was removed and the extent of RBC lysis was determined by measuring absorbance at 405 nm. Controls for the assay were provided by complete RBC lysis with water in the absence of C lq- depleted serum, and by inhibition of lysis caused by Clq-depleted serum by 100 μΜ small molecule fD inhibitor 3,4-dichloroisocoumarin.
[00194] C1-C3 identified in Example 1, a non-specific control oligo (C8), and one anti-fD Fab antibody fragment as described in Example 1 (AFD) were incubated with Clq-depleted human serum to allow binding to fD present in the serum, then assayed for the ability to inhibit independent lysis of rabbit red blood cells (FIG. 14). The endogenous concentration of fD was expected to be about 9.6 nM in 10% C lq-depleted human serum (Loyet, Good, Davancaze et al. (2014) Complement inhibition in cynomolgus monkeys by anti-factor D antigen-binding fragment for the treatment of an advanced form of dry age-related macular degeneration. J. Pharm. Exp. Ther. 351, 527-537), so compounds that bound fD with significantly better affinity, such as less than 1 nM, were expected to bind nearly stoichiometrically to the fD present in the assay. This appeared to be the case for AFD (FIG. 14; Table 8), which was reported to have a low pM affinity for fD (20 pM, Loyet et al. 2014). IC50 values for C 1-C3, C8 and AFD are depicted in Table 8.
Table 8. IC50 values for C1-C3, C8 and AFD inhibiting alternative complement in human serum
Figure imgf000167_0001
Example 3. Factor D esterase activity assay.
[00195] In some cases, a fD esterase activity assay may be used to test the activity of putative anti-fD aptamers. In some cases, inhibition of esterase activity may suggest that the anti-fD aptamer is binding to the catalytic cleft, the associated substrate binding specificity pockets, or sterically occluding access to the active site. In some cases, an enhancement of esterase activity may suggest that the anti-fD aptamer is binding to the exosite in a manner which causes allosteric activation, such as observed for an anti-fD Fab having an amino acid sequence of heavy chain variable region according to SEQ ID NO: 7 and a light chain variable region according to SEQ ID NO: 8. In yet other cases, no effect on esterase activity in combination with inhibition of hemolysis may suggest that the anti-fD aptamer is binding the exosite in manner that does not cause allosteric activation, or is binding to neither the exosite or catalytic cleft. Cleavage of a modified peptide substrate of fD, such as Z-lys-S-Bzl, may be monitored by measuring the amount of reduced 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB). FD may have a lower catalytic rate than other complement proteases when using peptide thioester substrates, and one such substrate Z-lys-SBzl was found to be cleaved by fD and useful as a synthetic substrate (fD is called protein D in Kam, McRae et al. (1987) Human complement proteins D, C2, and B. I Biol. Chem. 262, 3444-3451).
[00196] In one aspect a molecule that binds fD could block catalytic activity by binding in the catalytic cleft to sterically prevent access of the peptide substrate to the catalytic residues of fD (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892). In another aspect a molecule that binds fD could block catalytic activity by an allosteric mechanism that induces structural changes in the enzyme. In a further aspect, a molecule that binds fD could bind to the fD exosite region to sterically inhibit binding of the physiologic substrate protein fB, but not of the synthetic modified peptide substrate Z-Lys-SBzl (Katschke, Wu, Ganesan, et al. (2012)
Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892).
[00197] In a further aspect where a molecule inhibits fD binding and proteolytic cleavage of fB but not Z-Lys-SBzl, the binding could be similar to how anti-factor D FAb antibody fragment binds to the exosite and induces a subtle conformational change that increases fD cleaving Z- Lys-S-Bzl (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892).
[00198] Briefly, in flat bottom 96-well plates, the following reagents were added to a final volume of 200 μΐ,: IX Veronal buffer with 0.1% gelatin and 10 mM MgCl2; anti-fD antibody (AFD), aptamers (C1-C3, see Example 1) or a non-specific oligo control (C8); and a final concentration of fD at or within 5% of 10 nM, 20 nM, 40 nM, 80 nM, or 160 nM. After incubating for 10 min. at room temperature, Z-Lys-SBzl was added at or within 5% of 94 μΜ, 188 μΜ, 375 μΜ, or 750 μΜ and DTNB at or within 5% of 5 μΜ, 20 μΜ, or 40 μΜ. In some cases, fD was added at 41.7 nM, Z-Lys-SBzl at 375 μΜ, and DTNB at 20.0 μΜ. The absorbance was immediately read in a plate reader at 405 nm for 1.5 hours with a read every 30 seconds and a 3 second plate shaking before each read.
[00199] Results of the assay are depicted in Table 9 and FIG. 15. Briefly, C3 was determined to be an active site inhibitor based on having inhibitory activity comparable to a known active site inhibitor of fD, dichloroisocoumarin (DIC). When run in this assay under these conditions, fD activity in this assay was reduced to 29±15 8% (mean±SD), which established that C3 was a potent fD inhibitor, operating via the catalytic or active site cleft. The data further established that C2 bound the exosite in a manner similar to that of AFD. The data also established that CI either worked by a different mechanism of action than C2 and C3, or it functioned like C2 via the exosite, but did not affect fD in exactly the same way to cause allosteric activation of fD.
Table 9. Impact of CI, C2, C3, C8 and AFD on fD Esterase activity.
Figure imgf000169_0001
Example 4. Identification of I'D inhibitors in reconstituted enzymatic I I) assay
[00200] In some cases, the disclosure provides for the identification of fD inhibitors in a reconstituted biochemical fD activity assay which is composed of purified proteins fD, fB, and C3b. When fD binds to the complex of fB and C3b (C3bB), fB is cleaved by fD into fragments Ba and Bb (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J. Biol. Chem. 287, 12886-12892). The activity of fD can be monitored by the rate of fB cleavage and Ba fragment production using an ELISA that uses an antibody that specifically binds Ba (Quidel, A033).
[00201] The fB convertase assay mixture is 0.1% gelatin Veronal buffer and 10 mM MgCl2 with complement proteins fD at or within 5% of 7.5 nM, 15 nM, 30 nM, 60 nM, 120 nM, 240 nM (0.125 μΜ), factor B (fB) at 125 nM, 250 nM, 500 nM, or 1 μΜ and C3b at 125 nM, 250 nM, 500 nM, or 1 μΜ and antibodies or aptamers.
[00202] In one example, the concentrations of fB and C3b are equal so they form a 1 : 1 complex which can then bind fD and allow enzymatically active fD to cleave fB to fragments Ba and Bb. In another example, the fB:C3b complex is present in 4-fold excess of fD. For example, final reaction concentrations of fD of 125 nM and 0.5 μΜ aptamer (or a concentration range) are mixed for 15 minutes, then 0.5 μΜ fB and 0.5 μΜ of C3b are added to the FD/inhibitor mixture and incubated for 30 minutes at 37°C, then 10 mM EDTA in 0.1% gelatin Veronal buffer is added to stop the reaction. Example 5. Identification of inhibitors of fD binding to C3bB
[00203] In some aspects, the disclosure provides for the identification of inhibitors of fD binding to ffi in complex with C3b. fD is the rate-limiting enzyme in the alternative complement pathway, and converts the proconvertases C3bB and C3b2B to form the active C3 convertase C3bBb or the active C5 convertase C3b2Bb (Katschke et al 2012). For surface plasmon resonance (SP ) to detect fD in a stable complex with fB, in some cases catalytically inactive fD (S195A) can be used so that it does not cleave the fB upon binding to the fB:C3b complex (Katschke, Wu, Ganesan, et al. (2012) Inhibiting alternative pathway complement activation by targeting the Factor D exosite. J Biol. Chem. 287, 12886-12892). In other cases, wild type fD can be catalytically inactivated by the covalent inhibitor 3,4-dichloroisocoumarin (DIC) (Harper, Hemmi, Powers (1985) Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor
Biochemistry 24, 1831-1841).
[00204] When C3b is amine-coupled to a CM5 chip, SPR detects binding of fB to C3b as increased mass, and binding of fD to the resultant C3b:fB complex as a further increase in mass. fB, 3,4-dichloroisocoumarin (DlC)-inactivated fD and fD binding compounds in assay buffer (Veronal buffer, 1 mM NiCl2, and 0.05% surfactant P-20) are flowed over the SPR chip at a flow rate of 10, 20, 30, 40, 50, or 60 μΙνΊηίη, 90 μΐ,. fB is flowed over the immobilized C3b at 0.25, 0.5, 1, 2, or 4 μΜ, then fB and fD are co-injected at 0.25, 0.5, 1, 2, or 4 μΜ fB and DIC- inactivated fD at 2-fold dilutions concentration range of 7.8 nM to 8 μΜ. In some cases, the flow rate is 30 μΙ ιηιη and the fB concentration is 1 μΜ, and complexes formed are allowed to dissociate in assay buffer for 5 minutes.
[00205] In one example, fD binding compounds are co-injected with a mixture of fB and fD. For example, 1 μΜ fB and 1 μΜ 3,4-dichloroisocoumarin (DlC)-inactivated fD are co-injected with aptamers at a 2-fold dilution range of 1 μΜ to 128 μΜ. In one aspect, the fD binding compounds are aptamers that bind fD and prevent fD binding to fB:C3b as determined by a reduced mass detected by SPR.
Example 6. Inhibition of I I) in Cell-based Model Complement Pathology in Stargardt Disease
[00206] Retinal pigment epithelial (RPE) cells undergo cell death early during the progress of Stargardt disease, and evidence points toward the involvement of the alternative complement pathway (AP) in RPE cell death (Berchuck, Yang, et al (2013) All-trans-retinal (atRal) sensitizes human RPE cells to alternative complement pathway-induced cell death. Invest Ophthalmol Vis Sci 54, 2669-2677). ARPE-19 cells are a spontaneously arising RPE cell line derived from the normal eyes of a 19-year-old male. The ARPE-19 cell line, established using the cuboidal basal cell layer cultured in specific culture media, expresses the RPE-specific markers cellular retinaldehyde binding protein and RPE-65.
[00207] Stargardt disease is a hereditary juvenile macular degeneration that occurs in patients with homozygous mutations in the ABCA4 genes, which encode a protein that is responsible for removal of bisretinoid fluorophores, which can include N-retinylidene-N-retinyethanolamine (A2E), all-trans-retinal and related photo-oxidation products of vitamin A aldehyde which together constitute lipofuscin, from photoreceptor cells (Molday (2007) ATP -binding cassette transporter ABCA4: molecular properties and role in vision and macular degeneration. J.
Bioenerg Biomembr 39, 507-517). An ABCA4 and RDH8 mouse model of Stargardt disease presents with retinal pathology caused by accumulated atRal, and ABCA4 mutations are present in 16% of AMD patients, suggesting that elevated atRal may contribute to Stargardt disease and AMD disease progression (Berchuck et al 2013).
[00208] Mechanistically, atRal decreased expression of CD46 and CD59 on RPE cells in vitro, which increased susceptibility to cell lysis mediated by alternative complement in response to anti-RPE antibody binding to the RPE cell membranes (Berchuck et al 2013).
[00209] In some cases, the disclosure provides for the identification of fD inhibitors that inhibit alternative complement-mediated lysis of human retinal pigmented epithelial cells. Briefly, human RPE cells (ARPE-19 cells, ATCC, Manassas, Virginia, USA) are grown in 1 : 1 mixture (vol/vol) of Dulbecco's modified Eagle's and Ham's nutrient mixture F-12; (Invitrogen-Gibco, Carlsbad, California, USA), non-essential amino acids 10 mM, 0.37% sodium bicarbonate, 0.058% L-glutamine, 10% fetal bovine serum, and antibiotics (penicillin G 100 U/mL, streptomycin sulfate 0.1 mg/mL, gentamicin 10 g/mL, amphotericin-B 2.5 μg/mL). Cells are incubated at 37°C in 5% C02 and 95% relative humidity.
[00210] ARPE-19 cells are plated on six -well plates for determining cell viability in an in vitro model of Stargardt disease. 5 χ 105 cells in 2 mL of culture media per well are plated and incubated in standard conditions for 24 hours. To sensitize cells to complement mediated lysis by atRal, ARPE-19 cells are treated with atRal for 90 minutes or 24 hours. To activate the fD- dependent alternative complement pathway, cells are incubated with 24% sheep anti-RPE antibody for 30 minutes and then treated with 6% Clq-depleted human serum. After 90 minutes at 37°C, the supernatant is collected in a 96-well plate and replaced with fresh medium. LDH release is measured in the supernatant using a Cytotoxicity Detection Kit. The effect of fD- neutralizing aptamers is determined in the A -induced cytotoxicity assay using defined doses (control— no drug, 1/2*, l x, 2* and 10x) of all drugs.
Example 7. Treatment of geographic atrophy with anti-fD aptamer.
[00211] In this example, a patient is diagnosed with geographic atrophy secondary to AMD. The patient is treated with a therapeutically effective dose of a PEGylated-anti-fD aptamer by intravitreal administration. The aptamer targets the exosite of fD and prevents binding and cleavage of the C3bB complex. The patient is treated once every 4 weeks or once every 8 weeks. After six months of treatment, one year of treatment, and every six months thereafter, the patient is assessed for stabilization of geographic atrophy. The patient shows significantly greater stabilization when compared to an untreated patient and comparable or greater stabilization when compared to a patient who has been treated with an anti-fD antibody fragment therapy once every 4 weeks.
Example 8. Isolation of aptamers with pseudoknot secondary structures
[00212] Example 1 identified C2 as a unique inhibitor of fD activity with a mechanism of action directed towards binding the exosite of fD so as to prevent association of fD with its substrate, the C3bB complex. Further examination of the sequences isolated in the selection of aptamers to fD described in Example 1 identified aptamers C4 and C6 as potentially related to C2 based upon similarities in the sequence derived from the 3 ' portion of the randomized region of the library, including a U-rich sequence, followed by an AAG, separated by approximately one nucleotide from the 3' portion of the engineered stem sequence (see Table 10). C4 and C6 are high affinity ligands to fD, with Kd's of approximately 3.2 and 5.0 nM, respectively, as measured in the bead-immobilized fD flow cytometry assay, and are similarly potent inhibitors of fD, with IC5o' s in the alternative complement dependent hemolysis assay of 8-12 nM.
Table 10. fD Aptamer Sequences
Figure imgf000172_0001
SEQ ID NO: 1 1 C6 GGGAGUGUGUACGAGGCAUUAGGCC with modifications GCCGAAGUCUAAUGGCUCGGGUGUU
CUAAGUUCGGCGGCUUUGAUACUUG
AUCGCCCUAGAAGC;
where G is 2'F and A, C, and U are 2'OMe modified RNA.
SEQ ID NO: 12 C2ml C6SH- with modifications GAGGCAUUAGUCCGCCGAAGUCUUU
UGGCUCGGUUUUUUCAAGGUCGGCG GCUUU-idT;
where G is 2'F and A, C, and U are 2'OMe modified RNA; C6SH represents a six- carbon thiol containing linker, and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 13 C6ml C6SH- with modifications GAGGCAUUAGGCCGCCGAAGUCUAA
UGGCUCGGGUGUUCUAAGUUCGGCG GCUUU-idT;
where G is 2'F and A, C, and U are 2'OMe modified RNA; C6SH represents a six- carbon thiol containing linker, and idT represents a 3 ' inverted deoxythymidine residue.
SEQ ID NO: 14 C4ml C6SH-
GAGGCAUUAGGCCGCCGAAGUCUAA
with modifications
UGUCCUCGGCGCUGAAAGUUCGGCG GCUUU-idT;
where G is 2'F and A, C and U are 2'OMe modified RNA; C6SH represents a six- carbon thiol containing linker, and idT represents a 3 ' inverted deoxythymidine residue.
[00213] Neither visual examination nor folding algorithms yielded a consensus secondary structure consistent with the primary sequences of C2, C4 and C6. Further, attempts to truncate each of these molecules by designing compounds with the engineered stem as the terminal stem were unsuccessful, indicating either the engineered stem did not form, or that additional sequences originating from the 5' or 3' constant regions were required for folding of the aptamers into their active, fD inhibitory structures. As an initial attempt to identify the portion of the C2 sequence responsible for fD binding and inhibition, the 5' and 3' boundaries were probed by annealing complementary oligos to portions of the respective constant regions and testing for binding to fD in the flow-cytometry binding assay. This analysis identified C2ml, a 55 nucleotide truncate of C2 as containing the active aptamer portion of C2, including the complete random region derived sequences as well as portions of sequence derived from the 5' and 3' constant regions.
Example 9. Determination of the secondary structures of exosite directed aptamer inhibitors of I D
[00214] To define the secondary structure of this class of aptamers, as well as to potentially identify fD aptamers with increased potency, a secondary selection was performed utilizing a partially randomized library consisting of 70% C2ml parental sequence +10% of the other 3 nucleotides at each position within C2ml, flanked by 5' and 3' constant regions. Additionally, a secondary selection was performed utilizing a partially randomized library consisting of 70% C6ml parental sequence +10% of the other 3 nucleotides at each position within C6ml, flanked by 5' and 3' constant regions. Six rounds of selection against fD were conducted using these libraries in independent selections, after which each library possessed a greater binding activity than C2ml or C6ml to fD, respectively. Clones from rounds 3 through 6 of the secondary selections were sequenced. Close examination of the sequences obtained from the secondary selections identified a common secondary structure shared by aptamers obtained from both secondary selections. This consensus secondary structure as shown in FIGs. 4A-C and Tables 11 and 12 is an H-H type pseudoknot that consists of three non-nested stem regions j oined by four loop regions, which together define a pseudoknot secondary structure. Interestingly, the engineered stem is indeed formed in these aptamers as the second stem, S2. Representative examples of the secondary structure of this family of exosite directed aptamer fD inhibitors are shown in FIGs. 5A-G and FIGs. 6A-G.
[00215] As defined in the multiple sequence alignments presented in Tables 11 and 12 and as depicted in FIGs. 4A-C, FIGs. 5A-G, and FIGs. 6A-G, C2, C4 and C6 define a pseudoknot family of aptamers. The anti-fD aptamer may have: (1) a terminal stem (S I); (2) a first loop (LI) joining the 5' side of S I to the 5' end of a second stem (S2); (3) a second stem (S2); (4) a second loop (L2) j oining the 5 ' side of S2 to the 3 ' side of S 1 ; (5) a third loop (L3) j oining the 3 ' side of SI to the 5' side of S3; (6) a third stem (S3); and (7) a fourth loop (L4) joining the 5' side of S3 to the 3 ' side of S2
[00216] The anti-fD aptamer may include: (1) a terminal stem (S I). SI may include from 3-9 base pairs In some cases, S I is not highly conserved in sequence identity. The anti-fD aptamer may further include: (2) a first loop (LI) joining the 5' side of SI to the 5' side of S2. LI may have two to five nucleotides. In some case, LI has 2 nucleotides. In some cases, LI has a nucleic acid sequence comprising 5'-GU-3 ' for C2-related sequences and 5'-GG-3 ' for C4- and C6-related sequences. The anti-fD aptamer may further include: (3) a second stem (S2). S2 may have 6-7 base pairs. In some cases, S2 is not highly conserved in sequence identity. The anti-fD aptamer may further comprise: (4) a second loop (L2) joining the 5' side of S2 to the 3 ' side of SI . L2 may have 4-6 nucleotides. In some cases, L2 contains a highly conserved 5'-AGUC-3 ' sequence. The anti-fD aptamer may further include: (5) a third loop (L3) joining the 3 ' side of SI to the 5' side of S3. L3 may have three to fourteen nucleotides. In some cases, L3 has a nucleic acid sequence that is highly variable. In some cases, L3 has a nucleic acid sequence that is enriched in uridine. The anti-fD aptamer may further include: (6) a third stem (S3). S3 may have 3 to 4 base pairs. In some cases, S3 has a minimal consensus sequence of 5'- MHG-3', where N is any nucleotide, M is A or C, and H is A, C, or U. The anti-fD aptamer may further include: (7) a fourth loop (L4) joining the 5' side of S3 to the 3' side of S2. L4 may have from zero to two nucleotides. In some cases, L4 has one nucleotide. In some cases, L4 has a nucleic acid sequence having a G in C2-related sequences or a U for C4- and C6-related sequences. Additionally, single or multiple mispairs are observed in S I (mispaired nucleotides within the proposed stem 1 are underlined in Tables 11-13).
Table 11. Multiple sequence alignment of exosite directed aptamer inhibitors of fD obtained from C2ml secondary selection
SI LI S2 L2 SI L3 S3 L4 S2 S3 c2ml GAGGCAUUA GU CCGCCGA AGUC UUUUGGCUC GGUUUUUUC AAG G UCGGCGG CUU rd4 - 43 GAGGUAUAA GU CCGCGGA AGUC UUUUGUCUC GGGUAUAU GCAG G UCCGCGG CUGU rd4 - 14 GGUAGUA GUC CACCGA AGUC UACUGGC UCGGUUAUAU ACAG G UCGGUG CUGU rd4 - 16 GAGCAAUUA GUA CUCCGA AGUC UAUUUGCCC GGUUCACAC AUG G UCGGAG CAU rd4 - 02 GAGGCAUAA GU CCGCCGA AGUC UUAUGGCUC GGUUUAUUU AUG G UCGGCGG CAU rd4 - 08 GGGCAUGA CU ACGCCGA AGUC UCUUGUCU CGGCUUUCGU " AUG U UCGGCGU CAU rd4 - 19 GAGGCAUCA GU CCGCCGA AGUC UUGUGCCUU GGUUUGUU AUG G UCGGCGG CAU rd4 - 03 CAUAA GU C CACCGA AGUC UUAUG GAUCGGAUUUUUC AUG A UCGGUGG CAU rd4 - 31 GCAUCA GU CCUCCGA AGUCUUU UGGUUC GGAUUUUGU CUG G UCGGGGG CAG rd4 - 36 GAGGCAUUA GU CUGCCGA AGUCUUU UGGCUCGGU UUAUUC AUG U UCGGCAG CAU rd4 - 23 GAGCCAUUG GU C CACCGA AGUC CUAUGA.CUC GUUUAAUU AAUG U UCGGCGG CAUU rd4 - 37 GAUGCAUUG GU CCGCCGA AGUC CAAUGUAUC CGUUUCCUC AUG u UCGGCGG CAU rd4 - 04 UUA GU CCGCUGA AGUCUUU UGG CUCGGGUUUUU GAUG u UCGGCGG CAUU rd4 - 21 CCAUUA GU CGGGGGA AGUUUUU UGGCUGG AUUAUUUC ACG G UCCCCCG CGU rd4 - 29 AUUA GU CCGCCGA AGUCUUU UGGU UACGUUUAUAC : ACG G UCGGCGG CGU rd4 - 15 CCAUUA GU CCGCCGG AGUC UAUUGG GUACGUCAUUUC AUG G UCGGCGG CAU rd4 - 35 GCCAAUA GU CCGUCGA AGUC UUUUGGC CCUGUUAUUUU AUG G UCGGCGG CAU rd4 - 26 GCCAUUA GU CCGCCGA AGUC UAUUGGC CGGUUGCUU AAUG G UCGGCGG CAUU rd4 - 01 GUGCCAUUA GU CCGCGGA AGUC UACUGUCAC GGUAUCUU GAUG G UCCGCGG CAUU rd4-05 UUA GU CCGCCGA AGUCUUU UAG CUCGUUUUCUUC AUG G UCGGCGG CAU rd4- 10 GAUGUCAA CGU CCGUCGA AGUCU UUGGCAUC GGUUUUUUC AUG U UCGGCGG CAU rd4- 28 UAA GU CCGCCGA AGUCUU UUG CUUAAGCUCCCGC : AUG G UCGGCGG CAU rd4- 32 GACGCAUUA GU CCGCCGA AGUC UCUUGCGUC AGUUUUUUUC : AUG G UCGGCGG CAU rd4- 22 UUA GU CCGCAUA AGUCUUU UGG AUCGCUUUGUUC : AUG U UGUGCGG CAU rd4- 34 GAGGAAGUU GU UCGCAGA AGUC AACUGUCUC GGAAUUUUC AAG G UCUGCGG CUU rd4- 06 AAGGCAUGU GU CCGCAGA AGUC AUAUGGACUU GAUUUUUUC AUG G UCUGCGG CAU rd4- 39 GAUACAUUA GUG CGCUGA AGUC UAAUGAAUC AGUUUUUU CACC G UCGGCG GGUG rd4- 40 AUGCAUUA GU CGGCCGA AGUC UGUUGUCU CGGUGUUUUC : ACG G UCGGCCG CGU rd4- 20 GAGGCAUUA GU CAGCCGA AGUC UGGUGUCUC AGUUUGUUU ACG G UCGGCUG CGU rd4- 17 GAGACAUUU GU CCGCCGA AGUC AUCUGUCUC GGUUUGUUC ACG G UCGGCGG CGU rd4- 13 GUGGCAUCA GU CCGACGA AGUC UUUUGCCA UUUAUGUUC AAG G UCGUCGG CUU rd4- 18 CAGGCAUUA GU CAGCCGA AGUC UUUUGCCUG GGAUUUUC GAAG G UCGGCUG CUUU rd4- 11 GGGCAAUG GU CCGCCGA AGUC CAUUGUCC GGGAAUGUU GAUG A UCGGCGG CUUU rd4- 44 GGGGCAAUG GU CCACCGA AGUC CGUUGGCUCC GUAUUUUC AAG G UCGGUGG CUU rd4- 27 GGCAAUA GU CCGCCGA AGUC UUUUGCC ACGUAUUCUUC AAG G UCGGCGG CUU rd4- 45 GGGCACUG GU CCGCCGA AGUC CUUUGACUC GGUUUAUUC AUG G UCGGCGG CAU rd4- 42 GAGGCAAUG GU CCGCCCA AGUC CUUUGCCUC AGUUUAUUC AAG G UGGGCGG CUU rd4- 30 GAGUCAUGA GU CCGCCGA AGUC UCAUGGCUC GGUUUUCU GCAG G UCGGCGG CUGU rd4- 25 UUA GU ACGCCGA AGUCUUA UGG CUCUAUUC CAG G UCGGCGU CUG rd4- 24 GAGUCAUAA GU CCACCGA AGUC UUUUGGCUC UGUUUUCUC CAG G UCGGUGG CUG rd4- 12 AUAA GU CUGCCGA AGUCUU UUGU CAGUGUUUAUUC : CGG G UCGGCAG CCG rd4- 38 UUA GU CCGGCGG AGUCU AUU GUUUCGGUUUUUUC : CAG G UCGCCGG CUG rd4- 07 GAGGAAUUA GU CCGCAGA AGUC UCUUUCCUC GGUUGGUUC CAG G UCUGCGG CUG rd4- 46 GAGGCAUGA GU CCGCCGA AGUC UCAUGUUUC GGUUUCCUC AAG G UCUGCGG CUU rd4- 41 GAGUGACUA GU CCGGCCA AGUC UAUUCGCUC GGUUUCUUU AC AG UGGCCGG G GU rd5- 09 GAGCCUUUA GU CCGUCGA AGUC UUUUAGCUC GGAUUUAUC AUG G UCGGCGG CAU rd6- 58 UAGGCAUUA GU CAGCCGA AGUC UUUUGCCUG GAUUUAUUUC GUG G UCGGCUG CAC rd6- 35 GGGACAUCA GU CCGACGA AGUC UGAUGGCUC GGCUUACUC AUG U UCGUCGG CAU rd6- 59 CAGGCAGUA GU CCACCGA AGUC UACUGGCUC GGUUAUAUA CAG G UCGGUGG UUG rd6- 05 AGGCAGUA GU CCACCGA AGUC UACUGGCU CGGUUAUAU CAG G UCGGUGG CUG rd6- 30 GAGCCAUAA GU CCACCGA AGUC UUUUGGCAC GUUUGGUU AAUG G UCGGUGG CAUU rd6- 43 GAGACAUAA GU CAGCCGA AGUC UUCUGGCAC GUUUGGUU AAUG U UCGGUGG CAUU rd6- 61 CCAUUA GU CCGCCGA AGUC UAUUGG GUACGUCAUUUU AUG G UCGGCGG CAU rd6- 32 GCCAUUA GU ACGGCGA AGUC UCUUGGU GCGUCCUUUUU AUG G UCGGCGU CAU rd6- 06 GAAGCAAGA GUU CGCCGA AGUC UCUUGCCUC GGUAUAU CAC UG UCGGCG U GUG rd6- 29 GACGCAUA CGUA CGCCGA AGUCA UAUGGUUC GGUAUUUU CACU G UCGGCG GGUG rd5- 10 GAGGCAUUA GU CAGCCGA AGUC UAUUGGCUC GGUUUGUA CAAG G UCGGCUG CUGU rd6- 26 GAGGCAUGA GU CCACCGA AGUC UCAUGUCUC GGUAUGAUC AUG G UCGGUGG CAU rd6- 21 GAGGCAUGA GU CCACCGA AGUC UCAUGUCUC GGUAUGAUC AUG UCGGUGG CAUU rd6- 02 GAGGCUUA GU CCACCGA AGUCU UUUGCCUC GGUUUGUU GAUG G UCGGUGG CAUU rd6- 53 AGGCAUUA GU CCGCAGA AGUCU UUUGCCU CGGUUUUUUU GAUG G UGUGCGU CAUU rd6- 39 GAGGCAUUA GU CCGCCGA AGUC UUUUGCCUC GGUAUUUUC AUG G UCGGCGG CAU rd6- 42 GAGACAUCA GU CCACCGA AGUC UUCUGCCUC GGUUUGUUC AUG G UGGGUGG CAU rd6- 12 GGCAAUU GU CAGCCGA AGUC AUUUGCC ACGUUCCUUUC AUG G UCGGCUG CAU rd6- 10 GAGGCAUUA GU CCGCCGA AGUC UUUUGGCUC GGUUUUUUU GAUG G UCGGCGG CAUU rd6- 01 GAGGCAGCU GU CCGCCGA AGUC AUUUGGCUC GGUUUUAU GAUG G UGGCGG CAUU rd5- 16 GAGGCAUCG GG CAGCCGA AGUC CUUUGGCUC GGUAUUUUU GCUG A UCGGCUG CAGU rd5- 03 AG CCA UCAGU CCUCCGA AGUCCUU UUGCU CGGCAUUUU GACG G UAG GAG G CAUU rd6- 38 GAGUCGAUA GU CCACCGA AGUC UCUCGGCUC GGUUUGGUU CUG G UCGGUUG CAG rd5- 17 GAGGCAAAA GU CCACCGA AGUC UUUUGGUUC GAUUCUUUC AUG G UCGGUGG CAU rd6- 27 GCCACUA GU CGACCGA AGUC UUUUGGC UUGGUUAUUUC ACG G UCGGUCG CGU rd6- 04 GAGACAA CAU UAGCCGA AUUCUU UUGUCAC GGUUUUUUC AUG G UCGGCUG CAU rd6- 14 GGCAAUA GU UAGCUGG AGUC UAUUCGCC CUGUUAUGU ACAG U UCGGCUG CUGU rd6- 50 GAGUCAUAA GU UAGCCGA AGUC UUUUGGCUC GAGGACGU AUAG G UCGGCUG CUGU rd6- 62 GACGCACGA GU CAGCCGA AGUC UCCUGUGUC GGUCCUUAC AUG G UCGGCUG CAU rd6- 31 AGCUAUUA UU CAGCCAA AGUC UAUUAGCU CCGUUCAUUC AAG G UCGGCUG CUU rd5- 20 GGCAUUAA GU CAGCCGA AGUC UUAUGGGUC CUUUGCUC ACG G UCGGCUG CGU rd6- 40 GAGACAUAA GU CCGUCCA AGUC UUGUGUCUU CGUUUGUUC ACG G UGGGCGG CGU rd6- 47 GGCAUCA GUCC ACGGA AGUC UGUUGGU UCGAUUCCUUU AUG G UCCGU u CAU rd6- 08 GUGACAUAU GU CCGCCGA UGUC AUAUGUCUC GAGUCCUUG AAG G UCGGCGU CAUU rd6- 64 GAGACAUUU GUA CGCGGA AGUC AUCUGUCUC GGUUACUU AAUG G UCUGCG G CAUU rd5- 18 AGCCUUAG GU ACACCGA AGUC UUAUGGCU GGGUUUUAUC AUG G UCGGUGU CAU rd6- 16 CAGCCAUUG GC ACACCGA AGUC CUUUGGAUG GUUCUAUUC ACG G UCGGUGU CGU rd6- 63 GUGGCAUUG GU ACGCCGA AGUC UAAUGUCAC GCCUUAUUC ACG G UCGGCGU CGU rd6- 17 GAGACAUUG GU ACGCCGA AGUC CUCUGGCUC GGUUUGUUC ACG G UCGGCGU CGU rd6- 44 GUGGCAUAA GU UCGCCGA AGUC UUAUGGCUC GGUUUUGUC AUG G UCGGUGG CAU rd6- 09 GUGGCAUUA GU GCGCCGA AGUC UAAUGGCUC GGUGUUUUC AUG G UCCGCGG CAU rd6- 54 GAUGCAUUA GU CGGCCGA AGUC UCUGCUUC GUCUGUUU AUG G UCGGCCG CAU rd6- 13 GCAUCA GU CCGCAGA AGUC UGUUGC UUCGGUUUUUUC AUG G UCUGCGG CAU rd6- 45 GAAGCAUUA GU CCGCCGA AGU UAUUGGUUC GGAUGUUG AAUG G UCGGGGG CAUU rd5- 26 GAACCAUGA GU CCGCCGA AGUC UUAUGGCUC GUUUGUUG GUUG G UCGGCGG CAAU rd5- 25 GAGCCAUAA GU CUGCAGA AGUC UUAUGGGUU GGUGUUUU GAUG G UCUGCGG CAUU rd6- 60 GAGACAUUA GU CCGCCGA AGUC UUUUGUCUC GGUUUUUUAC AUG U UCGGCGG CAU rd6- 07 GAGCCAUUA GU CCGUCGA AGUC UAUUGGCUC GGUUUGUAC AUG U UCGGCGG CAU rd6- 37 GAGGAAAUA GU CCGACGA AGUC UAUUGCCUC GUUUCCCUC AUG U UCGUCGG CAU rd6- 34 GAGGCAUUA GU CCGCGGA AGUG UAUUGUCUC GUUUCCUUC AAG U UCAGUGG CUU rd6- 28 AGUUACUA CU CCGCCGA AGUC UUUUGGCU GGGAUCAUUC AUG G UCGGCGG CAU rd6- ■49 GCCACUA GU CUGCCGA AGUC UUUUGGC GCGGUAUAUUC AUG G UCGGCAG CAU rd5- 11 CAAUA GU UCGCCGA AGUC UUUUC GCGCUGUUAUUUC AUG G UCGGCGG CAU rd5- 04 GAGCCUUAA GU CCGCGUA AGUC UUUUGCCUC UGUCUAUUC AUG G UCGGCGG CAU rd5- 06 GAGUCAUAG GU CCGCCG AAGUC CUUUGCUC UGUUCCUUC AUG G UGGCGG CAU rd6- 48 GAGGCAUAA GU CCGCCGA AGUC UUUUGGCUC GGUUCAUUC AUG G UCGGCGG CAU rd6- 24 GAGUCAUAA GU CCGCCGA AGUC UUUUGACUC GUGUUUUUC AUG G UCGGCGG CUU rd5- 07 GAGCCAUUA GU CCGCCGA AGUC UAUUCGCUC GGUUUUUC AAG G UCGGCGG CUU rd6- 55 GAGCCAUUA GU CCACCGA AGUC UUAUGGCCC GGUUUUAUC CAG G UCGGUGG CUG rd5- 22 GGGGCAUAA GU CCACCGA AGUC UUUUGGCCC GGGAUUUU GCAG G UCGGUGG CUGU rd6- 36 GACGCAUU GU CCACCGA AGUC CUCUGCCUC GGUCCUGU AUAG U UCGGUGG UUGU rd5- 02 GGGGCAUUG GU ACACCGA AGUC CACUGGUAC CGUCUUUU ACAG G UCGGUGU CUGU rd6- 57 GAGGCAUAA GU CCACCGA GUC UUAUGGCUC GGUACUUU CAUG G UCGGUGG CUG rd5- 19 GUGGCAUU GU CCGAGGA AGUC CAUUGUCAC GGUUUAUAC CAG G UCCUCGG CUG rd5- 01 AGUCAUUU GU CCGCGGA AGUC AUUUGGCU ACGUUGUUAU CAG G UCCGUGG CUG rd5- 21 UUGCCAUAA GU CCGUCGA AGUC UUCUGGCUA GUUAAUAU GUAG G UCGGCGG UUGU rd6- 51 UAGCCAUUA GU CCGGCGA AGUC UUCUGGCUA GGUUAUUA ACGG G UCGUCGG CUGU rd5- 23 GAGGCAUUA GU CCGUAGA AGUC UAAUGGCAC GAAUAUUUC CAG G UCUACGG CUG rd6- 22 GAGGCAUAA GU CGCAGA AGUC UUAUGUCAC GGUGUCAUC CAG G UCUGCG G CUG rd6- 20 AGUCAUUA GU CCGCAGA AGUC UAUUGUCU UGGAUUUUU CAG G UCUGCGG CUG rd6- 56 GAGUCAUUA GU CCGCAGA AGUC UGAUGGUUC GGUUUUUG GCGG G UCUGCGG CCGU rd5- 29 AGGCAGUA GU CCGACCA AGUC UCCUGUCU GUUUGUUUU CAG G UGGUCGG CUG rd6- 52 GGG CAAUA GU CCGACGA AGUC UUUUGUCC CGGUUUUAUC CAG G UCGUCGG GUG rd5- 08 GGGGCAUUA GU CCGCCGA AGUC UAAUGGCCC AGUUUGUUC CAG U UCGGCGG CUGU rd5- 13 GAGCCAUUA GU CCGCCGA AGUC UUUUGGCUC GGUUGAUU GCAG U UCGGCGG CUGU rd5- 12 GGCAUUA GU CCGCCGA AGUC UUUUGCC UUGGUAUUCU ACAG G UCGGCGG CUGU
Table 12. Multiple sequence alignment of exosite directed aptamer inhibitors of fD obtained from C6ml secondary selection
SI LI S2 L2 SI L3 S3 L4 S2 S3
C6ml GAGGCAUUA GG CCGCCGA AGUC UAAUGGCUC GGGUGUUCU AAG U UCGGCGG CUU
C4ml GAGGCAUUA GG CCGCCGA AGUC UAAUGUCCUC GGCGCUG AAAG U UCGGCGG cuuu rd4 - 11 GGGGCAUUA GG CCGCUGA AGUC UAAUCGCCCU GAUGUUCA AUG u UCGGCGG CAU rd4 - 16 GGCAUUA GG CCGUCGA AGUC UAAUGCUU ACAG G GAU CU AUG u UCGGCGG CAU rd4 - 21 AGGCAUUA GG CCGUCGA AGUC UAAUGCUU ACAG G GAU CU AUG u UCGGCGG CAU rd4 - 15 AGUA GG CCGCUGA AGUC UACU UGACUGGGAGAUCU AUG u UCGGCGG CAU rd4 - 26 AGUA GG CCGCUGA AGUC UACU UGACUGGGAGAUCU AUG u UCGGCGG CAU rd4 - 27 GAGCCAGUA GG UCGCCGA UGUC UUCUGGCU GGGGAUUCAU ACG u UCGGCGG CGU rd4 - 37 CAUUA GG CAGCCGA AGUC UAAUG GCUCGGGUAUACU ACG u UCGGCUG CGU rd4 - 06 AGUA GG CCGACGG GUCUAG UGCU UGGGCUGUUUU CAG u UCGUCGG CUG rd4 - 01 GGGGCAUU GGU CAGACGA AGUCC AUUGCCUC GGGUAACCU CAG G UCGUCUG CUG rd4 - 24 GAGCCAUAA GG CCACCGA AGUC UAAUGGCUC GGGUACUCU CAG u UCGGCGG CUG rd4 - 35 GAGUCAUUA GG CCGGCGA AGUC UAAUGGCUC GGGUAUUCU CAG u UCGGCGG CUG rd4 - 39 GGGCCAUUA GG GCUGCGA AGUC UAAUGGCUC GAGUGUUGU CAG u UCGCCGC CUG rd4 - 31 GAGACAUUA GG CCGACGA AGUC UAAUGGCUC UUAUGGUCU CAG u UCGUCGG CUG rd4 - 09 GAG CAUUA GG CCGCCGA AGUC UAAUGUCU CGGGAGUUCU CAG u UCGGUGG CUG rd4 - 33 GUGGCAUUA GG CCGUCGA AGUC UAAUGUCUC GGCGGUUUC CAG u UCGGCGG CUG rd4 - 05 AUUA GG CUGCAGA GUC UAAU GGCUUGUGUGUUUC CAG u UCUGCAG CUG rd4 - 17 AGACAUUA GG CCGUCGA AGUC UAAUGUCU ACGGUGUUCU AAG u UCGGCGG CUU rd4 - 30 GAGACGUUA GC CCGCCGA AGUC UAAUGUCUC GGGUCUUGU CAG u UCGGCGG CUG rd4 - 04 GAGGCAUUA GU CCGCCGA AGUC UAAUGGCUC GUGUUUUCU AAG u UCGGCGG CUU rd4 - 19 AGGCAUUC GU CCGCCGG AGUC GAAUCGCCU GGGUAUACU CUG u UCGGCGG CAG rd4 - 14 AUUG GGC CGCCGG AGUC CAAU GCCUCGGAAGUCC AAUG u UCGGCG A CAUU rd4 - 40 GAUG CAUUA GG CCGGCGA AGUC UAAUGCUUC GGUUGUUCU AUG u UCGUCGG CAU rd4 - 28 GGCACAUAA GG UCCUCGA AGUC UUAUGUGUC GGCUGUUCU AUG u UCGGGGA CAU rd4 - 10 GUGUCAAAA GG CCGUCGA AGUC UUUUGGCUC UGGUUUUGU AUG u UCGGCGG CAU rd4 - 18 GUGUCAUUA GG CUACCGA AGUC UAAUGGCUC GGAUAUUCU AUG c UCGGUGG CAU rd4 - 08 GUGGCAUUA GG CCACCGA AGUC UAAUGGCUC GGAUUUUC AAUG u UCGGUGG CAUU rd4 - 38 UGGCAUUA GG CCGUCGA AGUC UAAUGGCUA GGAUCUUCU AUG u UCGGCGG CAU rd4 - 13 GCUAUUA GG CCGCCGG AGUC UAAUAGC UAGGUUUUACC AUG u UCGGCGG CAU rd4 - 12 GUGCCAUUG GG CCGGCGA AGUC UAAUGCCUC GGGUGUUCU AUG u UCGGCGG CAU rd4 - 36 GUGGCAUUA GG CCGUCGA AGUC UAAUGUCCC AGGUGUCCU AUG u UCGGCGG CAU rd4 - 29 GUGGCAUUA GG CCGUCGA AGUC UAAUGUCCC AGGUGUCCU AUG u UCGGCGG CAU rd4 - 23 GUGGCAUUA GG CCGUCGA AGUC UAAUGUCCC AGGUGUCCU AUG u UCGGCGG CAU rd4 - 02 GAGGCAUUA GG CCGUCGA AGUC UAAUGUCUC GAGUGUGAU AUG u UCGGCGG CAU rd6- 04 GAGGCAUUA GG CCGACGG AGUC UAAUGGCUC GGGUUUCCC AUG u UCGUCGG CAU rd6- 09 GAGGCAUA GGC CGACGG AGUC UAAUGGCUC GGGUUUCCC AUG u UCGUCG U CAU rd5- 34 GAGGCAUUA GG CCGACGG AGUC UAAUGGCUC GGGUUUCCC AUG u UCGUCGG CAU rd6- 03 GAGGCAUUA GG UCGACGG AGUC UAAUGGCUC GGGUUUCCC AUG u UCGUCGG CAU rd6-26 AGGCAUUA GG CCGACGG GGUC UAAUGGCU AGGGUUUCAC AUG U UCGUCGG CAU rd5- 15 GUGGCAUUA GG CCGACGG AGUC UAAUGGCUC GGUUUUCCC AUG U UCGUCGG CAU rd6- 13 GGCAUU AGG CCGACGG AGUCC AAUGGUU CGGGUUUCCC AUG U UCGUAGG CAU rd6- 02 GAGGCAUUA GG CCGUCGG AGUC UAAUGGUUC GGGUUUCCC AUG U UCGUCGG CAU rd6- 05 GAGCCGUUA GG CCGACGG AGUC UAAUGGCUC GGGUGUCCC AUG U UCGUCGG CAU rd6- 52 GAGCCAUUA GG CCGACGG AGUC UAAUGGCUC GUGUUUCCC AUG U UCGUCGG CAU rd6- 53 GAGUCAUUA GG CCGAUGG AGUC UAAUGGCUC GGGUUUCCC AUG U UCGUCGG CAU rd5- 10 GUGUCAUUA GG CCACCGG AGUC UAAUGGCAC UGGUGUCU GCAG U UCGGUGG CUGU rd5- 12 GGGUCAUAA GG CCACCGG AGUC UUAUGGCCC UGGAAGUCU AUG U UCGGUGG CAU rd6- 12 GUGUCAUUA GG CCACCGG AGUC UAAUGGCUC GGGUAAUCU AUG U UCGGUGG CAU rd6- 54 GUGUUAUUA GG CCACCGG AGUC UAAUGGCAC UGUUGUCU GCG U UCGGUGG CUGU rd5- 23 GAGUCAUUA GG CCGCCGG AGUC UAAUGGCUC GUGUGGUCU ACG U UCGGCGG CGU rd5- 13 GAGCCAUUA GG CCGCCGG AGUC UAGUGGUUC GCGUAUUC AAUG U UCGGCGG CAUU rd6- 45 GAGGCAUUA GG CCGCCGG AGUC UAAUGGUUC GUGUGUACU AUG U UCGGCGG CAU rd5- 37 GAGGCAUUA GG CCGCCGG AGUC UAAUGGCUC GUGUGUCCU AUG U UCGGCGG CAU rd6- 46 GGCAUUA GG CCGCCGG AGUC UAAUGGC CGUGUUUCCU AUG U UCGGCGG CAU rd6- 25 GGCAUUA GG CCGACGA AGUC UAAUGUC UGGGGGGUUGU CUG U UCGUCGG CAG rd5- 30 GAGGCAUUA GG UCGCAGA AGUC UAAUGCGUG GGGGAUUCU UUG U UCGGCGG CAG rd6- 43 UAGGCAUUA GG CCGACGG AGUC UAAUGGCUC GGUUACUGU AUG U UCGUCGG CAU rd5- 27 GAGGCAUUA GU CCGCCGG AGUC UAAUACCUC GUGUGUCUU ACG U UCGGCGG CGU rd6- 16 AGGGAUUA GG CCGCCGG AGUC UAACCCCU AGAGUGUCUU AUG U UCGGCGG CAU rd5- 07 GUGGCAUUA GG CAGUCUA AGUC UAAUGCUUC GGUAGUUU ACG U UCGGCUG CGU rd6- 17 GAGGCAUUA GG CCACCUA AGUC UAAUGUUUC GCUUGAUGU AUG u UCGGCGG UAU rd5- 20 GAGGCAUUA GG CCGUCGA AGUC UAAUGUCUC GGGUGUGUU AUG u UCGGCGG CAU rd5- 16 GAUGCGUUA GG CCGCCGG AGUC UAACGAAUC GGGUCUUGU AUG u UCGGCGG CAU rd5- 19 GAUG CAU CA GG CCGGCGA AGUC UAAUGCAUC GAGUGUUCU AUG u UCGACGG CAU rd6- 36 GAUGCAUUA GG CCGCCGG AGUC UAAUGCAUC GGUUGUCCC AUG u UCGGCGG CAU rd6- 34 AGUGCAUUA GG CCGUCGA AGUC UAAUGCAUU GGUUGUCCU AUG u UCGGUGG CAU rd6- 48 GGGGUAUUA GG CCGUCGA AGUG UAAUGCCUU GAGUUACC AUG UCGGCUG CAU rd6- 19 GUGGCAUUA GG CCGUUGG AGUC UAAUACCAG GAUUGUCC GAUG c UCGGCUG CAUU rd6- 08 GAGGCAUUA GG CCGCUGA AGUC UAAUACCUC GACAGUUCU AUG u UCGGUGG CAU rd5- 08 GAGGCAUUA GG CCGUCGA AGUC UAAUGGCUC GUUAGUUCU AUG u UCUGCGG CAU rd5- 11 GAGGCAUUA GG CCGUCGA AGUC UAUUGGCUC GGGAAUUCU AUG u UCGGCGG CAU rd5- 17 GAGGCAUUA GG CCGGCGA AGUU UAAUGGCUC AGGAAUCCU AUG u UCGGGGG CAU rd6- 06 GAGGCAUUA GG CCGCCGG AGU UAAUGGCUC GGAUGUU GAUG u UCGGCGG CAUU rd5- 29 GAGGCAUUA GG CCGCCGG AGUC UAAUGGCUC GGAUGUCU GAUG u UCGGCGG CAUU rd6- 07 GAGGCAUUA GG CCGACGG AGUC UAAUGGCUC GUGUUCACU GAG u UCGUCGG cue rd5- 40 GGGUCAUUA GG CCGGCGG AGUC UAAUGACGC GGUUGUACU CAU u UCGCCGG AUG rd5- 28 GUGUCAUUA GG CCGGCGG AGUC UAAUGGCUC GGGUGUUAU CAG u UCGCCGG CUG rd5- 35 GGGCAUUA GG CUGGCGG AGUC UAAUCCCU CGGUUGUUAU CUG u UCGCCAG CAG rd6- 29 GAGA.CAUUA GG CCAUCGG AGUC UAAUGCCUC GGACGUACU CAG u GCGGUGG CUG rd6- 11 GAGAUUA GG CCACCGG AGUC UAAUGCC UCGGACGUAUU CAG u UCGGUGG CUG rd6- 10 GAGUCAUU GG CCACCGG AGUC UAAUGUCU CGGACGUACU CAG u UCGGUGG CUG rd5- 32 GUACCAUUA GG CCACCGA AGUC UAAUGGUUC GAGUGUUAUC AG u UCGGUGG CU rd5- 03 GUACCAUUA GGC CACCGA AGUC UAAUGCUUC GAGUGUUAU CAG u UCGGUG CUG rd5- 18 GAGCCAAUA GG CCACCGG AGUC UAUUGGCUG GGUUGUCCU CAG u UCGGUGG CUG rd6- 18 GAGCCAAUA GG CUACCGG AGUC UAUUGGCUG GGUUGUCU CAG u UCGGUGG CUG rd6- 30 UAGCCAAUA GG CCAUCGG AGUC UAUUGGCUG GGUUGUCCU CAG u UCGGUGG CUG rd6- 37 GAGCCAAUA GG CCACCGG AGUC UAUUGGCUG GGUUCUCUU CAG u UCGGUGG CUG rd6- 50 GAGCCAAUA GG UCACCGG AGUC UAUUGUCUG GGUUGUCCU CAG u UCGGUGG CUG rd6- 24 GAGGCAUUA GG CCUCUGA AGUC UAAUGGCUC GUAAUUUCU CAG u UCGGUGG CUG rd6- 32 GAUGCAUUA GU CCGCCGA AGUC UAAUGCGUC GGGUCUUCU CAG u UCGGCGG CUG rd5- 31 GGUGCAUUA GU CCGCCGG AGUC UAAUGUAUC GGGUCGUCU CAG u UCGGCGG CUG rd6- 51 GUGGCAUUA GG CUGCCGA AGUC UAAUGUCAC GGUUUAUCU CAG u UCGGCAG CUG rd6- 15 GUGGCAUUA GG CUGCCGA AGUC UAAUGUCAC GGGUGAUUU CAG u UCGGCAG CUG rd6- 23 GUGGUAUUA GGC CGUCGA AGUC UAAUGCCUC GGUUGUUC GCAG u UCGGG CUGU rd6- 21 CAGUCAUUA GG GCGUAGA AGUC UAAUGUCUA GAGUGUUCU CCG u UCUGCGC CGG rd5- 24 CAAGCAGUA GG CCGACGA AGUC UACUGUCUU GGAUGUUGU CAG u UCGGCGG CUG rd5- 22 GAGGCAUUA GG CCGACGG AGUC UAUUGCCUC GGGUGUUCU CAG u UCGUCGG CUG rd5- 06 GAGGCAUUA GG CCGACGG AGUC UAAUGUCCC GGACUUCC CAG u UCGGCAG CUG rd5- 36 GAGUCAUUA GG CCGACGG AGUC UAAUGCCUC GGAACUUU A CAG u UCGUCGG CUGU rd5- 02 GAGGCACUA GG CCGCCGA AGUC UAUUGGCUC GGGUGUUCU CAU u UCGGCGG AUG
Example 10. Structure activity relationship of aptamers
[00217] To test the proposed consensus secondary structures defined by the multiple sequence alignment which defines the secondary structures of C2, C4 and C6, selected variants were chemically synthesized and evaluated in competition binding assays and the alternative complement dependent hemolysis assay. Aptamers selected for testing are shown in Table 13. Table 13. fD Aptamer Sequences
SEQ ID NO: Aptamer Sequence (5' to 3')
Number
SEQ ID NO: Aptamer 41 C6NH2-
GAGUCAUGAGUCCGCCGAAGUCUCAUG
463
GCUCGGUUUUCUGCAGGUCGGCGGCUG
with U-idT
modifications
SEQ ID NO: Aptamer 42 C6NH2-
GAUGCAUUGGUCCGCCGAAGUCCAAUG
464
UAUCCGUUUCCUCAUGUUCGGCGGCAU- with idT
modifications
SEQ ID NO: Aptamer 43 C6NH2-
GAGCCUUUAGUCCGUCGAAGUCUUUUA
465
GCUCGGAUUUAUCAUGGUCGGCGGCAU
with U-idT
modifications
SEQ ID NO: Aptamer 44 C6NH2-
UAGGCAUUAGUCAGCCGAAGUCUUUUG
466
CCUGGAUUUAUUUCGUGGGUCGGCUGC
with AC-idT
modifications
SEQ ID NO: Aptamer 45 C6NH2-
CAGGCAGUAGUCCACCGAAGUCUACUGG
467
CU CGGUUAUAUCAGUCGGUGGCUGU- with idT
modifications
SEQ ID NO: Aptamer 46 C6NH2-
GAGGCAUUAGGCCGCCGAAGUCUAAUG
468
GCUCGGGUGUUCUAAGUUCGGCGGCUU- with idT
modifications
SEQ ID NO: Aptamer 47 C6NH2-
GAGGC AUUAGGCC GACGGAGUCUAAUG
469
GCU
with CGGGUUUCCCAUGUUCGUCGGCAU-idT modifications
SEQ ID NO: Aptamer 48 C6NH2-
GUGUCAUUAGGCCACCGGAGUCUAAUG
470
GCACUGGUGUCUGCAGUUCGGUGGCUG
with U-idT modifications
SEQ ID NO: Aptamer 49 C6NH2-
GAGACGUUAGCCCGCCGAAGUCUAAUG
471
UCUCGGGUCUUGUCAGUUCGGCGGCUG- with idT
modifications
SEQ ID NO: Aptamer 50 C6NH2-
GGCACAUAAGGUCCUCGAAGUCUUAUG
472
UGUCGGCUGUUCUAUGUUCGGGGACAU- with idT
modifications
SEQ ID NO: Aptamer 51 C6NH2-
GAGGCAUUAGGCCGCCGAAGUCUAAUG
473
UCCUCGGCGCUGAAAGUUCGGCGGCUUU
with -idT
modifications
SEQ ID NO: Aptamer 52 C6NH2-
GAGGCAUUAGUCCGCCGAAGUCUUUUG
474
GCU
with CGGUUUUUUCAAGGUCGGCGGCUUU-idT
modifications
where G is 2Ί F and A, C and U are 2'OMe modified RNA;
C6NH2 repres ent a six-carbon amino containing linker; and idT
represents a 3 ' inverted deoxythymidine residue.
[00218] Aptamers 41-52 were initially assessed for fD binding activity in a bead-based competition binding assay, in which Aptamer 52 (C2ml containing a 5' hexylamino linker and a 3' idT) was fluorescently labeled, and incubated with bead-immobilized fD at 200 nM Aptamer 52 in the presence of 0, 200 and 400 nM of unlabeled Aptamers 41 -52 As shown in FIG. 16, all of the truncated aptamer sequences designed based on the pseudoknot structures bound to fD, confirming the secondary structure model for this family of aptamers. Several of the sequences, including Aptamers 41, 42, 46 (C6ml containing a 5' hexylamino linker and a 3' idT), 47, 48, 49, 50 and potentially 51 (C4ml containing a 5' hexylamino linker and a 3 ' idT) bind with higher affinity to fD than C2ml . Additional competition binding was performed with Aptamers 41, 42, 46, and 48 to better define the relative increase in affinity of these aptamers for fD as compared to C2ml (Aptamer 52). As show in FIG. 17, each of these aptamers have substantially higher affinity for fD than C2ml, showing an approximate 10 to 75 fold increase in affinity, with Aptamer 41 exhibiting the highest affinity for fD. Of interest, Aptamer 41 (FIG. 5D), as compared to C2ml, contains features expected to stabilize the consensus pseudoknot secondary structure, including a fully paired nine base pair S I, a 4 base pair S3 and a relatively shorter L2.
[00219] Aptamers 41-52 were further evaluated for fD inhibitory activity in the alternative complement dependent hemolysis assay at 10 and 100 nM aptamer concentrations. As shown in FIGs. 18A & B, each aptamer except for Aptamer 45 exhibited inhibition of fD activity. Given the low affinity of Aptamer 45, it is likely that a 100 nM concentration is insufficient to bind to and inhibit an amount of fD necessary to show an effect in this assay. Potency in this assay correlated with affinity, with Aptamer 41 exhibiting the most potent fD inhibitory activity. The potency of selected aptamers was further characterized in the alternative complement dependent hemolysis assay. As shown in Table 14, potency correlated well with relative affinity, with Aptamers 41 and 42 exhibiting the greatest potency, an approximately 2 fold increase over C6ml, which itself showed significantly better affinity to fD than C2ml .
Table 14. IC50 values for various fD aptamers
Figure imgf000181_0001
[00220] Together, the multiple sequence alignments of the secondary selections performed on C2ml and C6ml, combined with the synthesis and binding and activity profile of truncated compounds designed to test the secondary structure model, confirm that this family of exosite- directed aptamer inhibitors adopt an H-H type pseudoknot structure, as described herein.
Example 11. Minimization and optimization of pseudoknot variants
[00221] Modeling of aptamer variants identified from the doped selection suggested that the improved molecules displayed increased pairing within S I and S2 of the aptamer structure. Two of the best performing molecules, Aptamer 41 and Aptamer 42, demonstrated complete pairing in this region (FIG. 5D and FIG. 5F).
[00222] If the pseudoknot structure proposed for the exo-site directed fD aptamers is accurate, portions of the structure could be shortened/removed while still maintaining aptamer function. Aptamer 41 was chosen as a model for these additional characterizations (and minimizations) using two strategies: shortening of linker region L3 and truncation of stem S I . A summary of the approach, along with the predicted secondary structure of Aptamer 52 (C2ml) and Aptamer 41 are shown in FIG. 19.
[00223] Because the length of both Linker 3 (L3) and Stem 1 (SI) are likely related, we generated and tested a series of Aptamer 41 variants to evaluate the relationship between these regions (FIG. 20). For these constructs, Linker 3 (L3) was replaced with non-nucleotide spacers ranging from two spacer 18 linkers (Spl8Spl8) which approximate the length of the 8 nucleotides in Loop 3 (L3) that the linker replaces (~ 4 nm) down to a single spacer 3 linker (Sp3), which provides spacing roughly equivalent to a single nucleotide (-0.5 nm). In the case of Stem 1 (SI), we tested molecules bearing the full length, 9 base pair stem as well as compounds generated bearing both a 7 and 5 base pair stem (FIG. 20). A total list of the compounds synthesized and their activity as inhibitors in hemolysis assays are shown in Table 15
Table 15. fD Aptamer Sequences
Figure imgf000182_0001
SEQ ID NO: 544 Aptamer 123 C6NH2- ++ with modifications GAGUCAUGAGUCCGCCGA
AGUCUCAUGGCUC(Sp9)GC AGGUCGGCGGCUGU-idT
SEQ ID NO: 545 Aptamer 124 C6NH2- with modifications GAGUCAUGAGUCCGCCGA
AGUCUCAUGGCUC(L6)GC
AGGUCGGCGGCUGU-idT
SEQ ID NO: 546 Aptamer 125 C6NH2- ++ with modifications GAGUCAUGAGUCCGCCGA
AGUCUCAUGGCUC(Sp3)GC AGGUCGGCGGCUGU-idT
SEQ ID NO: 547 Aptamer 126 C6NH2- ++ with modifications CAUGAGUCCGCCGAAGUC
UCAUG(Sp 18 Sp 18)GC AGGU CGGCGGCUGU-idT
SEQ ID NO: 548 Aptamer 127 C6NH2- ND with modifications CAUGAGUCCGCCGAAGUC
UCAUG(Sp 18)GCAGGUCGG CGGCUGU-idT
SEQ ID NO: 549 Aptamer 128 C6NH2- ND with modifications CAUGAGUCCGCCGAAGUC
UCAUG(Sp9)GCAGGUCGGC GGCUGU-idT
SEQ ID NO: 550 Aptamer 129 C6NH2- ND with modifications CAUGAGUCCGCCGAAGUC
UCAUG(L6)GCAGGUCGGC
GGCUGU-idT
SEQ ID NO:551 Aptamer 130 C6NH2- with modifications CAUGAGUCCGCCGAAGUC
UCAUG(Sp3)GCAGGUCGGC GGCUGU-idT
SEQ ID NO: 552 Aptamer 131 C6NH2- with modifications GUCAUGAGUCCGCCGAAG UCUC AUGGC(Sp 18)GCAGG
UCGGCGGCUGU-idT
SEQ ID NO: 553 Aptamer 132 C6NH2- ND
with modifications GUCAUGAGUCCGCCGAAG
UCUCAUGGC(Sp9)GCAGGU
CGGCGGCUGU-idT
SEQ ID NO: 554 Aptamer 133 C6NH2- with modifications GUCAUGAGUCCGCCGAAG
UCUCAUGGC(L6)GCAGGU
CGGCGGCUGU-idT
SEQ ID NO: 555 Aptamer 134 C6NH2- with modifications GUCAUGAGUCCGCCGAAG
UCUCAUGGC(Sp3)GCAGGU
CGGCGGCUGU-idT
where G is 2'F and A, C and U are 2'OMe modified RNA; idT represents a 3 ' inverted deoxythymidine residue, Spl8 represents a spacer 18 linker, Sp9 represents a Spacer 9 linker, Sp3 represents a Spacer 3 linker, and L6 represents a 6-carbon linker.
Activity Key: ++++ = IC50 <10 nM; +++ = IC50 -10 nM; ++ = IC50 between 10 and 100 nM; + = IC50 of -100 nM; - = > 100 nM; ND = not determined
[00224] In summary, 8 nucleotides in Loop 3 (L3) can be replaced with two spacer 18 modifications with little effect on aptamer function (Table 15; compare Aptamer 41 with Aptamer 1736). Shorter linkers have a modest effect on activity suggesting that the spacing and flexibility of this region is important for optimal function. Additionally, Stem 1 (S I) can be truncated to at least 5 base pairs in length without complete loss of aptamer function. Together these data further strengthen our structure model for this class of aptamer (FIGs. 4A-C), and provide a starting point for the future development of additional aptamer variants (truncates) with improved potency and activity.
[00225] Consistent with the multiple sequence alignments presented in Tables 11 and 12 and as depicted in FIGs. 4A-C, FIGs. 5A-G, and FIGs. 6A-G, the full-length sequences of clone C2 (Table 6) with that of clones C4 and C6 (Table 10), these new data further strengthen and define the secondary structure for this pseudoknot family of aptamers which may be composed of: (1) a terminal stem (SI); (2) a first loop (LI) joining the 5' side of S I to the 5' end of a second stem (S2); (3) a second stem (S2); (4) a second loop (L2) joining the 5' side of S2 to the 3' side of SI ; (5) a third loop (L3) joining the 3' side of S I to the 5' side of S3; (6) a third stem (S3); and (7) a fourth loop (L4) joining the 5 ' side of S3 to the 3' side of S2. [00226] The terminal stem (S I) may include from 3-9 base pairs and in some cases, is not highly conserved in sequence identity across aptamers in the family. The first loop (LI) may have two to five nucleotides. The second stem (S2) may have 6-7 base pairs. In some cases, S2 is not highly conserved in sequence identity. The second loop (L2) may have 4-6 nucleotides and in some cases, L2 contains a highly conserved 5'-AGUC-3' sequence. The third loop (L3) may have three to fourteen nucleotides, can be highly variable in sequence, and can have a nucleic acid sequence that is enriched in uridine. This loop can also be removed and replaced with a non-nucleoside linker. The third stem (S3) may have 3 to 4 base pairs. In some cases, S3 has a minimal consensus sequence of 5'-NMHG-3 ', where N is any nucleotide; M is A or C; and H is A, C, or U. The fourth loop (L4) may have from zero to two nucleotides. In some cases, L4 has one nucleotide. In some cases, L4 has a nucleic acid sequence having a G in C2-related sequences or a U for C4- and C6-related sequences.
Example 12. Anti-fD aptamers block association of I D with the C3bB substrate complex
[00227] In some cases, surface plasmon resonance (SPR) may be used to assemble fD in complex with the natural substrate fB when fB is in complex with C3b (C3bB), where C3b is immobilized to a solid surface, incubated with fB to allow the C3bB complex to form, and then further incubated with enzymatically inactivated fD (*fD) to allow *fD to bind to the C3bB substrate without proteolytically cleaving fB. This assay may be used to test whether putative anti-fD aptamers compete for binding to fD with C3bB. In some cases, an anti-fD aptamer may bind and inhibit fD enzyme activity by binding at or near the active site without interfering with C3bB substrate binding. In some cases, an anti-fD aptamer may bind fD and inhibit C3bB substrate binding.
[00228] In one aspect, lack of inhibition of fD binding to C3bB may suggest that the anti-fD aptamer is binding to fD at the catalytic site without interfering, or competing, with C3bB binding to fD. In one aspect, inhibition of fD binding to C3bB may suggest that the anti-fD aptamer is binding to fD on a shared portion of the interface between fD and C3bB such that the aptamer interferes, or competes, with C3bB binding to fD in a substrate competitive mechanism.
[00229] Briefly, human fD was pre-incubated with 50 μΜ of the covalent fD inhibitor, 3,4- dichloroisocoumarin (DIC), for lh, which completely inactivated fD esterase activity (referred to hereafter as *fD). 25 μg/mL human C3b in 10 mM sodium acetate pH 4.0 was amine coupled to an EDC/NHS activated dextran chip surface for 0.5 - 10 minutes, then blocked with 1 M ethanolamine pH 8.5, resulting in 800 - 2,242 RIU immobilized, then *fD was injected as a negative control. 1 μΜ fB was injected for 3 minutes to allow the C3bB complex to form, then 1 μΜ fB preincubated with 1 μΜ *fD plus up to 2 μΜ aptamer was injected for 3 minutes to allow the C3bB:fD complex to form. Following each injection of fB/*fD/aptamer, the complexes were dissociated from the bound C3b with 2 x 60-second injections of 3M NaCl in 50 mM sodium acetate pH 5.0.
[00230] Results of the assay are depicted in FIG. 21, which has the results presented as non- ligand channel subtracted from the C3b-ligand channel. fB was bound to immobilized C3b, and this was further bound by *fD (fD (DO)). When fB plus c2ml in which the 5' thiol moiety had been capped by reaction with N-ethylmaleimide (capped C2ml) was added, there was no increased signal above fB alone. In contrast to fB plus *fD, when fB plus *fD was injected after being preincubated with capped c2ml, there was no increased signal above fB alone. This indicates that in all conditions, fB bound to the immobilized C3b, which could then bind *fD, but the *fD binding to C3bB was inhibited by capped c2ml .
[00231] Aptamer inhibition of C3bB:*fD complex assembly was further demonstrated by titrating 2-fold capped c2ml from 4 μΜ down to 31.3 nM (FIG. 22). A dose-dependent inhibition of C3bB: *fD complex assembly was observed throughout the dose-response curve, consistent with capped c2ml binding *fD and inhibiting *fD binding to substrate C3bB (FIG. 22).
[00232] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. An aptamer having a nucleic acid sequence, wherein said aptamer forms a pseudoknot secondary structure which specifically binds to complement Factor D (fD).
2. The aptamer of claim 1, wherein the nucleic acid sequence does not comprise any one of SEQ ID NOs:475-534
3. The aptamer of any of the preceding claims, which specifically binds to an exosite of fD.
4. The aptamer of any of the preceding claims, wherein said nucleic acid sequence comprises 30-90 nucleotides.
5. The aptamer of any of the preceding claims, wherein said pseudoknot secondary structure is an H-H type pseudoknot secondary structure.
6. The aptamer of any of the preceding claims, wherein said pseudoknot secondary structure comprises up to four loops and up to three base-paired stems.
7. The aptamer of claim 6, wherein each base-paired stem of said up to three base-paired stems comprises up to 15 base pairs.
8. The aptamer of claims 6 or 7, wherein each loop of said up to four loops comprises up to 12 nucleotides.
9. The aptamer of any of the preceding claims, wherein said pseudoknot secondary structure comprises, in a 5' to 3' direction, a first base-paired stem, a first loop, a second base-paired stem, a second loop, a third loop j oining said first base-paired stem with a third base-paired stem, said third base-paired stem, and a fourth loop.
10. The aptamer of claim 9, wherein said first base-paired stem has from 2 to 12 base pairs.
11. The aptamer of claim 9, wherein said first base-paired stem has from 3 to 9 base pairs.
12. The aptamer of claim 9, wherein said first base-paired stem has 5 base pairs.
13. The aptamer of claim 9, wherein said first base-paired stem has 7 base pairs.
14. The aptamer of claim 9, wherein said first base-paired stem has one or more mismatched base pairs.
15. The aptamer of claim 9, wherein said second base-paired stem has from 2 to 9 base pairs
16. The aptamer of claim 9, wherein said second base-paired stem has 6 or 7 base pairs.
17. The aptamer of claim 9, wherein said third base-paired stem has from 2 to 6 base pairs.
18. The aptamer of claim 9, wherein said third base-paired stem has 3 or 4 base pairs.
19. The aptamer of claim 9, wherein said third base-paired stem comprises a nucleic acid sequence of 5'-NMHG-3 ', where N is any nucleotide; M is A or C; and H is A, C, or U.
20. The aptamer of claim 9, wherein said first loop has from 1 to 5 nucleotides.
21. The aptamer of claim 9, wherein said first loop has from 2 to 5 nucleotides.
22. The aptamer of claim 9, wherein said first loop has 2 nucleotides.
23. The aptamer of claim 9, wherein said first loop comprises, in a 5' to 3' direction, GU.
24. The aptamer of claim 9, wherein said first loop comprises, in a 5' to 3' direction, GG.
25. The aptamer of claim 9, wherein said second loop has from 2 to 9 nucleotides.
26. The aptamer of claim 9, wherein said second loop has from 4 to 6 nucleotides.
27. The aptamer of claim 9, wherein said second loop comprises, in a 5' to 3' direction,
AGUC
28. The aptamer of claim 9, wherein said third loop has from 2 to 12 nucleotides.
29. The aptamer of claim 9, wherein said third loop has from 3 to 14 nucleotides.
30. The aptamer of claim 9, wherein said third loop comprises one or more non-nucleotidyl spacers.
31. The aptamer of claim 9, wherein said fourth loop has 0, 1, or 2 nucleotides.
32. The aptamer of claim 9, wherein said fourth loop has a single nucleotide.
33. The aptamer of claim 32, wherein said single nucleotide is G or U.
34. The aptamer of any of the preceding claims, wherein said aptamer is an RNA aptamer or a modified RNA aptamer.
35. The aptamer of any of the preceding claims, wherein said aptamer is a DNA aptamer or a modified DNA aptamer.
36. The aptamer of any of the preceding claims, wherein said aptamer comprises at least one modified nucleotide.
37. The aptamer of claim 36, comprising a nuclease-stabilized nucleic acid backbone.
38. The aptamer of any of the preceding claims, wherein said aptamer specifically binds to fD with a Kd of less than about 50nM.
39. The aptamer of any of the preceding claims, wherein said aptamer increases esterase activity of fD.
40. The aptamer of any of the preceding claims, wherein said aptamer inhibits fD in an alternative complement dependent hemolysis assay with an IC50 of less than about 50nM.
41. The aptamer of any of the preceding claims, conjugated to a polyethylene glycol (PEG) molecule.
42. The aptamer of claim 41, wherein said PEG molecule has a molecular weight of 80 kDa or less.
43. The aptamer of any of the preceding claims, wherein said nucleic acid sequence comprises nucleotides having ribose in a β-D-ribofuranose configuration.
44. The aptamer of any of the preceding claims, wherein at least 50% of said nucleic acid sequence comprises nucleotides having ribose in a β-D-ribofuranose configuration.
45. An aptamer having a nucleic acid sequence comprising any one of SEQ ID NOs:l-3, 10- 474, and 543-556 or a nucleic acid sequence as described in Table 2, or having at least 80% sequence identity to any one of SEQ ID NOs:l-3, 10-474, and 543-556 or a nucleic acid sequence as described in Table 2.
46. A method for modulating complement Factor D (fD) in a biological system, said method comprising: administering to said biological system, an aptamer according to any of the preceding claims, thereby modulating fD in said biological system.
47. The method of claim 46, wherein said modulating comprises inhibiting a function associated with fD.
48. The method of claim 46 or 47, wherein said biological system is a subject.
49. The method of claim 48, wherein said subject is a human.
50. An aptamer according to any one of claims 1-45, for use in a method of therapy; for use in a method of treatment that benefits from modulating fD; for use in a method of treatment that benefits from inhibiting a function associated with fD; or for use in a method for the treatment of ocular diseases.
51. A pharmaceutical composition or medicament comprising a plurality of aptamers according to any one of claims 1-45 and a pharmaceutically acceptable carrier, excipient, or diluent.
52. The pharmaceutical composition or medicament of claim 51, wherein greater than 90% of said plurality of aptamers comprise nucleotides having ribose in a β-D-ribofuranose configuration.
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