WO2019040590A1 - Modulation of soluble fas expression - Google Patents

Modulation of soluble fas expression Download PDF

Info

Publication number
WO2019040590A1
WO2019040590A1 PCT/US2018/047473 US2018047473W WO2019040590A1 WO 2019040590 A1 WO2019040590 A1 WO 2019040590A1 US 2018047473 W US2018047473 W US 2018047473W WO 2019040590 A1 WO2019040590 A1 WO 2019040590A1
Authority
WO
WIPO (PCT)
Prior art keywords
dts
das
oligonucleotide
dgs
dcs
Prior art date
Application number
PCT/US2018/047473
Other languages
French (fr)
Inventor
Caroline WOO
Original Assignee
Translate Bio Ma, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Translate Bio Ma, Inc. filed Critical Translate Bio Ma, Inc.
Publication of WO2019040590A1 publication Critical patent/WO2019040590A1/en

Links

Classifications

    • 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
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • 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
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the invention relates in part to compositions and methods for modulating gene expression, e.g., in the context of cell based therapies.
  • Adoptive cell transfer is a therapy that generally involves the transfer of cells into a subject for modulating one or more biological responses or functions in the subject.
  • Cells used in an adoptive cell transfer made be obtained from the patient (autologous cells) or from another individual (allogenic cells).
  • cells derived from the immune system of a subject e.g., monocytes, T cells, etc.
  • T cells may be obtained from the subject, modified (e.g., to express an engineered receptor) and/or expanded in culture and returned to the same subject.
  • T cell populations prepared according to methods provided herein are useful for cancer treatment via adoptive transfer, which may result in cancer regression or remission in subjects receiving the transfer.
  • T cells are prepared for adoptive transfer such that the make-up of the T cell population is controlled in order to optimize efficacy of treatment.
  • methods are provided for controlling the make-up of a T cell population (e.g., a CAR T cell population) in order to maintain or increase the percentage of naive T cells in the T cell population.
  • methods for adoptive transfer of specific T cell populations containing naive CD8-positive T cells are provided. Further aspects of the disclosure generally relate to methods for producing and/or maintaining cell populations containing naive CD8-positive T cells. In some embodiments,
  • oligonucleotides e.g., gapmers
  • other molecules are utilized to modulate the expression of genes that control the differentiation state of T cells, particularly naive CD8-positive T cells.
  • a method of maintaining or increasing the number of naive T cells in a T cell population comprising delivering ex vivo an oligonucleotide that inhibits the interaction of FAS-AS 1 with RBM5 to a T cell population comprising naive T cells.
  • delivering the oligonucleotide results in an increase in soluble Fas (sFas) expression in the naive T cells in the T cell population compared to sFas expression in control naive T cells in a control T cell population to which the oligonucleotide has not been delivered.
  • sFas soluble Fas
  • the T cell population is a CD4 + T cell population.
  • the method further comprises isolating T cells from a sample obtained from a donor subject; and selecting CD4 + T cells from the isolated T cells, thereby producing the T cell population comprising naive T cells to which the oligonucleotide is delivered.
  • the method further comprises administering the T cell population to a host subject after the oligonucleotide has been delivered to the T cell population.
  • the donor subject and the host subject are the same. In some embodiments, the donor subject and the host subject are different.
  • the method further comprises transfecting the T cell population with an expression construct encoding a chimeric antigen receptor (CAR).
  • the transfection occurs before delivery of the oligonucleotide to the T cell population. In some embodiments, the transfection occurs after delivery of the CAR.
  • the CAR is specific for a tumor antigen.
  • the host subject has cancer.
  • the T cell population is a human T cell population.
  • the oligonucleotide comprises a region of complementarity that is complementary with at least 8 nucleotides of FAS-AS 1. In some embodiments, the oligonucleotide reduces the level of FAS -AS 1. In some embodiments, the oligonucleotide is a gapmer, an siRNA, a ribozyme or an aptamer that causes degradation of FAS -AS 1. In some embodiments, the oligonucleotide is single stranded. In some embodiments, the oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a sequence selected from SEQ ID NOs: 2 to 1833.
  • the oligonucleotide sterically interferes with the interaction of FAS-AS 1 with RBM5.
  • the oligonucleotide is single stranded.
  • the oligonucleotide is a mixmer. In some embodiments, the
  • oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a sequence selected from SEQ ID NOs: 2 to 1833.
  • the oligonucleotide is a gapmer comprising a region of complementarity that is complementary with at least 8 nucleotides of FAS -AS 1.
  • the gapmer comprises the general formula:
  • each instance of X , X 3 is independently a modified or unmodified nucleotide, wherein m and o are independently integers in a range of 1 to 10, reflecting the number of instances of X 1 and X 3 , respectively, linked consecutively together through internucleotide linkages, wherein each instance of X 2 is a deoxyribonucleotide, wherein n is an integer in a range of 6 to 20, reflecting the number of instances of X 2 linked consecutively together through internucleotide linkages.
  • at least one of X 1 , X 3 is a 2'- modified nucleotide.
  • the 2'-modified nucleotide is a 2'-0,4'-C- bridged nucleotide. In some embodiments, 2'-modified nucleotide is a 2'-0,4'-C- methylene bridged nucleotide. In some embodiments, the a 2'-modified nucleotide is a 2'-0-methyl nucleotide. In some embodiments, the oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a sequence set forth in Table 1 or Table 3 (e.g., a sequence selected from SEQ ID NOs: 2 to 1833).
  • each instance of X 1 , X 3 is a LNA nucleotide and each of the nucleotides in the oligonucleotide are linked by phosphorothioate linkages.
  • each instance of X 1 ⁇ 3 is a LNA nucleotide, m and o are each 3, n is 9, and each of the nucleotides in the oligonucleotide are linked by phosphorothioate linkages.
  • any cytosine LNAs in the oligonucleotide are 5-methylcytosine LNAs.
  • any oligonucleotide disclosed herein having one or more 5-methylcytosine LNAs may have any one or more of the 5-methylcytosine LNAs replaced with a cytosine LNA.
  • FIG. 1A is a diagram showing alternative splicing of the FAS gene in the presence of FAS-AS 1 and RBM5.
  • FIG. IB is a diagram showing FAS signaling between naive T cells and memory T cells and FAS-mediated precocious differentiation of naive T cells.
  • FIG. 1C is a diagram showing blocking of FAS signaling upon production of soluble Fas (sFas). See also genevisible.com/tissues/HS/Gene%20Symbol/FAS.
  • FIG. 2 is a graph showing expression of RMB5 in naive T cells and other cell types.
  • the adoptive transfer of specific T cell populations can increase the probability of cancer cell regression and remission (see, e.g., Cieri, N. et al. (2013) IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood 121, 573-584;
  • Naive T (TN) cells are the precursors of T stem cell memory cells (TSCM), T effector memory cells (TEMX and T central memory cells (TCM)-
  • TSCM and TCM populations may increase antitumor, antibacterial, and antiviral responses following adoptive cell transfer in preclinical models.
  • Interaction between memory T cells (T Me m) and T N cells may contribute to proliferation and expansion of the T N cells (see, e.g., Klebanoff, C. A. et al. (2016) Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J Clin Invest 126, 318-334).
  • TMem cells may induce precocious differentiation of the T N cells, thereby limiting the number of the desired T SCM and T CM populations that can be produced from the depleted T N cell population.
  • This precocious differentiation is mediated through FasL (CD95L) and Fas (CD95) interaction on the TMem and T cells, respectively.
  • Blocking of this interaction through a recombinant leucine zipper- dimerized FasL results in an increase of T N cells with proliferative capacity and to increase tumor regression upon adoptive transfer (see, e.g., Klebanoff, C. A. et al. (2016) Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J Clin Invest 126, 318-334).
  • the FAS gene encodes both a membrane-bound form and a soluble form of Fas (mFas and sFas, respectively).
  • the form of Fas protein that is produced in a cell is mediated by alternative splicing of exon 6 of the FAS mRNA. Inclusion of exon 6 results in the production of mFas, whereas exclusion of exon 6 results in the production of sFAS (FIG. 1A).
  • FALS FasL to mFas mediates the differentiation of T N cells described above (FIG. IB and 1C).
  • FAS-AS 1 a long noncoding RNA (IncRNA) that is antisense to the FAS gene, binds to RBM5 (RNA-binding protein 5), which is involved in alternative splicing of exon 6 of FAS.
  • RBM5 RNA-binding protein 5
  • FAS-AS 1 binding inhibits RBM5 activity and results in the inclusion of exon 6 and the production of mFAS (see, e.g., Sehgal, L. et al. (2014) FAS-antisense 1 IncRNA and production of soluble versus membrane Fas in B-cell lymphoma. Leukemia 28, 2376-2387).
  • compositions and methods for maintaining or increasing the number of T N cells in a T cell population by inhibiting the interaction of FAS-ASl with RBM5.
  • the interaction is inhibited using an oligonucleotide as described herein.
  • inhibiting the interaction of FAS-AS l with RBM5 in T N cells will lead to the production of increased levels of sFAS and/or decreased levels of mFAS based on the change in exon 6 splicing.
  • Both RBM5 and FAS-AS l are expressed in T cells (FIGs. 2 and 3). Consequently, in some embodiments, less mFAS is produced by the T N cells, which may result in less FAS-mediated signaling and less precocious differentiation of the T cells.
  • increased levels of sFAS may act as an inhibitor of FasL signaling from the T Mem population, again resulting in less precocious differentiation of the T N cells.
  • inhibiting the interaction of FAS-AS 1 with RBM5 will cause a greater number of T N cells to be present in a T cell population, which, if used in adoptive T cell transfer for cancer treatment, may result in a more effective antitumor response.
  • treatment of a T cell population ex vivo with an oligonucleotide as described herein may result in effects that are limited in time, as the oligonucleotide may be diluted out as the T cell population expands, e.g., once the T cell population is administered to a subject.
  • this temporary window effectiveness may be advantageous, as mFas/FasL signaling is important for in vivo T cell differentiation into effector cells and apoptosis of T cells, which may result in greater efficacy of adoptive T cell transfer in vivo as well as prevent unwanted negative effects caused by T cells that are resistant to apoptosis.
  • the disclosure provides methods for maintaining or increasing the number of naive T cells and/or increasing soluble Fas cell surface death receptor (sFas) in a T cell population.
  • the method comprises administering to a T cell population an oligonucleotide as described herein, e.g., that inhibits the interaction of FAS- AS 1 with RBM5.
  • the administration of the oligonucleotide is ex vivo.
  • the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of oligonucleotide delivered to the T cell population is 0.5 ⁇ to 10 ⁇ , 1 ⁇ to 20 ⁇ , or 0.01 ⁇ to 50 ⁇ . In some embodiments, the concentration of
  • the concentration of oligonucleotide delivered to the T cell population is up to 1 ⁇ , up to 5 ⁇ , up to 10 ⁇ , up to 20 ⁇ , up to 50 ⁇ , or up to 100 ⁇ . It is understood that any reference to uses of compounds (e.g., oligonucleotides, expression vectors, inhibitors) throughout the description contemplates use of the compound in preparation of a pharmaceutical composition or medicament for use in the treatment of condition or a disease (e.g., cancer) where maintaining or increasing the number of naive T cells in a T cell population is therapeutically beneficial, e.g., in adoptive T cell transfer.
  • a disease e.g., cancer
  • delivering an oligonucleotide as described herein results in an increase in sFas expression in the T cell population (e.g., in the naive T cells) compared to a control level of sFas expression, such as sFas expression in a control T cell population (e.g., in control naive T cells) to which the oligonucleotide has not been delivered.
  • a level of sFas expression may be determined using any suitable assay known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001;
  • the sFas expression level may be an mRNA level or a protein level.
  • sequences of FAS mRNAs and proteins known in the art see, e.g., NCBI Transcript IDs: NM_000043.5, NM_001320619.1,
  • NM_152871.3, and NM_152872.3, and NCBI Protein IDs: NP_000034.1, NP_001307548.1, NP_690610.1, and NP_690611.1) may used to design suitable reagents and assays for measuring an sFas expression level.
  • an appropriate control level of sFas expression may be, e.g., a level of sFas expression in a cell or population of cells to which an oligonucleotide has not been delivered or to which a negative control has been delivered (e.g., a scrambled oligo, a carrier, etc.).
  • an appropriate control level of sFas expression may be a predetermined level or value, such that a control level need not be measured every time.
  • the predetermined level or value can take a variety of forms. It can be single cut-off value, such as a median or mean.
  • increasing sFas expression in a cell includes a level of sFas expression that is, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more above an appropriate control level of sFas.
  • the appropriate control level may be a level of sFas expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein.
  • administration of an oligonucleotide as described herein results in a decreased level of FAS -AS 1 or steric interference with the interaction of FAS- AS 1 with RBM5.
  • decreasing a level of FAS-AS 1 includes a level of FAS- AS 1 that is, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than an appropriate control level of FAS-AS 1.
  • the appropriate control level may be a level of FAS-AS 1 expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein.
  • a T cell population is obtained and an oligonucleotide as described herein is delivered ex vivo to the T cell population.
  • the T cell population is obtained by isolating T cells from a sample (e.g., a blood sample) obtained from a donor subject (e.g., a human donor subject).
  • the T cell population is further enriched, e.g., by selecting T cells expressing certain cell surface markers, such as CD4 and/or CD8.
  • the T cell population is enriched for naive T cells, e.g., by selection for CD4-positive T cells. The selection of T cells may be accomplished using any method known in the art or described herein, e.g., by fluorescence activated cell sorting or magnetic cell sorting.
  • a T cell population to which an oligonucleotide as described herein has been delivered is administered to a host subject (e.g., a human host subject). In some embodiments, this process is referred to as adoptive T cell transfer.
  • the T cell population administered to the subject may further be engineered, prior to administration to the subject, to express a recombinant receptor such as a chimeric antigen receptor (CAR) as described herein.
  • a recombinant receptor such as a chimeric antigen receptor (CAR) as described herein. Suitable administration routes for delivery of the T cell population to a subject are described herein.
  • oligonucleotides are provided for maintaining or increasing the number of naive T cells in a T cell population, e.g., by inhibiting the interaction of FAS-AS 1 with RBM5.
  • expression of soluble Fas (sFas) is upregulated or increased.
  • the oligonucleotide comprises a region of complementarity that is complementary with FAS- AS 1.
  • An exemplary sequence of FAS-AS 1 sequence is provided below:
  • Single stranded oligonucleotides may include secondary structures, e.g., a loop or helix structure.
  • the oligonucleotide comprises at least one modified nucleotide or modified internucleoside linkage as described herein.
  • the oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine
  • oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
  • the oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene.
  • a threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.
  • the oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, greater than 70% G-C content, or greater than 80% G-C content.
  • the oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content.
  • all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of FAS-AS 1 are cytosine or guanosine nucleotides.
  • the sequence of the FAS-AS l to which the oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.
  • the region of complementarity of the oligonucleotide is complementary with 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of FAS-AS l.
  • the region of complementarity is complementary with at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive nucleotides of FAS-AS l, optionally wherein the oligonucleotide is 8 to 30 nucleotides in length.
  • Complementary refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an
  • oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of FAS-AS l, then the oligonucleotide and FAS-AS l are considered to be complementary to each other at that position.
  • the oligonucleotide and FAS-AS l are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases.
  • complementary is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and FAS-ASl . For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of FAS-AS l, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
  • the oligonucleotide may be at least 70% complementary to (optionally one of at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of FAS-AS 1.
  • the oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of FAS -AS 1.
  • the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
  • a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a target molecule.
  • a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable or specific for the target molecule when binding of the sequence to the target molecule (e.g., FAS-AS 1) interferes with the normal function of the target (e.g., FAS-AS 1) to cause a loss of activity (e.g., inhibiting the interaction with RBM5) or expression (e.g., degrading the FAS-AS 1) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays, ex vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • the oligonucleotide is up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 35, up to 40, up to 45, or up to 50 nucleotides in length.
  • the oligonucleotide is 5 to 50, 6 to 50, 7 to 50, 8 to 50, 9 to 50, 10 to 50, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15 nucleotides in length.
  • the oligonucleotide is 8 to 30 nucleotides in length.
  • Base pairings may include both canonical Watson-Crick base pairing and non- Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa.
  • GC content of the oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.
  • the oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof.
  • the oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; or have improved endosomal exit.
  • oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g., a cleavable linker.
  • a linker e.g., a cleavable linker.
  • Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • nucleic acid sequences of the invention may include a phosphorothioate at least the first, second, or third internucleoside linkage at the 5' or 3' end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'- deoxy, 2'-deoxy-2'-fluoro, 2 -O-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0- DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0--N-methylacetamido (2'-0— NMA).
  • a 2'-modified nucleotide e.g., a 2'- deoxy, 2'-deoxy-2'-fluoro, 2 -O-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl
  • the nucleic acid sequence can include at least one 2'-0- methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0- methyl modification.
  • the nucleic acids are "locked,” i.e., comprise nucleic acid analogues in which the ribose ring is "locked” by a methylene bridge connecting the 2'-0 atom and the 4'-C atom.
  • any of the modified chemistries or formats of oligonucleotides described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
  • an oligonucleotide may comprise one or more modified nucleotides (also referred to herein as nucleotide analogs).
  • the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and/or at least one bridged nucleotide.
  • the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide.
  • LNA locked nucleic acid
  • cEt constrained ethyl
  • ENA ethylene bridged nucleic acid
  • the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes.
  • the oligonucleotide may have one or more 2' O-methyl nucleotides.
  • the oligonucleotide may consist entirely of 2' O-methyl nucleotides.
  • the oligonucleotide has one or more nucleotide analogues.
  • the oligonucleotide may have at least one nucleotide analogue that results in an increase in T m of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an
  • the oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in T m of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.
  • the oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.
  • the oligonucleotide may consist entirely of bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides).
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides.
  • the oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides.
  • the oligonucleotide may have a 5' nucleotide that is a bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide).
  • the oligonucleotide may have a 5' nucleotide that is a deoxyribonucleotide.
  • the oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the oligonucleotide may comprise
  • deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the 3' position of the oligonucleotide may have a 3' hydroxyl group.
  • the 3' position of the oligonucleotide may have a 3' thiophosphate.
  • the oligonucleotide may be conjugated with a label.
  • the oligonucleotide may be conjugated with a label.
  • oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • a biotin moiety cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • the oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
  • the oligonucleotides are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • beneficial properties such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target
  • Chimeric oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
  • the oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, preferably a 2'-0-alkyl, 2'-0-alkyl-0-alkyl or 2'-fluoro- modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
  • modified oligonucleotides include those comprising modified backbones, for example, modified internucleoside linkages such as
  • oligonucleotides may have phosphorothioate backbones; heteroatom backbones, such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see
  • PNA peptide nucleic acid
  • Phosphorus -containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos.
  • the oligonucleotide is an oligonucleotide mimetic.
  • Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001;
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
  • PMO phosphorodiamidate morpholino oligomer
  • Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos.
  • Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
  • Arabinonucleosides are stereoisomers of ribonucleo sides, differing only in the configuration at the 2'-position of the sugar ring.
  • a 2'-arabino modification is 2'-F arabino.
  • the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
  • ENAs ethylene -bridged nucleic acids
  • Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene-bridged nucleic acids. Examples of LNAs are described in WO/2008/043753 and include compounds of the following general formula.
  • -CH CH-, where R is selected from hydrogen and Ci_ 4 -alkyl; Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.
  • the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
  • Y is -0-, -S-, -NH-, or N(R ); Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety, and RH is selected from hydrogen and Ci_ 4 -alkyl.
  • the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
  • the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O) 2 -O-, -0-P(0,S)-0-, -0-P(S) 2 -O-, -S-P(0) 2 -0-, -S-P(0,S)-0-, -S-P(S) 2 -0-, -0-P(0) 2 -S-, -0-P(0,S)-S-, -S-P(0) 2 -S-, -0-PO(R H )-0-, o- PO(OCH 3 )-0-, -0-PO(NR H )-0-, -0-PO(OCH 2 CH 2 S-R)-O-, -0-PO(BH 3 )-0-, -0-PO(NHR H )- 0-, -0-P(0) 2 -NR H -, -NR H -P(0) 2 -0-, -NR H -CO-0-0-
  • LNA units are shown below:
  • thio-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH 2 -S-.
  • Thio-LNA can be in both beta-D and alpha-L-configuration.
  • amino-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH 2 -N(H)-, and -CH 2 -N(R)- where R is selected from hydrogen and Ci_ 4 -alkyl.
  • Amino-LNA can be in both beta-D and alpha-L-configuration.
  • oxygen-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH 2 -0-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
  • ena-LNA comprises a locked nucleotide in which Y in the general formula above is -CH 2 -0- (where the oxygen atom of -CH 2 -0- is attached to the 2'-position relative to the base B).
  • LNAs are described in additional detail herein.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0(CH 2 )n CH 3 , 0(CH 2 )n NH 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; OCF 3 ; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a
  • a preferred modification includes 2'-methoxyethoxy [2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486).
  • Other preferred modifications include 2'- methoxy (2'-0-CH 3 ), 2'-propoxy (2'-OCH 2 CH 2 CH 3 ) and 2'-fluoro (2'-F). Similar
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5- Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2- thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine,
  • both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with modified groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
  • Oligonucleotides can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • base any nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • “unmodified” or “natural” nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo -uracil), 4-thiouracil, 8-halo, 8- amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-sub
  • nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, Chapter 15, Antisense Research and Applications," pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ⁇ 0>C (Sanghvi, et al., eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos.
  • the oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • one or more oligonucleotides, of the same or different types can be conjugated to each other; or oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type.
  • moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg.
  • a thioether e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
  • an aliphatic chain e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate
  • a phospholipid e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate
  • conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thioether,
  • oligonucleotide modification includes modification of the 5' or 3' end of the oligonucleotide.
  • the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate.
  • additional molecules e.g. a biotin moiety or a fluorophor
  • the oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
  • the oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxynbonucleotides and 2'- fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxynbonucleotides and 2'-0-methyl nucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides.
  • LNA locked nucleic acids
  • ENA modified nucleotides 2'-0-methyl nucleotides
  • 2'-fluoro-deoxyribonucleotides In some embodiments, the oligonucleotide comprises alternating deoxynbonucleotides and 2'- fluor
  • the oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the oligonucleotide comprises alternating locked nucleic acid nucleotides and 2'-0-methyl nucleotides.
  • the 5' nucleotide of the oligonucleotide is a
  • the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide.
  • the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5' and 3 ' ends of the deoxyribonucleotides.
  • the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group or a 3' thiophosphate.
  • the oligonucleotide comprises phosphorothioate internucleoside linkages.
  • the oligonucleotide comprises
  • the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleotides.
  • oligonucleotide can have any combination of modifications as described herein.
  • an oligonucleotide described herein may be a mixmer or comprise a mixmer sequence pattern.
  • the term 'mixmer' refers to oligonucleotides which comprise both naturally and non-naturally occurring nucleotides or comprise two different types of non-naturally occurring nucleotides.
  • Mixmers are generally known in the art to have a higher binding affinity than unmodified oligonucleotides and may be used to specifically bind a target molecule, e.g., to block a binding site on the target molecule. Generally, mixmers do not recruit an RNAse to the target molecule and thus do not promote cleavage of the target molecule.
  • the mixmer comprises or consists of a repeating pattern of nucleotide analogues and naturally occurring nucleotides, or one type of nucleotide analogue and a second type of nucleotide analogue.
  • the mixmer need not comprise a repeating pattern and may instead comprise any arrangement of nucleotide analogues and naturally occurring nucleotides or any arrangement of one type of nucleotide analogue and a second type of nucleotide analogue.
  • a pattern in general, refers to a pattern of modifications or lack thereof, and not to a pattern of A, T, C, G, or U nucleotides.
  • the repeating pattern may, for instance be every second or every third nucleotide is a nucleotide analogue, such as LNA, and the remaining nucleotides are naturally occurring nucleotides, such as DNA, or are a 2' substituted nucleotide analogue such as 2'-0-methyl, 2'MOE or 2' fluoro analogues, or any other nucleotide analogues described herein. It is recognized that the repeating pattern of nucleotide analogues, such as LNA units, or 2'-0-methyl, 2'MOE or 2' fluoro analogues, may be combined with nucleotide analogues at fixed positions— e.g. at the 5' or 3 ' termini.
  • the mixmer does not comprise a region of more than 5, more than 4, more than 3, or more than 2 consecutive naturally occurring nucleotides, such as DNA nucleotides.
  • the mixmer comprises at least a region consisting of at least two consecutive nucleotide analogues, such as at least two consecutive LNAs.
  • the mixmer comprises at least a region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNAs.
  • the mixmer does not comprise a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleotide analogues, such as LNAs. It is to be understood that the LNA units may be replaced with other nucleotide analogues, such as those referred to herein.
  • the mixmer comprises at least one nucleotide analogue in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx and xxxxxX, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occurring nucleotide, such as DNA or RNA.
  • the mixmer comprises at least two nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of XXxxxx, XxXxxx, XxxXxx, xXXxxx, xXxXxx, xXxxxX, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxXx, xxxXxX and xxxxXX, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x” denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the substitution pattern for the nucleotides may be selected from the group consisting of XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXxxxX, xxXxXx, xxXxxX and xxxXxX.
  • the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX.
  • the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx and xxXxXx.
  • the substitution pattern for the nucleotides is xXxXxx.
  • the mixmer comprises at least three nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXX, XxxxXX, XxxxXX, xXxXXx, xXxxXXX, xxXXX, xXxXxX and XxXxXx, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x” denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the substitution pattern for the nucleotides is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxxxXX, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx.
  • the substitution pattern for the nucleotides is selected from the group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX. n some embodiments, the substitution pattern for the nucleotides is xXxXxX or XxXxXx. In some embodiments, the substitution pattern for the nucleotides is xXxXxX.
  • the mixmer comprises at least four nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of xXXXX, xXxXXX, xXXxXX, xXXXxX, xXXXx, XxxXXX, XxXxX, XxXXxX, XxXXx, XXxxXX, XXxXxX, XXxXx, XXxxX, XXXxXx and XXXXxx, wherein "X” denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the mixmer comprises at least five nucleotide analogues in one or more of six consecutive nucleotides.
  • the substitution pattern for the nucleotides may be selected from the group consisting of xXXXXX, XxXXXX, XXxXXX, XXXxXX,
  • XXXXxX and XXXXx wherein "X” denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
  • the oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
  • the mixmer contains a modified nucleotide, e.g., an LNA, at the 5' end. In some embodiments, the mixmer contains a modified nucleotide, e.g., an LNA, at the first two positions, counting from the 5' end.
  • the mixmer is incapable of recruiting RNAseH.
  • Oligonucleotides that are incapable of recruiting RNAseH are well known in the literature, in example see WO2007/112754, WO2007/112753, or PCT/DK2008/000344.
  • Mixmers may be designed to comprise a mixture of affinity enhancing nucleotide analogues, such as in non- limiting example LNA nucleotides and 2'-0-methyl nucleotides.
  • the mixmer comprises modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • a mixmer is 4 to 40 nucleotides (e.g., 4 to 40, 4 to 35, 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10), in length having the general formula:
  • each instance of X 4 is a modified or unmodified nucleotide described herein (e.g., a modified or unmodified ribonucleotide described herein), wherein each instance of X s is a deoxyribonucleotide, wherein p and q are independently 0 or 1, reflecting the number of instances of X 1 and X 2 , respectively, wherein at least one of X 1 and X 2 is present in each instance of the unit, ⁇ X p — X q wherein r is an integer from 2 to 20 reflecting the number of instances of the unit, ⁇ X p — X ⁇ , linked together through internucleotide linkages, wherein the protecting oligonucleotide or region does not contain a sequence of more than 5 consecutive deoxyribonucleotides, and wherein the symbol "— " denotes an internucleotide linkage.
  • a mixmer may be produced using any method known in the art or described herein.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. patent publication Nos. US20060128646,
  • the oligonucleotide is a gapmer.
  • the gapmer has a sequence following the general formula:
  • each instance of X , X 3 is independently a modified or unmodified nucleotide described herein (e.g., a modified or unmodified ribonucleotide described herein), wherein m and o are independently integers in a range of 1 to 10 (e.g., 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 1 to 6, 2 to 6, 3 to 6, or 4 to 6) reflecting the number of instances of X 1 and X 3 , respectively, linked consecutively together through internucleotide linkages, wherein each instance of X 2 is a deoxyribonucleotide, wherein n is an integer in a range of 6 to 20 (e.g., 6 to 20, 6 to 15, 6 to 10, 7 to
  • a gapmer oligonucleotide may also have the formula 5'-X-Y-Z-3', with X and Z as flanking regions around a gap region Y.
  • the Y region is a contiguous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides, which are capable of recruiting an RNAse, such as RNAseH.
  • RNAseH an RNAse
  • the Y region is flanked both 5' and 3' by regions X and Z comprising high-affinity modified nucleotides, e.g., 1 - 6 modified nucleotides.
  • exemplary modified oligonucleotides include, but are not limited to, 2' MOE or 2'OMe or Locked Nucleic Acid bases (LNA).
  • the flanks X and Z may be have a of length 1 - 20 nucleotides, preferably 1-8 nucleotides and even more preferred 1 - 5 nucleotides.
  • the flanks X and Z may be of similar length or of dissimilar lengths.
  • the gap-segment Y may be a nucleotide sequence of length 5 - 20 nucleotides, preferably 6-12 nucleotides and even more preferred 6 - 10 nucleotides.
  • the gap region of the gapmer oligonucleotides of the invention may contain modified nucleotides known to be acceptable for efficient RNase H action in addition to DNA nucleotides, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-configured nucleotides.
  • the gap region comprises one or more unmodified internucleosides.
  • flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • the gap region and two flanking regions each independently comprise modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • a gapmer may be produced using any method known in the art or described herein.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. patent publication Nos. US20090286969, US20100197762, and US20110112170; and PCT publication Nos.
  • oligonucleotides provided herein may be in the form of small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA.
  • siRNA is a class of double- stranded RNA molecules, typically about 20-25 base pairs in length that target nucleic acids (e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway in cells. Specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are generally less than 30 to 35 base pairs in length to prevent the triggering of non-specific RNA interference pathways in the cell via the interferon response, although longer siRNA can also be effective.
  • siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence can be designed and prepared using any method known in the art (see, e.g., PCT Publication Nos. WO08124927A1 and WO 2004/016735; and U.S. Patent
  • a number of commercial packages and services are available that are suitable for use for the preparation of siRNA molecules. These include the in vitro transcription kits available from Ambion (Austin, TX) and New England Biolabs (Beverly, MA) as described above; viral siRNA construction kits commercially available from Invitrogen (Carlsbad, CA) and Ambion (Austin, TX), and custom siRNA construction services provided by Ambion (Austin, TX), Qiagen (Valencia, CA), Dharmacon (Lafayette, CO) and Sequitur, Inc (Natick, MA).
  • a target sequence can be selected (and a siRNA sequence designed) using computer software available commercially (e.g.
  • an siRNA may be designed or obtained using the RNAi atlas (available at the RNAiAtlas website), the siRNA database (available at the Swedish Bioinformatics Website), or using DesiRM (available at the Institute of Microbial
  • the siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single- stranded (i.e. a ssRNA molecule comprising just an antisense strand).
  • the siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense strands.
  • Double- stranded siRNA may comprise RNA strands that are the same length or different lengths.
  • Double-stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single- stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
  • Small hairpin RNA (shRNA) molecules thus are also contemplated herein. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3' end and/or the 5' end of either or both strands).
  • shRNA Small hairpin RNA
  • a spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double- stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or the 5' end of either or both strands).
  • a spacer sequence is may be an unrelated nucleotide sequence that is situated between two complementary nucleotide sequence regions which, when annealed into a double-stranded nucleic acid, comprise a shRNA.
  • the overall length of the siRNA molecules can vary from about 14 to about 200 nucleotides depending on the type of siRNA molecule being designed. Generally between about 14 and about 50 of these nucleotides are complementary to the RNA target sequence, i.e. constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double- or single- stranded siRNA, the length can vary from about 14 to about 50 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 40 nucleotides to about 200 nucleotides.
  • siRNA molecule may comprise a 3' overhang at one end of the molecule, The other end may be blunt-ended or have also an overhang (5' or 3') ⁇
  • the siRNA molecule of the present invention comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule.
  • an oligonucleotide may be a microRNA (miRNA).
  • MicroRNAs are small non-coding RNAs, belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on a target RNA transcript.
  • miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre- miRNAs, which fold into imperfect stem- loop structures.
  • pri-miRNAs large RNA precursors
  • pre-miRNAs typically undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.
  • miRNAs including pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of mature miRNA.
  • the size range of the miRNA can be from 21 nucleotides to 170 nucleotides, although miRNAs of up to 2000 nucleotides can be utilized. In one embodiment the size range of the miRNA is from 70 to 170 nucleotides in length. In another embodiment, mature miPvNAs of from 21 to 25 nucleotides in length can be used.
  • a miRNA is expressed from a vector.
  • the vector may include a sequence encoding a mature miRNA.
  • the vector may include a sequence encoding a pre-miRNA such that the pre-miRNA is expressed and processed in a cell into a mature miRNA.
  • the vector may include a sequence encoding a pri-miRNA.
  • the primary transcript is first processed to produce the stem-loop precursor miRNA molecule. The stem-loop precursor is then processed to produce the mature microRNA.
  • oligonucleotides provided herein may be in the form of aptamers.
  • An "aptamer” is any nucleic acid that binds specifically to a target, such as a small molecule, protein, nucleic acid, cell, tissue or organism.
  • the aptamer is a DNA aptamer or an RNA aptamer.
  • a nucleic acid aptamer is a single- stranded DNA or RNA (ssDNA or ssRNA). It is to be understood that a single- stranded nucleic acid aptamer may form helices and/or loop structures.
  • the nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleotides, modified nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, or a combination of thereof.
  • hydrocarbon linkers e.g., an alkylene
  • a polyether linker e.g., a PEG linker
  • nucleic acid aptamers may be accomplished by any suitable method known in the art, including an optimized protocol for in vitro selection, known as SELEX (Systemic Evolution of Ligands by Exponential enrichment). Many factors are important for successful aptamer selection. For example, the target molecule should be stable and easily reproduced for each round of SELEX, because the SELEX process involves multiple rounds of binding, selection, and amplification to enrich the nucleic acid molecules. In addition, the nucleic acids that exhibit specific binding to the target molecule have to be present in the initial library. Thus, it is advantageous to produce a highly diverse nucleic acid pool. Because the starting library is not guaranteed to contain aptamers to the target molecule, the SELEX process for a single target may need to be repeated with different starting libraries.
  • SELEX Systemic Evolution of Ligands by Exponential enrichment
  • Exemplary publications and patents describing aptamers and method of producing aptamers include, e.g., Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; 5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCT application WO 99/31275, each incorporated herein by reference.
  • oligonucleotides provided herein may be in the form of a ribozyme.
  • a ribozyme ribonucleic acid enzyme
  • Ribozymes are molecules with catalytic activities including the ability to cleave at specific phosphodiester linkages in RNA molecules to which they have hybridized, such as mRNAs, RNA-containing substrates, IncRNAs, and ribozymes, themselves.
  • Ribozymes may assume one of several physical structures, one of which is called a "hammerhead.”
  • a hammerhead ribozyme is composed of a catalytic core containing nine conserved bases, a double- stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA flanking regions the catalytic core. The flanking regions enable the ribozyme to bind to the target RNA specifically by forming double- stranded stems I and III.
  • Cleavage occurs in cis (i.e., cleavage of the same RNA molecule that contains the hammerhead motif) or in trans (cleavage of an RNA substrate other than that containing the ribozyme) next to a specific ribonucleotide triplet by a transesterification reaction from a 3', 5'-phosphate diester to a 2', 3 '-cyclic phosphate diester.
  • this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
  • Ribozyme oligonucleotides can be prepared using well known methods (see, e.g., PCT Publications W09118624; W09413688; WO9201806; and WO 92/07065; and U.S.
  • Patents 5436143 and 5650502 can be purchased from commercial sources (e.g., US Biochemicals) and, if desired, can incorporate nucleotide analogs to increase the resistance of the oligonucleotide to degradation by nucleases in a cell.
  • the ribozyme may be synthesized in any known manner, e.g., by use of a commercially available synthesizer produced, e.g., by Applied Biosystems, Inc. or Milligen.
  • the ribozyme may also be produced in recombinant vectors by conventional means. See, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current edition).
  • the ribozyme RNA sequences maybe synthesized conventionally, for example, by using RNA polymerases such as T7 or SP6.
  • the oligonucleotide is an oligonucleotide mimetic, such as a morpholino -based oligomeric compound, a cyclohexenyl nucleic acid oligonucleotide mimetics, or peptide nucleic acid (PNA) compound.
  • oligonucleotide mimetic such as a morpholino -based oligomeric compound, a cyclohexenyl nucleic acid oligonucleotide mimetics, or peptide nucleic acid (PNA) compound.
  • methods are provided for producing candidate
  • oligonucleotides that are useful for, e.g., inhibiting the interaction of FAS-AS1 with RBM5.
  • the oligonucleotides are complementary to sequences in a target RNA, e.g., FAS- AS 1.
  • the oligonucleotides are designed by determining a region of a target RNA (FAS -AS 1); producing an oligonucleotide that has a region of complementarity that is complementary with a plurality of (e.g., at least 5) contiguous nucleotides of the region of the target RNA; and determining whether administering the oligonucleotide to a cell in which FAS-AS 1 and RBM5 are expressed results in inhibition of the interaction and/or increased levels of soluble Fas.
  • FAS -AS 1 target RNA
  • methods are provided for obtaining one or more
  • oligonucleotides for inhibiting the interaction of FAS-AS 1 with RBM5 that further involve producing a plurality of different oligonucleotides, in which each oligonucleotide has a region of complementarity that is complementary with a plurality of (e.g., at least 5) contiguous nucleotides in a target RNA (e.g., FAS-AS1); subjecting each of the different oligonucleotides to an assay that assesses whether delivery of an oligonucleotide to a cell harboring the target gene results in inhibition of the interaction and/or increased levels of soluble Fas in the cell; and obtaining one or more oligonucleotides that inhibit the interaction and/or increase levels of soluble Fas in the assay.
  • a target RNA e.g., FAS-AS1
  • a T cell population described herein e.g., comprising naive T cells
  • an expression construct encoding a CAR is transfected with an expression construct encoding a CAR.
  • CARs have been utilized to engineer T cells to target various antigens, such as tumor antigens.
  • CARs comprise an extracellular antigen-binding domain (e.g., a single chain variable fragment (scFv) from an antibody), a transmembrane domain (e.g., a transmembrane domain of any one of the following: alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, and CD154) and an intracellular domain comprising one or more signaling or co- stimulatory domains (e.g., one or more signaling domains of the ⁇ 3 ⁇ chain, 4-1BB (CD137) and CD28 and/or one or more co-stimulatory domains of 4-1BB, CD28, ICOS, DAP
  • CARs may further comprise a hinge region such as a IgGl, IgG4, and IgD or CD8 hinge.
  • a hinge region such as a IgGl, IgG4, and IgD or CD8 hinge.
  • Exemplary CARs and methods of making such CARs are known in the art (see, e.g., PCT publication numbers WO2014184744A1,
  • the CAR comprises (a) a scFv specific for an antigen (e.g., a tumor antigen), (b) a hinge region (e.g., an Ig hinge region), (c) a transmembrane domain (e.g., a CD3 ⁇ chain, CD4, CD8, ICOS, or CD28 transmembrane domain), (d) a CD3 ⁇ chain signaling domain and optionally (e) one or more co- stimulatory domains selected from ICOS, OX40 (CD134), CD28, 4-1BB (CD137), CD27, and DAP10.
  • an antigen e.g., a tumor antigen
  • a hinge region e.g., an Ig hinge region
  • a transmembrane domain e.g., a CD3 ⁇ chain, CD4, CD8, ICOS, or CD28 transmembrane domain
  • CD3 ⁇ chain signaling domain e.g., OX40 (CD134), CD28, 4-1BB (CD137),
  • the CAR is specific for a tumor antigen (e.g., contains a scFv specific for a tumor antigen).
  • a tumor antigen e.g., contains a scFv specific for a tumor antigen.
  • exemplary tumor antigens include CD 19, CD20, CD33, HER2, GD2 ganglioside, CD171, CAIX, a-folate receptor, IL13Ra2 and CEA.
  • an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross- species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A,” the presence of a molecule containing epitope "A” (or free, unlabeled "A”), in a reaction containing labeled "A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • transfection of the T cell with the CAR expression construct occurs before the T cell population is contacted with the oligonucleotide. In some embodiments, transfection occurs after the T cell population is contacted with the
  • transfection occurs at the same time that the T cell population is contacted with the oligonucleotide.
  • the T cell population is activated prior to transfection, e.g., by contacting with an activating agent such as an anti-CD3 and/or anti-CD28 antibody optionally immobilized on a solid substrate.
  • the T cell is activated after transfection, e.g., by contacting with an activating agent such as an anti-CD3 and/or anti-CD28 antibody.
  • transfection is achieved by lentiviral infection of the T cell population with the expression construct encoding the CAR.
  • the expression construct may comprise the coding sequence of the CAR optionally along with one or more regulatory sequences that drive expression of the coding sequence, e.g., a promoter and/or enhancer sequence.
  • the expression construct is a lentiviral construct comprising 5' and 3' long terminal repeats (LTRs).
  • Lentiviruses for use in transfecting T cell populations can be produced using any method known in the art or described herein.
  • 293FT cells may be co- transfected with lentiviral helper plasmids and a lentiviral construct comprising the coding sequence of the CAR optionally with regulatory sequences.
  • Virus supernatants can be isolated from the 293T cells and then concentrated, e.g., by ultracentrifugation.
  • the T cells for use in developing a CAR T cells may be obtained using any method known in the art or described herein (see, e.g., PCT publication numbers WO2014184744A1, WO2014184143A1, WO2014059173A2 and WO2015179801A1).
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells
  • PBMCs bone marrow, lymph node tissue, cord blood, thymus tissue and spleen tissue from a donor subject (e.g., a human donor subject).
  • PBMCs can be obtained, e.g., by FicollTM separation from blood obtained from the donor subject.
  • the T cells may be obtained from a T cell line.
  • a specific subpopulation of T cells, such as CD4 + T cells, can be further isolated by positive or negative selection techniques, such as by fluorescent activated cell sorting or magnetic cell sorting.
  • a T cell population transfected with a CAR expression construct is administered to a host subject (e.g., a human host subject).
  • a host subject e.g., a human host subject.
  • the subject has cancer and the CAR is specific for a tumor antigen expressed by the cancer in the subject.
  • Vectors include, but are not limited to, plasmids, viral vectors, other vehicles derived from viral or bacterial or other sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences for expressing an RNA transcript (e.g., mRNA).
  • expression vectors are provided that are engineered to express a chimeric antigen receptor (CAR) as described herein.
  • an expression vector may be engineered by incorporating a coding sequence for a gene of interest (e.g., a CAR) into a plasmid that is suitably configured with expression elements (e.g., a promoter) for expressing the gene of interest.
  • cDNA may be obtained or synthesized using a commercially available kit or any method known in the art.
  • a vector may comprise one or more expression elements.
  • “Expression elements” are any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient expression of an RNA transcript (e.g., mRNA).
  • the expression element may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter or a tissue specific promoter, examples of which are well known to one of ordinary skill in the art.
  • Constitutive mammalian promoters include polymerase promoters as well as the promoters for the following non- limiting genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, and beta-actin.
  • HPTR hypoxanthine phosphoribosyl transferase
  • adenosine deaminase pyruvate kinase
  • beta-actin beta-actin
  • Exemplary viral promoters which function constitutively in eukaryotic cells include promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
  • Other constitutive promoters may be used.
  • Inducible promoters are expressed in the presence of an inducing agent and include metal-inducible promoters and steroid-regulated promoters, for example. Other inducible promoters may be used.
  • Expression vectors may also comprise an origin of replication, a suitable promoter polyadenylation site, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • Viral vectors are generally based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the nucleic acid sequence of interest. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lines with plasmid, production of recombinant retroviruses by the packaging cell lie, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) may be used.
  • Viral and retroviral vectors that may be used include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as: Moloney murine leukemia virus; Murine stem cell virus, Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses such as any retrovirus.
  • retroviruses such as: Moloney murine leukemia virus; Murine stem cell virus, Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses
  • compositions e.g., T cell populations that have been contacted with an oligonucleotide
  • routes e.g., T cell populations that have been contacted with an oligonucleotide
  • Exemplary routes include: intrathecal, intraneural, intracerebral, intramuscular, oral, intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, or ocular.
  • therapeutically effective amount is the amount of active agent (e.g., oligonucleotide or T cell population) present in the composition that is needed to provide the desired level of sFas expression in the T cell population or to provide a treatment effect in the subject to be treated, e.g., treatment of cancer.
  • physiologically effective amount is that amount delivered to a subject to give the desired palliative or curative effect.
  • pharmaceutically acceptable carrier means that the carrier can be administered to a subject with no significant adverse toxicological effects to the subject.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically include one or more species of oligonucleotide or T cell population and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or
  • intraventricular administration The route and site of administration may be chosen to enhance targeting. For example, to target a tumor, intratumoral injection may be desirable.
  • Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject.
  • the most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface.
  • the most common topical delivery is to the skin.
  • the term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum.
  • Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition.
  • Topical administration can also be used as a means to selectively deliver compositions to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
  • Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics.
  • the dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin.
  • Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle
  • transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.
  • iontophoresis transfer of ionic solutes through biological membranes under the influence of an electric field
  • phonophoresis or sonophoresis use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea
  • optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
  • oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
  • GI gastrointestinal
  • compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek.
  • the sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
  • a pharmaceutical composition may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant.
  • the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
  • compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
  • carriers that can be used include lactose, sodium citrate and salts of phosphoric acid.
  • Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration.
  • parental administration involves administration directly to the site of disease (e.g., tumor).
  • Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
  • the total concentration of solutes should be controlled to render the preparation isotonic.
  • Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
  • Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers.
  • the delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • the types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
  • HSA human serum albumin
  • bulking agents such as carbohydrates, amino acids and polypeptides
  • pH adjusters or buffers such as sodium chloride
  • salts such as sodium chloride
  • Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
  • Pulmonary administration of a micellar oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non- CFC and CFC propellants.
  • Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
  • Other devices include non-vascular devices, e.g., devices implanted in the
  • the device can release a therapeutic substance in addition to an oligonucleotide.
  • unit doses or measured doses of a composition are dispensed by an implanted device.
  • the device can include a sensor that monitors a parameter within a subject.
  • the device can include pump, e.g., and, optionally, associated electronics.
  • T cell populations described herein can be treated with an oligonucleotide ex vivo and then administered or implanted in a subject.
  • the T cell population can be autologous, allogeneic, or xenogeneic to the subject.
  • Introduction of treated T cell populations, whether autologous or transplant, can be combined with other therapies.
  • the invention features a method of administering an oligonucleotide to a T cell population (e.g., a human T cell population comprising naive T cells).
  • a T cell population e.g., a human T cell population comprising naive T cells.
  • 1 to 40 micromolar (e.g., 1 to 20 micromolar) of oligonucleotide is
  • the invention features a method of administering a T cell population (e.g., a human T cell population comprising naive T cells that has been contacted with an oligonucleotide herein) to a subject (e.g., a human subject).
  • a T cell population e.g., a human T cell population comprising naive T cells that has been contacted with an oligonucleotide herein
  • the dosage in between 10 4 to 10 9 cells/kg body weight.
  • T cell populations may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
  • the defined amount can be an amount effective to upregulate sFas expression in naive T cells in a T cell population.
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder that would benefit from adoptive T cell transfer, such as cancer.
  • the unit dose is administered or delivered daily. In some embodiments, less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered or delivered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered or delivered a single time. In some embodiments, the unit dose is administered or delivered more than once a day, e.g., once an hour, two hours, four hours, eight hours, twelve hours, etc. Further, the treatment regimen for T cell populations may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient.
  • the subject can be monitored for changes in his condition and for alleviation of the symptoms of the disease state.
  • the dosage may either be increased in the event the subject does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
  • the effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semipermanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
  • a delivery device e.g., a pump, semipermanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
  • Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. It will also be appreciated that the effective dosage may increase or decrease over the course of a particular treatment.
  • Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models.
  • the administration of a composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, ocular, intraneuronal, intrathecal, or intracerebral.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • kits comprising a container housing a composition comprising an oligonucleotide as described herein.
  • the composition is a pharmaceutical composition comprising an oligonucleotide and a pharmaceutically acceptable carrier.
  • the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g., one container for oligonucleotides, and at least another for a carrier compound.
  • the kit may be packaged in a number of different configurations such as one or more containers in a single box.
  • the different components can be combined, e.g., according to instructions provided with the kit.
  • the components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition.
  • the kit can also include a delivery device.
  • Gapmer oligonucleotides were designed to target FAS-AS 1.
  • the sequence and chemistry of each gapmer oligonucleotide is shown in Table 1.
  • Table 2 provides a description of the nucleotide analogs, modifications and internucleoside linkages used for certain oligonucleotides described in Table 1.
  • GTGTAAGAAAATTGT 8 lnaGs;lnaTs;lnaGs;dTs;dAs;dAs;dGs;d
  • TTTATTGTCACACAG 26 lnaTs;lnaTs;lnaTs;dAs;dTs;dTs;dGs;dT s;dCs;dAs;dCs;dAs;lnaCs;lnaAs;lnaG
  • CTGCCTTGTCTCCCT 28 lnaCs;lnaTs;lnaGs;dCs;dCs;dTs;dTs;dG s;dTs;dCs;dTs;dCs;lnaCs;lnaCs;lnaT
  • TCCGGGAATTCTCTC 29 lnaTs;lnaCs;lnaCs;dGs;dGs;dGs;dAs;d
  • CTCGTTCCCACCGCA 42 lnaCs;lnaTs;lnaCs;dGs;dTs;dTs;dCs;dC s;dCs;dAs;dCs;dCs;lnaGs;lnaCs;lnaA
  • GCGCCTATTATTGGC 44 lnaGs;lnaCs;lnaGs;dCs;dCs;dTs;dAs;dT s;dTs;dAs;dTs;dTs;lnaGs;lnaGs;lnaC
  • TTTGAGTACCGGAGC 53 lnaTs;lnaTs;lnaTs;dGs;dAs;dGs;dTs;dA s;dCs;dCs;dGs;dGs;lnaAs;lnaGs;lnaC
  • AATTCCAAAACTCAG 72 lnaAs;lnaAs;lnaTs;dTs;dCs;dCs;dAs;dA s;dAs;dAs;dCs;dTs;lnaCs;lnaAs;lnaG
  • CCTTTCAGAAATAGT 80 lnaCs;lnaCs;lnaTs;dTs;dTs;dCs;dAs;dG s;dAs;dAs;dAs;dTs;lnaAs;lnaGs;lnaT
  • AATGATTCAAGATTG 85 lnaAs;lnaAs;lnaTs;dGs;dAs;dTs;dTs;dC s;dAs;dAs;dGs;dAs;lnaTs;lnaTs;lnaG
  • TTATACAACCTCAGG 87 lnaTs;lnaTs;lnaAs;dTs;dAs;dCs;dAs;dA s;dCs;dTs;dCs;lnaAs;lnaGs;lnaG
  • GCCACACTCTTCTCT 88 lnaGs;lnaCs;lnaCs;dAs;dCs;dAs;dCs;dT s;dCs;dTs;dCs;lnaTs;lnaCs;lnaT
  • GCTTTGAAAATCTCA 92 lnaGs;lnaCs;lnaTs;dTs;dTs;dGs;dAs;dA s;dAs;dAs;dTs;dCs;lnaTs;lnaCs;lnaA
  • GAACTTTTGTACCAA 98 lnaGs;lnaAs;lnaAs;dCs;dTs;dTs;dT s;dGs;dTs;dAs;dCs;lnaCs;lnaAs;lnaAs;lnaA
  • ACACACACGCATATG 104 lnaAs;lnaCs;lnaAs;dCs;dAs;dCs;dAs;d
  • GTAAATATTCATACA 105 lnaGs;lnaTs;lnaAs;dAs;dAs;dTs;dAs;dT s;dTs;dCs;dAs;dTs;lnaAs;lnaCs;lnaA CATTTATGTATATAT 106 lnaCs;lnaAs;lnaTs;dTs;dTs;dAs;dTs;dG s;dTs;dAs;dTs;dAs;lnaTs;lnaAs;lnaT
  • AACTTATATTTGTAT 109 lnaAs;lnaAs;lnaCs;dTs;dTs;dAs;dTs;dA s;dTs;dTs;dTs;dGs;lnaTs;lnaAs;lnaT
  • ATGATATATGGCCTA 110 lnaAs;lnaTs;lnaGs;dAs;dTs;dAs;dTs;dA s;dTs;dGs;dGs;dCs;lnaCs;lnaTs;lnaA
  • CTAGGAAATTAAGGC 111 lnaCs;lnaTs;lnaAs;dGs;dGs;dAs;dAs;d
  • CATAACTCTATCACC 116 lnaCs;lnaAs;lnaTs;dAs;dAs;dCs;dTs;dC s;dTs;dAs;dTs;dCs;lnaAs;lnaCs;lnaC
  • TCACCTAAGTAATCA 117 lnaTs;lnaCs;lnaAs;dCs;dCs;dTs;dAs;dA s;dGs;dTs;dAs;dAs;lnaTs;lnaCs;lnaA
  • TGCAGTTTATCTTCC 119 lnaTs;lnaGs;lnaCs;dAs;dGs;dTs;dTs;dT s;dAs;dTs;dCs;dTs;lnaTs;lnaCs;lnaC
  • CCATTTCTCCCCTCT 120 lnaCs;lnaCs;lnaAs;dTs;dTs;dTs;dCs;dT s;dCs;dCs;dCs;dCs;lnaTs;lnaCs;lnaT
  • CTTC ACG GTTATGTT 132 lnaCs;lnaTs;lnaTs;dCs;dAs;dCs;dGs;dG
  • oligonucleotides in Table 3 each have the structural motif of
  • InaNs are LNA nucleotides and dNs are DNA nucleotides and the nucleotides are all linked by phosphorothioate linkages as indicated by the "s" after each nucleotide.
  • InaCs are 5- methylcytosine LNAs. Table 3: Further gapmer oligonucleotides

Abstract

Provided herein are compositions and methods for maintaining or increasing the number of naive T cells in a T cell population, e.g., by delivering ex vivo an oligonucleotide that inhibits the interaction of FAS-AS l with RBM5 to a T cell population comprising naive T cells. Such compositions and methods are useful, e.g., in adoptive T cell therapies including chimeric antigen receptor (CAR) T cell therapies.

Description

MODULATION OF SOLUBLE FAS EXPRESSION
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/548,483, entitled "MODULATION OF SOLUBLE FAS EXPRESSION", filed August 22, 2017, the contents of which are incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention relates in part to compositions and methods for modulating gene expression, e.g., in the context of cell based therapies.
BACKGROUND OF THE INVENTION
Adoptive cell transfer is a therapy that generally involves the transfer of cells into a subject for modulating one or more biological responses or functions in the subject. Cells used in an adoptive cell transfer made be obtained from the patient (autologous cells) or from another individual (allogenic cells). For example, cells derived from the immune system of a subject (e.g., monocytes, T cells, etc.) may be used in an adoptive cell transfer therapy for purposes of improving immune function of the subject. In the context of immunotherapy, T cells may be obtained from the subject, modified (e.g., to express an engineered receptor) and/or expanded in culture and returned to the same subject.
SUMMARY OF THE INVENTION
Aspects of the disclosure relate to adoptive cell transfer of specific T cell populations, including engineered populations such as CAR T cells. In some embodiments, T cell populations prepared according to methods provided herein are useful for cancer treatment via adoptive transfer, which may result in cancer regression or remission in subjects receiving the transfer. In some embodiments, T cells are prepared for adoptive transfer such that the make-up of the T cell population is controlled in order to optimize efficacy of treatment. For example, in some embodiments, methods are provided for controlling the make-up of a T cell population (e.g., a CAR T cell population) in order to maintain or increase the percentage of naive T cells in the T cell population. Thus, in some embodiments, methods for adoptive transfer of specific T cell populations containing naive CD8-positive T cells are provided. Further aspects of the disclosure generally relate to methods for producing and/or maintaining cell populations containing naive CD8-positive T cells. In some embodiments,
oligonucleotides (e.g., gapmers) or other molecules are utilized to modulate the expression of genes that control the differentiation state of T cells, particularly naive CD8-positive T cells.
In some aspects, a method of maintaining or increasing the number of naive T cells in a T cell population is provided, the method comprising delivering ex vivo an oligonucleotide that inhibits the interaction of FAS-AS 1 with RBM5 to a T cell population comprising naive T cells.
In some embodiments, delivering the oligonucleotide results in an increase in soluble Fas (sFas) expression in the naive T cells in the T cell population compared to sFas expression in control naive T cells in a control T cell population to which the oligonucleotide has not been delivered.
In some embodiments, the T cell population is a CD4+ T cell population. In some embodiments, the method further comprises isolating T cells from a sample obtained from a donor subject; and selecting CD4+ T cells from the isolated T cells, thereby producing the T cell population comprising naive T cells to which the oligonucleotide is delivered.
In some embodiments, the method further comprises administering the T cell population to a host subject after the oligonucleotide has been delivered to the T cell population. In some embodiments, the donor subject and the host subject are the same. In some embodiments, the donor subject and the host subject are different.
In some embodiments, the method further comprises transfecting the T cell population with an expression construct encoding a chimeric antigen receptor (CAR). In some embodiments, the transfection occurs before delivery of the oligonucleotide to the T cell population. In some embodiments, the transfection occurs after delivery of the
oligonucleotide to the T cell population. In some embodiments, the CAR is specific for a tumor antigen. In some embodiments, the host subject has cancer.
In some embodiments, the T cell population is a human T cell population.
In some embodiments, the oligonucleotide comprises a region of complementarity that is complementary with at least 8 nucleotides of FAS-AS 1. In some embodiments, the oligonucleotide reduces the level of FAS -AS 1. In some embodiments, the oligonucleotide is a gapmer, an siRNA, a ribozyme or an aptamer that causes degradation of FAS -AS 1. In some embodiments, the oligonucleotide is single stranded. In some embodiments, the oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a sequence selected from SEQ ID NOs: 2 to 1833.
In some embodiments, the oligonucleotide sterically interferes with the interaction of FAS-AS 1 with RBM5. In some embodiments, the oligonucleotide is single stranded. In some embodiments, the oligonucleotide is a mixmer. In some embodiments, the
oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a sequence selected from SEQ ID NOs: 2 to 1833.
In some embodiments, the oligonucleotide is a gapmer comprising a region of complementarity that is complementary with at least 8 nucleotides of FAS -AS 1. In some embodiments, the gapmer comprises the general formula:
wherein each instance of X , X3 is independently a modified or unmodified nucleotide, wherein m and o are independently integers in a range of 1 to 10, reflecting the number of instances of X1 and X3, respectively, linked consecutively together through internucleotide linkages, wherein each instance of X2 is a deoxyribonucleotide, wherein n is an integer in a range of 6 to 20, reflecting the number of instances of X2 linked consecutively together through internucleotide linkages. In some embodiments, at least one of X1, X3 is a 2'- modified nucleotide. In some embodiments, the 2'-modified nucleotide is a 2'-0,4'-C- bridged nucleotide. In some embodiments, 2'-modified nucleotide is a 2'-0,4'-C- methylene bridged nucleotide. In some embodiments, the a 2'-modified nucleotide is a 2'-0-methyl nucleotide. In some embodiments, the oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a sequence set forth in Table 1 or Table 3 (e.g., a sequence selected from SEQ ID NOs: 2 to 1833). In some embodiments, each instance of X1, X3 is a LNA nucleotide and each of the nucleotides in the oligonucleotide are linked by phosphorothioate linkages. In some embodiments, each instance of X1^3 is a LNA nucleotide, m and o are each 3, n is 9, and each of the nucleotides in the oligonucleotide are linked by phosphorothioate linkages. In some embodiments, any cytosine LNAs in the oligonucleotide are 5-methylcytosine LNAs. In some embodiments, any oligonucleotide disclosed herein having one or more 5-methylcytosine LNAs may have any one or more of the 5-methylcytosine LNAs replaced with a cytosine LNA.
The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appending claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1A is a diagram showing alternative splicing of the FAS gene in the presence of FAS-AS 1 and RBM5.
FIG. IB is a diagram showing FAS signaling between naive T cells and memory T cells and FAS-mediated precocious differentiation of naive T cells.
FIG. 1C is a diagram showing blocking of FAS signaling upon production of soluble Fas (sFas). See also genevisible.com/tissues/HS/Gene%20Symbol/FAS.
FIG. 2 is a graph showing expression of RMB5 in naive T cells and other cell types.
FIG. 3 is a graph showing expression of FAS-AS 1 in naive T cells and other cell types. See also biogps.org/#goto=genereport&id=100302740.
DETAILED DESCRIPTION OF THE INVENTION
Based on mouse and preclinical human data, the adoptive transfer of specific T cell populations, such as naive CD8-positive T cells, can increase the probability of cancer cell regression and remission (see, e.g., Cieri, N. et al. (2013) IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood 121, 573-584;
Hinrichs, C. S. et al. (2009) Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity. Proc Natl Acad Sci U S A 106, 17469-1747; and Rosenberg, S. A. et al. (2011) Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res 17, 4550-4557). Ex vivo purification, engineering, and expansion of the appropriate cell types is useful in this context.
Naive T (TN) cells are the precursors of T stem cell memory cells (TSCM), T effector memory cells (TEMX and T central memory cells (TCM)- The TSCM and TCM populations may increase antitumor, antibacterial, and antiviral responses following adoptive cell transfer in preclinical models. Interaction between memory T cells (TMem) and TN cells may contribute to proliferation and expansion of the TN cells (see, e.g., Klebanoff, C. A. et al. (2016) Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J Clin Invest 126, 318-334). However, in some embodiments, TMem cells may induce precocious differentiation of the TN cells, thereby limiting the number of the desired TSCM and TCM populations that can be produced from the depleted TN cell population. This precocious differentiation is mediated through FasL (CD95L) and Fas (CD95) interaction on the TMem and T cells, respectively. Blocking of this interaction through a recombinant leucine zipper- dimerized FasL results in an increase of TN cells with proliferative capacity and to increase tumor regression upon adoptive transfer (see, e.g., Klebanoff, C. A. et al. (2016) Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J Clin Invest 126, 318-334).
The FAS gene encodes both a membrane-bound form and a soluble form of Fas (mFas and sFas, respectively). The form of Fas protein that is produced in a cell is mediated by alternative splicing of exon 6 of the FAS mRNA. Inclusion of exon 6 results in the production of mFas, whereas exclusion of exon 6 results in the production of sFAS (FIG. 1A). Without wishing to be bound by theory, it is believed that binding of FasL to mFas mediates the differentiation of TN cells described above (FIG. IB and 1C). FAS-AS 1, a long noncoding RNA (IncRNA) that is antisense to the FAS gene, binds to RBM5 (RNA-binding protein 5), which is involved in alternative splicing of exon 6 of FAS. FAS-AS 1 binding inhibits RBM5 activity and results in the inclusion of exon 6 and the production of mFAS (see, e.g., Sehgal, L. et al. (2014) FAS-antisense 1 IncRNA and production of soluble versus membrane Fas in B-cell lymphoma. Leukemia 28, 2376-2387).
In B cell malignancies where EZH2 is overexpressed or mutated, the promoter of FAS-AS 1 is targeted for epigenetic silencing. When EZH2-mediated repression of FAS-AS 1 is overcome, increased levels of FAS-AS 1 and correspondingly decreased expression of sFas are observed (see, e.g., Sehgal, L. et al. (2014) FAS-antisense 1 IncRNA and production of soluble versus membrane Fas in B-cell lymphoma. Leukemia 28, 2376-2387).
In view of the above, the disclosure, according to some aspects, provides methods for maintaining or increasing T cells in a T cell population. For example, provided herein are compositions and methods for maintaining or increasing the number of TN cells in a T cell population by inhibiting the interaction of FAS-ASl with RBM5. In some embodiments, the interaction is inhibited using an oligonucleotide as described herein.
In some embodiments, inhibiting the interaction of FAS-AS l with RBM5 in TN cells will lead to the production of increased levels of sFAS and/or decreased levels of mFAS based on the change in exon 6 splicing. Both RBM5 and FAS-AS l are expressed in T cells (FIGs. 2 and 3). Consequently, in some embodiments, less mFAS is produced by the TN cells, which may result in less FAS-mediated signaling and less precocious differentiation of the T cells. In addition, in some embodiments, increased levels of sFAS may act as an inhibitor of FasL signaling from the TMem population, again resulting in less precocious differentiation of the TN cells. As a result, in some embodiments, inhibiting the interaction of FAS-AS 1 with RBM5 will cause a greater number of TN cells to be present in a T cell population, which, if used in adoptive T cell transfer for cancer treatment, may result in a more effective antitumor response. Further, in some embodiments, treatment of a T cell population ex vivo with an oligonucleotide as described herein may result in effects that are limited in time, as the oligonucleotide may be diluted out as the T cell population expands, e.g., once the T cell population is administered to a subject. In some embodiments, this temporary window effectiveness may be advantageous, as mFas/FasL signaling is important for in vivo T cell differentiation into effector cells and apoptosis of T cells, which may result in greater efficacy of adoptive T cell transfer in vivo as well as prevent unwanted negative effects caused by T cells that are resistant to apoptosis.
Methods for Maintaining or Increasing Naive T Cells and/or Increasing Soluble Fas
Expression in a T Cell Population
In some aspects, the disclosure provides methods for maintaining or increasing the number of naive T cells and/or increasing soluble Fas cell surface death receptor (sFas) in a T cell population. In some embodiments, the method comprises administering to a T cell population an oligonucleotide as described herein, e.g., that inhibits the interaction of FAS- AS 1 with RBM5. In some embodiments, the administration of the oligonucleotide is ex vivo.
In some embodiments, the concentration of oligonucleotide delivered to the T cell population is 0.5 μΜ to 10 μΜ, 1 μΜ to 20 μΜ, or 0.01 μΜ to 50 μΜ. In some
embodiments, the concentration of oligonucleotide delivered to the T cell population is up to 1 μΜ, up to 5 μΜ, up to 10 μΜ, up to 20 μΜ, up to 50 μΜ, or up to 100 μΜ. It is understood that any reference to uses of compounds (e.g., oligonucleotides, expression vectors, inhibitors) throughout the description contemplates use of the compound in preparation of a pharmaceutical composition or medicament for use in the treatment of condition or a disease (e.g., cancer) where maintaining or increasing the number of naive T cells in a T cell population is therapeutically beneficial, e.g., in adoptive T cell transfer.
In some embodiments, delivering an oligonucleotide as described herein results in an increase in sFas expression in the T cell population (e.g., in the naive T cells) compared to a control level of sFas expression, such as sFas expression in a control T cell population (e.g., in control naive T cells) to which the oligonucleotide has not been delivered.
A level of sFas expression may be determined using any suitable assay known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001;
Current Protocols in Molecular Biology, Current Edition, John Wiley & Sons, Inc., New York; and Current Protocols in Protein Production, Purification, and Analysis, Current Edition, John Wiley & Sons, Inc., New York). The sFas expression level may be an mRNA level or a protein level. In some embodiments, the sequences of FAS mRNAs and proteins known in the art (see, e.g., NCBI Transcript IDs: NM_000043.5, NM_001320619.1,
NM_152871.3, and NM_152872.3, and NCBI Protein IDs: NP_000034.1, NP_001307548.1, NP_690610.1, and NP_690611.1) may used to design suitable reagents and assays for measuring an sFas expression level.
In some embodiments, an appropriate control level of sFas expression may be, e.g., a level of sFas expression in a cell or population of cells to which an oligonucleotide has not been delivered or to which a negative control has been delivered (e.g., a scrambled oligo, a carrier, etc.). In some embodiments, an appropriate control level of sFas expression may be a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. It can be single cut-off value, such as a median or mean.
As used herein, increasing sFas expression in a cell includes a level of sFas expression that is, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more above an appropriate control level of sFas. The appropriate control level may be a level of sFas expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein. In some embodiments, administration of an oligonucleotide as described herein results in a decreased level of FAS -AS 1 or steric interference with the interaction of FAS- AS 1 with RBM5. As used herein, decreasing a level of FAS-AS 1 includes a level of FAS- AS 1 that is, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than an appropriate control level of FAS-AS 1. The appropriate control level may be a level of FAS-AS 1 expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein.
In some embodiments, a T cell population is obtained and an oligonucleotide as described herein is delivered ex vivo to the T cell population. In some embodiments, the T cell population is obtained by isolating T cells from a sample (e.g., a blood sample) obtained from a donor subject (e.g., a human donor subject). In some embodiments, the T cell population is further enriched, e.g., by selecting T cells expressing certain cell surface markers, such as CD4 and/or CD8. In some embodiments, the T cell population is enriched for naive T cells, e.g., by selection for CD4-positive T cells. The selection of T cells may be accomplished using any method known in the art or described herein, e.g., by fluorescence activated cell sorting or magnetic cell sorting.
In some embodiments, a T cell population to which an oligonucleotide as described herein has been delivered is administered to a host subject (e.g., a human host subject). In some embodiments, this process is referred to as adoptive T cell transfer. The T cell population administered to the subject may further be engineered, prior to administration to the subject, to express a recombinant receptor such as a chimeric antigen receptor (CAR) as described herein. Suitable administration routes for delivery of the T cell population to a subject are described herein.
Oligonucleotides
In one aspect of the invention, oligonucleotides are provided for maintaining or increasing the number of naive T cells in a T cell population, e.g., by inhibiting the interaction of FAS-AS 1 with RBM5. In some embodiments, as a result of the inhibition, expression of soluble Fas (sFas) is upregulated or increased. In some embodiments, the oligonucleotide comprises a region of complementarity that is complementary with FAS- AS 1. An exemplary sequence of FAS-AS 1 sequence is provided below:
FAS-AS 1 cDNA TTCCAAGTAATTAGCACTTTGCATCTATTTTATTTATTGCTACTACTAAGAAAACATAACCGTGAAGGCA TAAAAGCAAACATTTTCTGTAGACACATGAGGTGACACAAGAGTGTTGAAACTTCTTTAAGGATAAAGGC CTGATGTTAGGCATGTCGTGAGCGCAAAACCAATTTCTGTGTAACTAAATTTAAAAGAGTCATATTAGAG GGGAGAAATGGAAGATAAACTGCAAAACGTAAACAATGATTACTTAGGTGATAGAGTTATGTCTGATCTT TGTATTTGACTCATCTGCATTTATAAATTTTATTTAATAAGCCTTAATTTCCTAGGCCATATATCATATA CAAATATAAGTTATACCTAACTTATAGGTATTATAATATGTATATATACATAAATGTATGAATATTTACA TATGCGTGTGTGTGTGTATTCTTTCAGAAAAGCAAAGTTTTCTTCGGGCTTTAAACACGCAGCCTCTGGT AAAGGTGCTATTGGTACAAAAGTTCAACAAGCATAGCTCCAAGCTTTGACAATTTTCTTACACTTTACTA ATACTGCTGTCCAAAAACTATGAGATTTTCAAAGCAGCCAACATTGCAGTGGTGCAATGGTTAATAACAA GAGAGAGAAGAGTGTGGCCTGAGGTTGTATAAATGTTTTCTCAATCTTGAATCATTAATTTTCTCCAAGG AAGTTTCTAAAGAATCCCAAATAGTGCACACTATTTCTGAAAGGAACAGGATTTTTTTTTCTAATCATAA AATGGACCCAGACATCTCAGCCTCTTGGTGTAATCTGGATATCAGATGCAATCAGCGAACAGCCTGAGTT TTGGAATTAGATCTGAGTTCAATCCCATATCTCCCATTTACTAGCTGTGTGATCTTGGCTGAATTACTGA AGCTCTCTGAACCTCATTTCGCCATCTGTAAAATGGGGATGTGGTTATCTTCCCCACTACATGGCTCTCG TGAGAATCCGCGGAGATCACATTTGTAAACACTTCTCTCGCTATGCCTGGCACTTTGTTGGGGCCCAATA AAATAAAAATCCATGGACTCTCAGAGGCTCCGGTACTCAAATGAGCCTCCTGGATCCACGTCTCTTTGAT TAGCCATCTGCAAGCTGGCATTTCTGAGCGAGGGACTTTTCAAAACAGGCTGCTCAAGTTTCTTGGCCAA TAATAGGCGCCGGGTTCTGTGCGGTGGGAACGAGTACCACCAACCCCAGCAGGAGACCAAGCAGAAATCA CCATGGGAGTGCAAGCTAAGAAAGGGCAAAAGAAGAAAGAGAAGGGCAGAAAAACAAAACAAAATGAAAC CACTCAGGCAGCGACTTACAGTCTTAAAGAGAGAATTCCCGGAAGGGAGACAAGGCAGTTTCTTTTTCTG TGTGACAATAAAAAACGGTAAACAAGCCTCCAGAAGCTCATTCAGCCCCCATATAACTTTTTCGAGAAAG AAAAGGTGCCGTTCTTCCGAGCCCTCCGGCTTAACCACTGCTTCGGTGCTGACTTATTTCCTACGTCTGA GAACTGCCAGAAAA (SEQ ID NO: 1) The oligonucleotide may be single stranded or double stranded. Single stranded oligonucleotides may include secondary structures, e.g., a loop or helix structure. In some embodiments, the oligonucleotide comprises at least one modified nucleotide or modified internucleoside linkage as described herein.
The oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine
nucleotides). In some embodiments, oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
The oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene. For example, an oligonucleotide may be designed to ensure that it does not have a sequence that maps to genomic positions encompassing or in proximity with all known genes (e.g., all known protein coding genes) other than FAS-AS l. The threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity. The oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, greater than 70% G-C content, or greater than 80% G-C content. The oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content. In some embodiments in which the oligonucleotide is 8 to 10 nucleotides in length, all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of FAS-AS 1 are cytosine or guanosine nucleotides. In some embodiments, the sequence of the FAS-AS l to which the oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.
In some embodiments, the region of complementarity of the oligonucleotide is complementary with 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of FAS-AS l. In some embodiments, the region of complementarity is complementary with at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive nucleotides of FAS-AS l, optionally wherein the oligonucleotide is 8 to 30 nucleotides in length.
Complementary, as the term is used in the art, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an
oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of FAS-AS l, then the oligonucleotide and FAS-AS l are considered to be complementary to each other at that position. The oligonucleotide and FAS-AS l are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases. Thus,
"complementary" is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and FAS-ASl . For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of FAS-AS l, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
The oligonucleotide may be at least 70% complementary to (optionally one of at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of FAS-AS 1. In some embodiments the oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of FAS -AS 1. In some embodiments, the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
It is understood in the art that a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a target molecule. In some embodiments, a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable or specific for the target molecule when binding of the sequence to the target molecule (e.g., FAS-AS 1) interferes with the normal function of the target (e.g., FAS-AS 1) to cause a loss of activity (e.g., inhibiting the interaction with RBM5) or expression (e.g., degrading the FAS-AS 1) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays, ex vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
In some embodiments, the oligonucleotide is up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 35, up to 40, up to 45, or up to 50 nucleotides in length. In some embodiments, the oligonucleotide is 5 to 50, 6 to 50, 7 to 50, 8 to 50, 9 to 50, 10 to 50, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15 nucleotides in length. In a preferred embodiment, the oligonucleotide is 8 to 30 nucleotides in length.
Base pairings may include both canonical Watson-Crick base pairing and non- Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are
complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3- nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa.
In some embodiments, GC content of the oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.
Oligonucleotide structure and modifications
The oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof. In addition, the oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; or have improved endosomal exit.
Any of the oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g., a cleavable linker.
Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. For example, nucleic acid sequences of the invention may include a phosphorothioate at least the first, second, or third internucleoside linkage at the 5' or 3' end of the nucleotide sequence. As another example, the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'- deoxy, 2'-deoxy-2'-fluoro, 2 -O-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0- DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0--N-methylacetamido (2'-0— NMA). As another example, the nucleic acid sequence can include at least one 2'-0- methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0- methyl modification. In some embodiments, the nucleic acids are "locked," i.e., comprise nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'-0 atom and the 4'-C atom.
Any of the modified chemistries or formats of oligonucleotides described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
In some embodiments, an oligonucleotide may comprise one or more modified nucleotides (also referred to herein as nucleotide analogs). In some embodiments, the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and/or at least one bridged nucleotide. In some embodiments, the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. Examples of such nucleotides are disclosed herein and known in the art. In some embodiments, the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes. The oligonucleotide may have one or more 2' O-methyl nucleotides. The oligonucleotide may consist entirely of 2' O-methyl nucleotides.
Often the oligonucleotide has one or more nucleotide analogues. For example, the oligonucleotide may have at least one nucleotide analogue that results in an increase in Tm of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an
oligonucleotide that does not have the at least one nucleotide analogue. The oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in Tm of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue. The oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues. The oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
The oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues. Optionally, the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.
The oligonucleotide may consist entirely of bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides). The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides. The oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues. The oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides. The oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides. The oligonucleotide may have a 5' nucleotide that is a bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide). The oligonucleotide may have a 5' nucleotide that is a deoxyribonucleotide.
The oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides. The oligonucleotide may comprise
deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the deoxyribonucleotides. The 3' position of the oligonucleotide may have a 3' hydroxyl group. The 3' position of the oligonucleotide may have a 3' thiophosphate.
The oligonucleotide may be conjugated with a label. For example, the
oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
Preferably the oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
In some embodiments, the oligonucleotides are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and
5,700,922, each of which is herein incorporated by reference.
In some embodiments, the oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, preferably a 2'-0-alkyl, 2'-0-alkyl-0-alkyl or 2'-fluoro- modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.
A number of nucleotide modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, modified internucleoside linkages such as
phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. In some embodiments, oligonucleotides may have phosphorothioate backbones; heteroatom backbones, such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see
Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleic acid (PNA) backbones (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus -containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5, 177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799; 5,587,361; and 5,625,050.
In some embodiments, the oligonucleotide is an oligonucleotide mimetic.
Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001;
Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216- 220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596, 086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
Arabinonucleosides are stereoisomers of ribonucleo sides, differing only in the configuration at the 2'-position of the sugar ring. In some embodiments, a 2'-arabino modification is 2'-F arabino. In some embodiments, the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
Other preferred modifications include ethylene -bridged nucleic acids (ENAs) (e.g., International Patent Publication No. WO 2005/042777, Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8: 144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171- 172, 2005; the disclosures of which are incorporated herein by reference in their entireties). Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene-bridged nucleic acids. Examples of LNAs are described in WO/2008/043753 and include compounds of the following general formula.
Figure imgf000020_0001
where X and Y are independently selected among the groups -0-,
-S-, -N(H)-, N(R)-, -CH2- or -CH- (if part of a double bond),
-CH2-0-, -CH2-S-, -CH2-N(H)-, -CH2-N(R)-, -CH2-CH2- or -CH2-CH- (if part of a double bond),
-CH=CH-, where R is selected from hydrogen and Ci_4-alkyl; Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.
In some embodiments, the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
Figure imgf000020_0002
wherein Y is -0-, -S-, -NH-, or N(R ); Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety, and RH is selected from hydrogen and Ci_4-alkyl.
In some embodiments, the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
In some embodiments, the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O)2-O-, -0-P(0,S)-0-, -0-P(S)2-O-, -S-P(0)2-0-, -S-P(0,S)-0-, -S-P(S)2-0-, -0-P(0)2-S-, -0-P(0,S)-S-, -S-P(0)2-S-, -0-PO(RH)-0-, o- PO(OCH3)-0-, -0-PO(NRH)-0-, -0-PO(OCH2CH2S-R)-O-, -0-PO(BH3)-0-, -0-PO(NHRH)- 0-, -0-P(0)2-NRH-, -NRH-P(0)2-0-, -NRH-CO-0-, where RH is selected from hydrogen and Ci_4-alkyl.
Specifically preferred LNA units are shown below:
Figure imgf000021_0001
The term "thio-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH2-S-. Thio-LNA can be in both beta-D and alpha-L-configuration.
The term "amino-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH2-N(H)-, and -CH2-N(R)- where R is selected from hydrogen and Ci_4-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.
The term "oxy-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH2-0-. Oxy-LNA can be in both beta-D and alpha-L-configuration. The term "ena-LNA" comprises a locked nucleotide in which Y in the general formula above is -CH2-0- (where the oxygen atom of -CH2-0- is attached to the 2'-position relative to the base B).
LNAs are described in additional detail herein.
One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3 ; OCF3; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy [2'-0-CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'- methoxy (2'-0-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar
modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
Oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5- Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2- thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine, 2-chloro-6- aminopurine and 2,6-diaminopurine or other diaminopurines. See, e.g., Kornberg, "DNA Replication," W. H. Freeman & Co., San Francisco, 1980, pp75-77; and Gebeyehu, G., et al. Nucl. Acids Res., 15:4513 (1987)). A "universal" base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. (Sanghvi, in Crooke, and Lebleu, eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and may be used as base substitutions.
It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be
incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
In some embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with modified groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
Oligonucleotides can also include one or more nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo -uracil), 4-thiouracil, 8-halo, 8- amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- deazaadenine and 3- deazaguanine and 3-deazaadenine.
Further, nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, Chapter 15, Antisense Research and Applications," pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, et al., eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos. 3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.
In some embodiments, the oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. For example, one or more oligonucleotides, of the same or different types, can be conjugated to each other; or oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxychole sterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). See also US patent nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486, 603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5, 565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941, each of which is herein incorporated by reference.
These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.
In some embodiments, oligonucleotide modification includes modification of the 5' or 3' end of the oligonucleotide. In some embodiments, the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate. It should be appreciated that additional molecules (e.g. a biotin moiety or a fluorophor) can be conjugated to the 5' or 3' end of the oligonucleotide. In some embodiments, the oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
In some embodiments, the oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxynbonucleotides and 2'- fluoro-deoxyribonucleotides. In some embodiments, the oligonucleotide comprises alternating deoxynbonucleotides and 2'-0-methyl nucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides. In some embodiments, the oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the oligonucleotide comprises alternating locked nucleic acid nucleotides and 2'-0-methyl nucleotides.
In some embodiments, the 5' nucleotide of the oligonucleotide is a
deoxyribonucleotide. In some embodiments, the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5' and 3 ' ends of the deoxyribonucleotides. In some embodiments, the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group or a 3' thiophosphate. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises
phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleotides.
It should be appreciated that the oligonucleotide can have any combination of modifications as described herein.
Oligonucleotide types
In some embodiments, an oligonucleotide described herein may be a mixmer or comprise a mixmer sequence pattern. The term 'mixmer' refers to oligonucleotides which comprise both naturally and non-naturally occurring nucleotides or comprise two different types of non-naturally occurring nucleotides. Mixmers are generally known in the art to have a higher binding affinity than unmodified oligonucleotides and may be used to specifically bind a target molecule, e.g., to block a binding site on the target molecule. Generally, mixmers do not recruit an RNAse to the target molecule and thus do not promote cleavage of the target molecule.
In some embodiments, the mixmer comprises or consists of a repeating pattern of nucleotide analogues and naturally occurring nucleotides, or one type of nucleotide analogue and a second type of nucleotide analogue. However, it is to be understood that the mixmer need not comprise a repeating pattern and may instead comprise any arrangement of nucleotide analogues and naturally occurring nucleotides or any arrangement of one type of nucleotide analogue and a second type of nucleotide analogue. It is to be understood that a pattern, in general, refers to a pattern of modifications or lack thereof, and not to a pattern of A, T, C, G, or U nucleotides. The repeating pattern, may, for instance be every second or every third nucleotide is a nucleotide analogue, such as LNA, and the remaining nucleotides are naturally occurring nucleotides, such as DNA, or are a 2' substituted nucleotide analogue such as 2'-0-methyl, 2'MOE or 2' fluoro analogues, or any other nucleotide analogues described herein. It is recognized that the repeating pattern of nucleotide analogues, such as LNA units, or 2'-0-methyl, 2'MOE or 2' fluoro analogues, may be combined with nucleotide analogues at fixed positions— e.g. at the 5' or 3 ' termini.
In some embodiments, the mixmer does not comprise a region of more than 5, more than 4, more than 3, or more than 2 consecutive naturally occurring nucleotides, such as DNA nucleotides. In some embodiments, the mixmer comprises at least a region consisting of at least two consecutive nucleotide analogues, such as at least two consecutive LNAs. In some embodiments, the mixmer comprises at least a region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNAs.
In some embodiments, the mixmer does not comprise a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleotide analogues, such as LNAs. It is to be understood that the LNA units may be replaced with other nucleotide analogues, such as those referred to herein.
In some embodiments, the mixmer comprises at least one nucleotide analogue in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx and xxxxxX, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occurring nucleotide, such as DNA or RNA.
In some embodiments, the mixmer comprises at least two nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of XXxxxx, XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXXxxx, xXxXxx, xXxxXx, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxX and xxxxXX, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA. In some embodiments, the substitution pattern for the nucleotides may be selected from the group consisting of XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX. In some embodiments, the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX. In some embodiments, the substitution pattern is selected from the group consisting of xXxXxx, xXxxXx and xxXxXx. In some embodiments, the substitution pattern for the nucleotides is xXxXxx.
In some embodiments, the mixmer comprises at least three nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA. In some embodiments, the substitution pattern for the nucleotides is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx. In some embodiments, the substitution pattern for the nucleotides is selected from the group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX. n some embodiments, the substitution pattern for the nucleotides is xXxXxX or XxXxXx. In some embodiments, the substitution pattern for the nucleotides is xXxXxX.
In some embodiments, the mixmer comprises at least four nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of xXXXX, xXxXXX, xXXxXX, xXXXxX, xXXXXx, XxxXXX, XxXxXX, XxXXxX, XxXXXx, XXxxXX, XXxXxX, XXxXXx, XXXxxX, XXXxXx and XXXXxx, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
In some embodiments, the mixmer comprises at least five nucleotide analogues in one or more of six consecutive nucleotides. The substitution pattern for the nucleotides may be selected from the group consisting of xXXXXX, XxXXXX, XXxXXX, XXXxXX,
XXXXxX and XXXXXx, wherein "X" denotes a nucleotide analogue, such as an LNA, and "x" denotes a naturally occuring nucleotide, such as DNA or RNA.
The oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,
(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx, (X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX, (X)xxxXXx, (X)xxxXxX and (X)xxxxXX,
(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx, (X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx
(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,
(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx,
(X)XxxXXXX, (X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx, (X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,
(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx, and (f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, in which "X" denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, and "x" denotes a DNA or RNA nucleotide unit. Each of the above listed patterns may appear one or more times within an oligonucleotide, alone or in combination with any of the other disclosed modification patterns.
In some embodiments, the mixmer contains a modified nucleotide, e.g., an LNA, at the 5' end. In some embodiments, the mixmer contains a modified nucleotide, e.g., an LNA, at the first two positions, counting from the 5' end.
In some embodiments, the mixmer is incapable of recruiting RNAseH.
Oligonucleotides that are incapable of recruiting RNAseH are well known in the literature, in example see WO2007/112754, WO2007/112753, or PCT/DK2008/000344. Mixmers may be designed to comprise a mixture of affinity enhancing nucleotide analogues, such as in non- limiting example LNA nucleotides and 2'-0-methyl nucleotides. In some embodiments, the mixmer comprises modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
In some embodiments, a mixmer is 4 to 40 nucleotides (e.g., 4 to 40, 4 to 35, 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10), in length having the general formula:
wherein each instance of X4 is a modified or unmodified nucleotide described herein (e.g., a modified or unmodified ribonucleotide described herein), wherein each instance of Xs is a deoxyribonucleotide, wherein p and q are independently 0 or 1, reflecting the number of instances of X1 and X2, respectively, wherein at least one of X1 and X2 is present in each instance of the unit, {Xp— Xq wherein r is an integer from 2 to 20 reflecting the number of instances of the unit, {Xp— X^ , linked together through internucleotide linkages, wherein the protecting oligonucleotide or region does not contain a sequence of more than 5 consecutive deoxyribonucleotides, and wherein the symbol "— " denotes an internucleotide linkage.
A mixmer may be produced using any method known in the art or described herein. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. patent publication Nos. US20060128646,
US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. patent No. 7687617.
In some embodiments, the oligonucleotide is a gapmer. In some embodiments, the gapmer has a sequence following the general formula:
wherein each instance of X , X3 is independently a modified or unmodified nucleotide described herein (e.g., a modified or unmodified ribonucleotide described herein), wherein m and o are independently integers in a range of 1 to 10 (e.g., 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 1 to 6, 2 to 6, 3 to 6, or 4 to 6) reflecting the number of instances of X1 and X3, respectively, linked consecutively together through internucleotide linkages, wherein each instance of X2 is a deoxyribonucleotide, wherein n is an integer in a range of 6 to 20 (e.g., 6 to 20, 6 to 15, 6 to 10, 7 to 20, 7 to 15, or 7 to 10), reflecting the number of instances of X2 linked consecutively together through internucleotide linkages. The deoxyribonucleotides of X2 may be substituted with, e.g., C4'- substituted nucleotides, acyclic nucleotides, or arabino-configured nucleotides.
A gapmer oligonucleotide may also have the formula 5'-X-Y-Z-3', with X and Z as flanking regions around a gap region Y. In some embodiments, the Y region is a contiguous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides, which are capable of recruiting an RNAse, such as RNAseH. Without wishing to be bound by theory, it is thought that the gapmer binds to the target nucleic acid, at which point an RNAse is recruited and can then cleave the target nucleic acid. In some embodiments, the Y region is flanked both 5' and 3' by regions X and Z comprising high-affinity modified nucleotides, e.g., 1 - 6 modified nucleotides. Exemplary modified oligonucleotides include, but are not limited to, 2' MOE or 2'OMe or Locked Nucleic Acid bases (LNA). The flanks X and Z may be have a of length 1 - 20 nucleotides, preferably 1-8 nucleotides and even more preferred 1 - 5 nucleotides. The flanks X and Z may be of similar length or of dissimilar lengths. The gap-segment Y may be a nucleotide sequence of length 5 - 20 nucleotides, preferably 6-12 nucleotides and even more preferred 6 - 10 nucleotides. In some aspects, the gap region of the gapmer oligonucleotides of the invention may contain modified nucleotides known to be acceptable for efficient RNase H action in addition to DNA nucleotides, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-configured nucleotides. In some embodiments, the gap region comprises one or more unmodified internucleosides. In some embodiments, one or both flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the gap region and two flanking regions each independently comprise modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
A gapmer may be produced using any method known in the art or described herein. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. patent publication Nos. US20090286969, US20100197762, and US20110112170; and PCT publication Nos.
WO2008049085 and WO2009090182, each of which is herein incorporated by reference in its entirety.
In some embodiments, oligonucleotides provided herein may be in the form of small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA. siRNA, is a class of double- stranded RNA molecules, typically about 20-25 base pairs in length that target nucleic acids (e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway in cells. Specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are generally less than 30 to 35 base pairs in length to prevent the triggering of non-specific RNA interference pathways in the cell via the interferon response, although longer siRNA can also be effective.
Following selection of an appropriate target RNA sequence, siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e. an antisense sequence, can be designed and prepared using any method known in the art (see, e.g., PCT Publication Nos. WO08124927A1 and WO 2004/016735; and U.S. Patent
Publication Nos. 2004/0077574 and 2008/0081791). A number of commercial packages and services are available that are suitable for use for the preparation of siRNA molecules. These include the in vitro transcription kits available from Ambion (Austin, TX) and New England Biolabs (Beverly, MA) as described above; viral siRNA construction kits commercially available from Invitrogen (Carlsbad, CA) and Ambion (Austin, TX), and custom siRNA construction services provided by Ambion (Austin, TX), Qiagen (Valencia, CA), Dharmacon (Lafayette, CO) and Sequitur, Inc (Natick, MA). A target sequence can be selected (and a siRNA sequence designed) using computer software available commercially (e.g.
OligoEngine™ (Seattle, Wash.); Dharmacon, Inc. (Lafayette, Colo.); Target Finder from Ambion Inc. (Austin, Tex.) and the siRNA Design Tool from QIAGEN, Inc. (Valencia, Calif.)). In some embodiments, an siRNA may be designed or obtained using the RNAi atlas (available at the RNAiAtlas website), the siRNA database (available at the Stockholm Bioinformatics Website), or using DesiRM (available at the Institute of Microbial
Technology website).
The siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single- stranded (i.e. a ssRNA molecule comprising just an antisense strand). The siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense strands.
Double- stranded siRNA may comprise RNA strands that are the same length or different lengths. Double-stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single- stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi. Small hairpin RNA (shRNA) molecules thus are also contemplated herein. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3' end and/or the 5' end of either or both strands). A spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double- stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or the 5' end of either or both strands). A spacer sequence is may be an unrelated nucleotide sequence that is situated between two complementary nucleotide sequence regions which, when annealed into a double-stranded nucleic acid, comprise a shRNA.
The overall length of the siRNA molecules can vary from about 14 to about 200 nucleotides depending on the type of siRNA molecule being designed. Generally between about 14 and about 50 of these nucleotides are complementary to the RNA target sequence, i.e. constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double- or single- stranded siRNA, the length can vary from about 14 to about 50 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 40 nucleotides to about 200 nucleotides.
An siRNA molecule may comprise a 3' overhang at one end of the molecule, The other end may be blunt-ended or have also an overhang (5' or 3')· When the siRNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be the same or different. In one embodiment, the siRNA molecule of the present invention comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule.
In some embodiments, an oligonucleotide may be a microRNA (miRNA).
MicroRNAs (referred to as "miRNAs") are small non-coding RNAs, belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on a target RNA transcript. Typically, miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre- miRNAs, which fold into imperfect stem- loop structures. These pre-miRNAs typically undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.
As used herein, miRNAs including pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of mature miRNA. In one embodiment, the size range of the miRNA can be from 21 nucleotides to 170 nucleotides, although miRNAs of up to 2000 nucleotides can be utilized. In one embodiment the size range of the miRNA is from 70 to 170 nucleotides in length. In another embodiment, mature miPvNAs of from 21 to 25 nucleotides in length can be used.
In some embodiments, a miRNA is expressed from a vector. In some embodiments, the vector may include a sequence encoding a mature miRNA. In some embodiments, the vector may include a sequence encoding a pre-miRNA such that the pre-miRNA is expressed and processed in a cell into a mature miRNA. In some embodiments, the vector may include a sequence encoding a pri-miRNA. In this embodiment, the primary transcript is first processed to produce the stem-loop precursor miRNA molecule. The stem-loop precursor is then processed to produce the mature microRNA.
In some embodiments, oligonucleotides provided herein may be in the form of aptamers. An "aptamer" is any nucleic acid that binds specifically to a target, such as a small molecule, protein, nucleic acid, cell, tissue or organism. In some embodiments, the aptamer is a DNA aptamer or an RNA aptamer. In some embodiments, a nucleic acid aptamer is a single- stranded DNA or RNA (ssDNA or ssRNA). It is to be understood that a single- stranded nucleic acid aptamer may form helices and/or loop structures. The nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleotides, modified nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, or a combination of thereof.
Selection of nucleic acid aptamers may be accomplished by any suitable method known in the art, including an optimized protocol for in vitro selection, known as SELEX (Systemic Evolution of Ligands by Exponential enrichment). Many factors are important for successful aptamer selection. For example, the target molecule should be stable and easily reproduced for each round of SELEX, because the SELEX process involves multiple rounds of binding, selection, and amplification to enrich the nucleic acid molecules. In addition, the nucleic acids that exhibit specific binding to the target molecule have to be present in the initial library. Thus, it is advantageous to produce a highly diverse nucleic acid pool. Because the starting library is not guaranteed to contain aptamers to the target molecule, the SELEX process for a single target may need to be repeated with different starting libraries.
Exemplary publications and patents describing aptamers and method of producing aptamers include, e.g., Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; 5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCT application WO 99/31275, each incorporated herein by reference.
In some embodiments, oligonucleotides provided herein may be in the form of a ribozyme. A ribozyme (ribonucleic acid enzyme) is a molecule, typically an RNA molecule, that is capable of performing specific biochemical reactions, similar to the action of protein enzymes. Ribozymes are molecules with catalytic activities including the ability to cleave at specific phosphodiester linkages in RNA molecules to which they have hybridized, such as mRNAs, RNA-containing substrates, IncRNAs, and ribozymes, themselves.
Ribozymes may assume one of several physical structures, one of which is called a "hammerhead." A hammerhead ribozyme is composed of a catalytic core containing nine conserved bases, a double- stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA flanking regions the catalytic core. The flanking regions enable the ribozyme to bind to the target RNA specifically by forming double- stranded stems I and III. Cleavage occurs in cis (i.e., cleavage of the same RNA molecule that contains the hammerhead motif) or in trans (cleavage of an RNA substrate other than that containing the ribozyme) next to a specific ribonucleotide triplet by a transesterification reaction from a 3', 5'-phosphate diester to a 2', 3 '-cyclic phosphate diester. Without wishing to be bound by theory, it is believed that this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
Modifications in ribozyme structure have also included the substitution or
replacement of various non-core portions of the molecule with non-nucleotidic molecules. For example, Benseler et al. (J. Am. Chem. Soc. (1993) 115:8483-8484) disclosed hammerhead-like molecules in which two of the base pairs of stem II, and all four of the nucleotides of loop II were replaced with non-nucleoside linkers based on hexaethylene glycol, propanediol, bis(triethylene glycol) phosphate, tris(propanediol)bisphosphate, or bis(propanediol) phosphate. Ma et al. (Biochem. (1993) 32: 1751-1758; Nucleic Acids Res. (1993) 21:2585-2589) replaced the six nucleotide loop of the TAR ribozyme hairpin with non-nucleotidic, ethylene glycol-related linkers. Thomson et al. (Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear, non-nucleotidic linkers of 13, 17, and 19 atoms in length. Ribozyme oligonucleotides can be prepared using well known methods (see, e.g., PCT Publications W09118624; W09413688; WO9201806; and WO 92/07065; and U.S. Patents 5436143 and 5650502) or can be purchased from commercial sources (e.g., US Biochemicals) and, if desired, can incorporate nucleotide analogs to increase the resistance of the oligonucleotide to degradation by nucleases in a cell. The ribozyme may be synthesized in any known manner, e.g., by use of a commercially available synthesizer produced, e.g., by Applied Biosystems, Inc. or Milligen. The ribozyme may also be produced in recombinant vectors by conventional means. See, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current edition). The ribozyme RNA sequences maybe synthesized conventionally, for example, by using RNA polymerases such as T7 or SP6.
In some embodiments, the oligonucleotide is an oligonucleotide mimetic, such as a morpholino -based oligomeric compound, a cyclohexenyl nucleic acid oligonucleotide mimetics, or peptide nucleic acid (PNA) compound.
Producing Candidate Oligonucleotides
In some embodiments, methods are provided for producing candidate
oligonucleotides that are useful for, e.g., inhibiting the interaction of FAS-AS1 with RBM5. Generally, the oligonucleotides are complementary to sequences in a target RNA, e.g., FAS- AS 1.
Typically, the oligonucleotides are designed by determining a region of a target RNA (FAS -AS 1); producing an oligonucleotide that has a region of complementarity that is complementary with a plurality of (e.g., at least 5) contiguous nucleotides of the region of the target RNA; and determining whether administering the oligonucleotide to a cell in which FAS-AS 1 and RBM5 are expressed results in inhibition of the interaction and/or increased levels of soluble Fas.
In some embodiments, methods are provided for obtaining one or more
oligonucleotides for inhibiting the interaction of FAS-AS 1 with RBM5 that further involve producing a plurality of different oligonucleotides, in which each oligonucleotide has a region of complementarity that is complementary with a plurality of (e.g., at least 5) contiguous nucleotides in a target RNA (e.g., FAS-AS1); subjecting each of the different oligonucleotides to an assay that assesses whether delivery of an oligonucleotide to a cell harboring the target gene results in inhibition of the interaction and/or increased levels of soluble Fas in the cell; and obtaining one or more oligonucleotides that inhibit the interaction and/or increase levels of soluble Fas in the assay.
Chimeric Antigen Receptors
Some embodiments of methods and compositions provided herein utilize chimeric antigen receptors (CARs ). In some embodiments, a T cell population described herein (e.g., comprising naive T cells) is transfected with an expression construct encoding a CAR.
CARs have been utilized to engineer T cells to target various antigens, such as tumor antigens. In some embodiments, CARs comprise an extracellular antigen-binding domain (e.g., a single chain variable fragment (scFv) from an antibody), a transmembrane domain (e.g., a transmembrane domain of any one of the following: alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, and CD154) and an intracellular domain comprising one or more signaling or co- stimulatory domains (e.g., one or more signaling domains of the Οϋ3ζ chain, 4-1BB (CD137) and CD28 and/or one or more co-stimulatory domains of 4-1BB, CD28, ICOS, DAP10, OX-40, BTLA, CD27, CD30, GITR, and HVEM). In some embodiments, CARs may further comprise a hinge region such as a IgGl, IgG4, and IgD or CD8 hinge. Exemplary CARs and methods of making such CARs are known in the art (see, e.g., PCT publication numbers WO2014184744A1,
WO2014184143A1, WO2014059173A2 and WO2015179801A1; and Dai et al. (2016) Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy. J Natl Cancer Inst 108 (7): djv439). In some embodiments, the CAR comprises (a) a scFv specific for an antigen (e.g., a tumor antigen), (b) a hinge region (e.g., an Ig hinge region), (c) a transmembrane domain (e.g., a CD3ζ chain, CD4, CD8, ICOS, or CD28 transmembrane domain), (d) a CD3ζ chain signaling domain and optionally (e) one or more co- stimulatory domains selected from ICOS, OX40 (CD134), CD28, 4-1BB (CD137), CD27, and DAP10.
In some embodiments, the CAR is specific for a tumor antigen (e.g., contains a scFv specific for a tumor antigen). Exemplary tumor antigens include CD 19, CD20, CD33, HER2, GD2 ganglioside, CD171, CAIX, a-folate receptor, IL13Ra2 and CEA.
By the term "specifically binds," as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross- species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A," the presence of a molecule containing epitope "A" (or free, unlabeled "A"), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.
In some embodiments, transfection of the T cell with the CAR expression construct occurs before the T cell population is contacted with the oligonucleotide. In some embodiments, transfection occurs after the T cell population is contacted with the
oligonucleotide. In some embodiments, transfection occurs at the same time that the T cell population is contacted with the oligonucleotide. In some embodiments, the T cell population is activated prior to transfection, e.g., by contacting with an activating agent such as an anti-CD3 and/or anti-CD28 antibody optionally immobilized on a solid substrate. In some embodiments, the T cell is activated after transfection, e.g., by contacting with an activating agent such as an anti-CD3 and/or anti-CD28 antibody. In some embodiments, transfection is achieved by lentiviral infection of the T cell population with the expression construct encoding the CAR. The expression construct may comprise the coding sequence of the CAR optionally along with one or more regulatory sequences that drive expression of the coding sequence, e.g., a promoter and/or enhancer sequence. In some embodiments, the expression construct is a lentiviral construct comprising 5' and 3' long terminal repeats (LTRs). Lentiviruses for use in transfecting T cell populations can be produced using any method known in the art or described herein. For example, 293FT cells may be co- transfected with lentiviral helper plasmids and a lentiviral construct comprising the coding sequence of the CAR optionally with regulatory sequences. Virus supernatants can be isolated from the 293T cells and then concentrated, e.g., by ultracentrifugation. The T cells for use in developing a CAR T cells may be obtained using any method known in the art or described herein (see, e.g., PCT publication numbers WO2014184744A1, WO2014184143A1, WO2014059173A2 and WO2015179801A1). For example, T cells can be obtained from a number of sources, including peripheral blood mononuclear cells
(PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue and spleen tissue from a donor subject (e.g., a human donor subject). PBMCs can be obtained, e.g., by Ficoll™ separation from blood obtained from the donor subject. Alternatively, the T cells may be obtained from a T cell line. A specific subpopulation of T cells, such as CD4+ T cells, can be further isolated by positive or negative selection techniques, such as by fluorescent activated cell sorting or magnetic cell sorting.
In some embodiments, a T cell population transfected with a CAR expression construct is administered to a host subject (e.g., a human host subject). In some
embodiments, the subject has cancer and the CAR is specific for a tumor antigen expressed by the cancer in the subject.
Expression vectors
It is to be appreciated that use of expression vectors to deliver oligonucleotides or any other appropriate nucleic acid (e.g., a mRNA encoding a CAR as described herein) is contemplated in any appropriate context. Vectors include, but are not limited to, plasmids, viral vectors, other vehicles derived from viral or bacterial or other sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences for expressing an RNA transcript (e.g., mRNA).
In some embodiments, expression vectors are provided that are engineered to express a chimeric antigen receptor (CAR) as described herein. In some embodiments, an expression vector may be engineered by incorporating a coding sequence for a gene of interest (e.g., a CAR) into a plasmid that is suitably configured with expression elements (e.g., a promoter) for expressing the gene of interest. In some embodiments, cDNA may be obtained or synthesized using a commercially available kit or any method known in the art.
In some embodiments, a vector may comprise one or more expression elements. "Expression elements" are any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient expression of an RNA transcript (e.g., mRNA). The expression element may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter or a tissue specific promoter, examples of which are well known to one of ordinary skill in the art. Constitutive mammalian promoters include polymerase promoters as well as the promoters for the following non- limiting genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, and beta-actin. Exemplary viral promoters which function constitutively in eukaryotic cells include promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters may be used. Inducible promoters are expressed in the presence of an inducing agent and include metal-inducible promoters and steroid-regulated promoters, for example. Other inducible promoters may be used.
Expression vectors may also comprise an origin of replication, a suitable promoter polyadenylation site, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
One of skill in the art can readily employ other vectors known in the art. Viral vectors are generally based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the nucleic acid sequence of interest. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lines with plasmid, production of recombinant retroviruses by the packaging cell lie, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) may be used. Viral and retroviral vectors that may be used include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as: Moloney murine leukemia virus; Murine stem cell virus, Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses such as any retrovirus. Route of Delivery
The compositions (e.g., T cell populations that have been contacted with an oligonucleotide) described herein can be delivered to a subject by a variety of routes.
Exemplary routes include: intrathecal, intraneural, intracerebral, intramuscular, oral, intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, or ocular. The term "therapeutically effective amount" is the amount of active agent (e.g., oligonucleotide or T cell population) present in the composition that is needed to provide the desired level of sFas expression in the T cell population or to provide a treatment effect in the subject to be treated, e.g., treatment of cancer. The term "physiologically effective amount" is that amount delivered to a subject to give the desired palliative or curative effect. The term "pharmaceutically acceptable carrier" means that the carrier can be administered to a subject with no significant adverse toxicological effects to the subject.
The oligonucleotides or T cell populations of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of oligonucleotide or T cell population and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or
intraventricular administration. The route and site of administration may be chosen to enhance targeting. For example, to target a tumor, intratumoral injection may be desirable.
Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver compositions to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle
(inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy. In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea), and optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
Both the oral and nasal membranes offer advantages over other routes of
administration. For example, oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
A pharmaceutical composition may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration. In some embodiments, parental administration involves administration directly to the site of disease (e.g., tumor).
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred. Pulmonary administration of a micellar oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non- CFC and CFC propellants.
Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
Other devices include non-vascular devices, e.g., devices implanted in the
peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to an oligonucleotide. In one embodiment, unit doses or measured doses of a composition are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.
T cell populations described herein can be treated with an oligonucleotide ex vivo and then administered or implanted in a subject. The T cell population can be autologous, allogeneic, or xenogeneic to the subject. Introduction of treated T cell populations, whether autologous or transplant, can be combined with other therapies.
Dosage
In one aspect, the invention features a method of administering an oligonucleotide to a T cell population (e.g., a human T cell population comprising naive T cells). In some embodiments, 1 to 40 micromolar (e.g., 1 to 20 micromolar) of oligonucleotide is
administered.
In another aspect, the invention features a method of administering a T cell population (e.g., a human T cell population comprising naive T cells that has been contacted with an oligonucleotide herein) to a subject (e.g., a human subject). In one embodiment, the dosage in between 104 to 109 cells/kg body weight. T cell populations may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
For oligonucleotides, the defined amount can be an amount effective to upregulate sFas expression in naive T cells in a T cell population. For T cell populations, the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder that would benefit from adoptive T cell transfer, such as cancer.
In some embodiments, the unit dose is administered or delivered daily. In some embodiments, less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered or delivered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered or delivered a single time. In some embodiments, the unit dose is administered or delivered more than once a day, e.g., once an hour, two hours, four hours, eight hours, twelve hours, etc. Further, the treatment regimen for T cell populations may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. Following treatment, the subject can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage may either be increased in the event the subject does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semipermanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. It will also be appreciated that the effective dosage may increase or decrease over the course of a particular treatment.
Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In one embodiment, the administration of a composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, ocular, intraneuronal, intrathecal, or intracerebral. Administration can be provided by the subject or by another person, e.g., a health care provider.
Kits
In certain aspects of the invention, kits are provided, comprising a container housing a composition comprising an oligonucleotide as described herein. In some embodiments, the composition is a pharmaceutical composition comprising an oligonucleotide and a pharmaceutically acceptable carrier. In some embodiments, the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g., one container for oligonucleotides, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting.
EXAMPLES Example 1 : Gapmers that target FAS-AS 1
Oligonucleotide design
Gapmer oligonucleotides were designed to target FAS-AS 1. The sequence and chemistry of each gapmer oligonucleotide is shown in Table 1. Table 2 provides a description of the nucleotide analogs, modifications and internucleoside linkages used for certain oligonucleotides described in Table 1.
Table 1 : Gapmer oligonucleotides
Base Sequence SEQ ID NO Oligo Chemistry
C AATGTTG G CTG CTT 2 lnaCs;lnaAs;lnaAs;dTs;dGs;dTs;dTs;dG
s;dGs;dCs;dTs;dGs;lnaCs;lnaTs;lnaT
GGCTGCTTTGAAAAT 3 lnaGs;lnaGs;lnaCs;dTs;dGs;dCs;dTs;dT
s;dTs;dGs;dAs;dAs;lnaAs;lnaAs;lnaT
TG AA AATCTC ATAGT 4 lnaTs;lnaGs;lnaAs;dAs;dAs;dAs;dTs;d
Cs;dTs;dCs;dAs;dTs;lnaAs;lnaGs;lnaT
A I AG I 1 1 1 I GGACAG 5 lnaAs;lnaTs;lnaAs;dGs;dTs;dTs;dTs;dT
s;dTs;dGs;dGs;dAs;lnaCs;lnaAs;lnaG
AC AG C AGTATT AGTA 6 lnaAs;lnaCs;lnaAs;dGs;dCs;dAs;dGs;d
Ts;dAs;dTs;dTs;dAs;lnaGs;lnaTs;lnaA
TTAGTAAAGTGTAAG 7 lnaTs;lnaTs;lnaAs;dGs;dTs;dAs;dAs;dA
s;dGs;dTs;dGs;dTs;lnaAs;lnaAs;lnaG
GTGTAAGAAAATTGT 8 lnaGs;lnaTs;lnaGs;dTs;dAs;dAs;dGs;d
As;dAs;dAs;dAs;dTs;lnaTs;lnaGs;lnaT
TGTCAAAGCTTGGAG 9 lnaTs;lnaGs;lnaTs;dCs;dAs;dAs;dAs;d
Gs;dCs;dTs;dTs;dGs;lnaGs;lnaAs;lnaG GCTATGCTTGTTGAA 10 lnaGs;lnaCs;lnaTs;dAs;dTs;dGs;dCs;dT s;dTs;dGs;dTs;dTs;lnaGs;lnaAs;lnaA
GAACTTTTGTACCAA 11 lnaGs;lnaAs;lnaAs;dCs;dTs;dTs;dTs;dT s;dGs;dTs;dAs;dCs;lnaCs;lnaAs;lnaA
ATAGCACCTTTACCA 12 lnaAs;lnaTs;lnaAs;dGs;dCs;dAs;dCs;d
Cs;dTs;dTs;dTs;dAs;;lnaCs;lnaCs;lnaA
TACCAGAGGCTGCGT 13 lnaTs;lnaAs;lnaCs;dCs;dAs;dGs;dAs;d
Gs;dGs;dCs;dTs;dGs;lnaCs;lnaGs;lnaT
TGTTTAAAGCCCGAA 14 lnaTs;lnaGs;lnaTs;dTs;dTs;dAs;dAs;dA s;dGs;dCs;dCs;dCs;lnaGs;lnaAs;lnaA
AAGAAAACTTTGCTT 15 lnaAs;lnaAs;lnaGs;dAs;dAs;dAs;dAs;d
Cs;dTs;dTs;dTs;dGs;lnaCs;lnaTs;lnaT
TTTTCTG G C AGTTCT 16 lnaTs;lnaTs;lnaTs;dTs;dCs;dTs;dGs;dG s;dCs;dAs;dGs;dTs;lnaTs;lnaCs;lnaT
CAGACGTAGGAAATA 17 lnaCs;lnaAs;lnaGs;dAs;dCs;dGs;dTs;d
As;dGs;dGs;dAs;dAs;lnaAs;lnaTs;lnaA
AAGTCAGCACCGAAG 18 lnaAs;lnaAs;lnaGs;dTs;dCs;dAs;dGs;d
Cs;dAs;dCs;dCs;dGs;lnaAs;lnaAs;lnaG
A AG C AGTG GTTA AG C 19 lnaAs;lnaAs;lnaGs;dCs;dAs;dGs;dTs;d
Gs;dGs;dTs;dTs;dAs;lnaAs;lnaGs;lnaC
CGGAGGGCTCGGAAG 20 lnaCs;lnaGs;lnaGs;dAs;dGs;dGs;dGs;d
Cs;dTs;dCs;dGs;dGs;lnaAs;lnaAs;lnaG
AALGGLALL 1 1 1 I U 21 lnaAs;lnaAs;lnaCs;dGs;dGs;dCs;dAs;d
Cs;dCs;dTs;dTs;dTs;lnaTs;lnaCs;lnaT
TTTCTCGAAAAAGTT 22 lnaTs;lnaTs;lnaTs;dCs;dTs;dCs;dGs;dA s;dAs;dAs;dAs;dAs;lnaGs;lnaTs;lnaT
TATATGGGGGCTGAA 23 lnaTs;lnaAs;lnaTs;dAs;dTs;dGs;dGs;d
Gs;dGs;dGs;dCs;dTs;lnaGs;lnaAs;lnaA
ATGAGCTTCTGGAGG 24 lnaA;lnaTs;lnaGs;dAs;dGs;dCs;dTs;dTs
;dCs;dTs;dGs;dGs;lnaAs;lnaGs;lnaG
L M G 1 1 l ALCG I 1 1 1 25 lnaCs;lnaTs;lnaTs;dGs;dTs;dTs;dTs;dA s;dCs;dCs;dGs;dTs;lnaTs;lnaTs;lnaT
TTTATTGTCACACAG 26 lnaTs;lnaTs;lnaTs;dAs;dTs;dTs;dGs;dT s;dCs;dAs;dCs;dAs;lnaCs;lnaAs;lnaG
CACAGAAAAAGAAAC 27 lnaCs;lnaAs;lnaCs;dAs;dGs;dAs;dAs;d
As;dAs;dAs;dGs;dAs;lnaAs;lnaAs;lnaC
CTGCCTTGTCTCCCT 28 lnaCs;lnaTs;lnaGs;dCs;dCs;dTs;dTs;dG s;dTs;dCs;dTs;dCs;lnaCs;lnaCs;lnaT
TCCGGGAATTCTCTC 29 lnaTs;lnaCs;lnaCs;dGs;dGs;dGs;dAs;d
As;dTs;dTs;dCs;dTs;lnaCs;lnaTs;lnaC
CTCTCTTTAAGACTG 30 lnaCs;lnaTs;lnaCs;dTs;dCs;dTs;dTs;dTs
;dAs;dAs;dGs;dAs;lnaCs;lnaTs;lnaG
CTGTA AGTCG CTG CC 31 lnaCs;lnaTs;lnaGs;dTs;dAs;dAs;dGs;dT s;dCs;dGs;dCs;dTs;lnaGs;lnaCs;lnaC
CCTGAGTGGTTTCAT 32 lnaCs;lnaCs;lnaTs;dGs;dAs;dGs;dTs;d
Gs;dGs;dTs;dTs;dTs;lnaCs;lnaAs;lnaT
I GG I 1 I LA I 1 1 I G I 1 33 lnaTs;lnaGs;lnaGs;dTs;dTs;dTs;dCs;dA s;dTs;dTs;dTs;dTs;lnaGs;lnaTs;lnaT 1 I b l 1 1 1 I U GO.U 34 lnaTs;lnaTs;lnaGs;dTs;dTs;dTs;dTs;dTs
;dCs;dTs;dGs;dCs;lnaCs;lnaCs;lnaT
GCCCTTCTCTTTCTT 35 lnaGs;lnaCs;lnaCs;dCs;dTs;dTs;dCs;dT s;dCs;dTs;dTs;dTs;lnaCs;lnaTs;lnaT
I U 1 1 I GO.U 1 I U 36 lnaTs;lnaCs;lnaTs;dTs;dTs;dTs;dGs;dC s;dCs;dCs;dTs;dTs;lnaTs;lnaCs;lnaT
CTTTCTTAGCTTGCA 37 lnaCs;lnaTs;lnaTs;dTs;dCs;dTs;dTs;dA s;dGs;dCs;dTs;dTs;lnaGs;lnaCs;lnaA
TGCACTCCCATGGTG 38 lnaTs;lnaGs;lnaCs;dAs;dCs;dTs;dCs;dC s;dCs;dAs;dTs;dGs;lnaGs;lnaTs;lnaG
GGTGATTTCTGCTTG 39 lnaGs;lnaGs;lnaTs;dGs;dAs;dTs;dTs;dT s;dCs;dTs;dGs;dCs;lnaTs;lnaTs;lnaG
TTG GTCTCCTG CTG G 40 lnaTs;lnaTs;lnaGs;dGs;dTs;dCs;dTs;dC s;dCs;dTs;dGs;dCs;lnaTs;lnaGs;lnaG
GGTTGGTGGTACTCG 41 lnaGs;lnaGs;lnaTs;dTs;dGs;dGs;dTs;d
Gs;dGs;dTs;dAs;dCs;lnaTs;lnaCs;lnaG
CTCGTTCCCACCGCA 42 lnaCs;lnaTs;lnaCs;dGs;dTs;dTs;dCs;dC s;dCs;dAs;dCs;dCs;lnaGs;lnaCs;lnaA
CGCACAGAACCCGGC 43 lnaCs;lnaGs;lnaCs;dAs;dCs;dAs;dGs;d
As;dAs;dCs;dCs;dCs;lnaGs;lnaGs;lnaC
GCGCCTATTATTGGC 44 lnaGs;lnaCs;lnaGs;dCs;dCs;dTs;dAs;dT s;dTs;dAs;dTs;dTs;lnaGs;lnaGs;lnaC
TTATTGGCCAAGAAA 45 lnaTs;lnaTs;lnaAs;dTs;dTs;dGs;dGs;dC s;dCs;dAs;dAs;dGs;lnaAs;lnaAs;lnaA
CTTG AG C AG CCTGTT 46 lnaCs;lnaTs;lnaTs;dGs;dAs;dGs;dCs;d
As;dGs;dCs;dCs;dTs;lnaGs;lnaTs;lnaT
TTTTGAAAAGTCCCT 47 lnaTs;lnaTs;lnaTs;dTs;dGs;dAs;dAs;dA s;dAs;dGs;dTs;dCs;lnaCs;lnaCs;lnaT
CTCGCTCAGAAATGC 48 lnaCs;lnaTs;lnaCs;dGs;dCs;dTs;dCs;dA s;dGs;dAs;dAs;dAs;lnaTs;lnaGs;lnaC
GCCAGCTTGCAGATG 49 lnaGs;lnaCs;lnaCs;dAs;dGs;dCs;dTs;dT s;dGs;dCs;dAs;dGs;lnaAs;lnaTs;lnaG
ATGGCTAATCAAAGA 50 lnaAs;lnaTs;lnaGs;dGs;dCs;dTs;dAs;d
As;dTs;dCs;dAs;dAs;lnaAs;lnaGs;lnaA
AAGAGACGTGGATCC 51 lnaAs;lnaAs;lnaGs;dAs;dGs;dAs;dCs;d
Gs;dTs;dGs;dGs;dAs;lnaTs;lnaCs;lnaC
CAGGAGGCTCATTTG 52 lnaCs;lnaAs;lnaGs;dGs;dAs;dGs;dGs;d
Cs;dTs;dCs;dAs;dTs;lnaTs;lnaTs;lnaG
TTTGAGTACCGGAGC 53 lnaTs;lnaTs;lnaTs;dGs;dAs;dGs;dTs;dA s;dCs;dCs;dGs;dGs;lnaAs;lnaGs;lnaC
CGGAGCCTCTGAGAG 54 lnaCs;lnaGs;lnaGs;dAs;dGs;dCs;dCs;d
Ts;dCs;dTs;dGs;dAs;lnaGs;lnaAs;lnaG
GAG I O.A I GGA I 1 1 1 55 lnaGs;lnaAs;lnaGs;dTs;dCs;dCs;dAs;d
Ts;dGs;dGs;dAs;dTs;lnaTs;lnaTs;lnaT
M A I M I A M GGGO. 56 lnaTs;lnaTs;lnaAs;dTs;dTs;dTs;dTs;dA s;dTs;dTs;dGs;dGs;lnaGs;lnaCs;lnaC
GGCCCCAACAAAGTG 57 lnaGs;lnaGs;lnaCs;dCs;dCs;dCs;dAs;d
As;dCs;dAs;dAs;dAs;lnaGs;lnaTs;lnaG CCAGGCATAGCGAGA 58 lnaCs;lnaCs;lnaAs;dGs;dGs;dCs;dAs;d
Ts;dAs;dGs;dCs;dGs;lnaAs;lnaGs;lnaA
GAGAAGTGTTTACAA 59 lnaGs;lnaAs;lnaGs;dAs;dAs;dGs;dTs;d
Gs;dTs;dTs;dTs;dAs;lnaCs;lnaAs;lnaA
CAAATGTGATCTCCG 60 lnaCs;lnaAs;lnaAs;dAs;dTs;dGs;dTs;d
Gs;dAs;dTs;dCs;dTs;lnaCs;lnaCs;lnaG
GCGGATTCTCACGAG 61 lnaGs;lnaCs;lnaGs;dGs;dAs;dTs;dTs;d
Cs;dTs;dCs;dAs;dCs;lnaGs;lnaAs;lnaG
GAGAGCCATGTAGTG 62 lnaGs;lnaAs;lnaGs;dAs;dGs;dCs;dCs;d
As;dTs;dGs;dTs;dAs;lnaGs;lnaTs;lnaG
AGTGGGGAAGATAAC 63 lnaAs;lnaGs;lnaTs;dGs;dGs;dGs;dGs;d
As;dAs;dGs;dAs;dTs;lnaAs;lnaAs;lnaC
AACCACATCCCCATT 64 lnaAs;lnaAs;lnaCs;dCs;dAs;dCs;dAs;dT s;dCs;dCs;dCs;dCs;lnaAs;lnaTs;lnaT
ATTTTACAGATGGCG 65 lnaAs;lnaTs;lnaTs;dTs;dTs;dAs;dCs;dA s;dGs;dAs;dTs;dGs;lnaGs;lnaCs;lnaG
AAATGAGGTTCAGAG 66 lnaAs;lnaAs;lnaAs;dTs;dGs;dAs;dGs;d
Gs;dTs;dTs;dCs;dAs;lnaGs;lnaAs;lnaG
AGCTTCAGTAATTCA 67 lnaAs;lnaGs;lnaCs;dTs;dTs;dCs;dAs;d
Gs;dTs;dAs;dAs;dTs;lnaTs;lnaCs;lnaA
TCAGCCAAGATCACA 68 lnaTs;lnaCs;lnaAs;dGs;dCs;dCs;dAs;d
As;dGs;dAs;dTs;dCs;lnaAs;lnaCs;lnaA
C AC AG CTAGTA AATG 69 lnaCs;lnaAs;lnaCs;dAs;dGs;dCs;dTs;d
As;dGs;dTs;dAs;dAs;lnaAs;lnaTs;lnaG
GGGAGATATGGGATT 70 lnaGs;lnaGs;lnaGs;dAs;dGs;dAs;dTs;d
As;dTs;dGs;dGs;dGs;lnaAs;lnaTs;lnaT
ATTGAACTCAGATCT 71 lnaAs;lnaTs;lnaTs;dGs;dAs;dAs;dCs;dT s;dCs;dAs;dGs;dAs;lnaTs;lnaCs;lnaT
AATTCCAAAACTCAG 72 lnaAs;lnaAs;lnaTs;dTs;dCs;dCs;dAs;dA s;dAs;dAs;dCs;dTs;lnaCs;lnaAs;lnaG
GCTGTTCGCTGATTG 73 lnaGs;lnaCs;lnaTs;dGs;dTs;dTs;dCs;dG s;dCs;dTs;dGs;dAs;lnaTs;lnaTs;lnaG
TG CATCTG ATATCC A 74 lnaTs;lnaGs;lnaCs;dAs;dTs;dCs;dTs;dG s;dAs;dTs;dAs;dTs;lnaCs;lnaCs;lnaA
AGATTACACCAAGAG 75 lnaAs;lnaGs;lnaAs;dTs;dTs;dAs;dCs;d
As;dCs;dCs;dAs;dAs;lnaGs;lnaAs;lnaG
AGGCTGAGATGTCTG 76 lnaAs;lnaGs;lnaGs;dCs;dTs;dGs;dAs;d
Gs;dAs;dTs;dGs;dTs;lnaCs;lnaTs;lnaG
GG I O.A I 1 1 I A I GA I 77 lnaGs;lnaGs;lnaTs;dCs;dCs;dAs;dTs;dT s;dTs;dTs;dAs;dTs;lnaGs;lnaAs;lnaT
TTATGATTAGAAAAA 78 lnaTs;lnaTs;lnaAs;dTs;dGs;dAs;dTs;dT s;dAs;dGs;dAs;dAs;lnaAs;lnaAs;lnaA
AAAAATCCTGTTCCT 79 lnaAs;lnaAs;lnaAs;dAs;dAs;dTs;dCs;d
Cs;dTs;dGs;dTs;dTs;lnaCs;lnaCs;lnaT
CCTTTCAGAAATAGT 80 lnaCs;lnaCs;lnaTs;dTs;dTs;dCs;dAs;dG s;dAs;dAs;dAs;dTs;lnaAs;lnaGs;lnaT
GTGTG C ACTATTTG G 81 lnaGs;lnaCs;lnaGs;dTs;dGs;dCs;dAs;d
Cs;dTs;dAs;dTs;dTs;lnaTs;lnaGs;lnaG TTGGGATTCTTTAGA 82 lnaTs;lnaTs;lnaGs;dGs;dGs;dAs;dTs;dT s;dCs;dTs;dTs;dTs;lnaAs;lnaGs;lnaA
AGAAACTTCCTTGGA 83 lnaAs;lnaGs;lnaAs;dAs;dAs;dCs;dTs;d
Ts;dCs;dCs;dTs;dTs;lnaGs;lnaGs;lnaA
CCTTGGAGAAAATTA 84 lnaCs;lnaCs;lnaTs;dTs;dGs;dGs;dAs;d
Gs;dAs;dAs;dAs;dAs;lnaTs;lnaTs;lnaA
AATGATTCAAGATTG 85 lnaAs;lnaAs;lnaTs;dGs;dAs;dTs;dTs;dC s;dAs;dAs;dGs;dAs;lnaTs;lnaTs;lnaG
AGATTGAGAAAACAT 86 lnaAs;lnaGs;lnaAs;dTs;dTs;dGs;dAs;d
Gs;dAs;dAs;dAs;dAs;lnaCs;lnaAs;lnaT
TTATACAACCTCAGG 87 lnaTs;lnaTs;lnaAs;dTs;dAs;dCs;dAs;dA s;dCs;dCs;dTs;dCs;lnaAs;lnaGs;lnaG
GCCACACTCTTCTCT 88 lnaGs;lnaCs;lnaCs;dAs;dCs;dAs;dCs;dT s;dCs;dTs;dTs;dCs;lnaTs;lnaCs;lnaT
CTCTCTCTTGTTATT 89 lnaCs;lnaTs;lnaCs;dTs;dCs;dTs;dCs;dT s;dTs;dGs;dTs;dTs;lnaAs;lnaTs;lnaT
TA ACC ATTG C ACC AC 90 lnaTs;lnaAs;lnaAs;dCs;dCs;dAs;dTs;dT s;dGs;dCs;dAs;dCs;lnaCs;lnaAs;lnaC
TG C A ATGTTG G CTG C 91 lnaTs;lnaGs;lnaCs;dAs;dAs;dTs;dGs;dT s;dTs;dGs;dGs;dCs;lnaTs;lnaGs;lnaC
GCTTTGAAAATCTCA 92 lnaGs;lnaCs;lnaTs;dTs;dTs;dGs;dAs;dA s;dAs;dAs;dTs;dCs;lnaTs;lnaCs;lnaA
LA I AG 1 1 1 1 I GACA 93 lnaCs;lnaAs;lnaTs;dAs;dGs;dTs;dTs;dT s;dTs;dTs;dGs;dGs;lnaAs;lnaCs;lnaA
C AG C AGT ATTAGT AA 94 lnaCs;lnaAs;lnaGs;dCs;dAs;dGs;dTs;d
As;dTs;dTs;dAs;dGs;lnaTs;lnaAs;lnaA
AGTGTAAGAAAATTG 95 lnaAs;lnaGs;lnaTs;dGs;dTs;dAs;dAs;d
Gs;dAs;dAs;dAs;dAs;lnaTs;lnaTs;lnaG
TGTCAAAGCTTGGAG 96 lnaTs;lnaGs;lnaTs;dCs;dAs;dAs;dAs;d
Gs;dCs;dTs;dTs;dGs;lnaGs;lnaAs;lnaG
CTATGCTTGTTGAAC 97 lnaCs;lnaTs;lnaAs;dTs;dGs;dCs;dTs;dT s;dGs;dTs;dTs;dGs;lnaAs;lnaAs;lnaC
GAACTTTTGTACCAA 98 lnaGs;lnaAs;lnaAs;dCs;dTs;dTs;dTs;dT s;dGs;dTs;dAs;dCs;lnaCs;lnaAs;lnaA
ATAGCACCTTTACCA 99 lnaAs;lnaTs;lnaAs;dGs;dCs;dAs;dCs;d
Cs;dTs;dTs;dTs;dAs;lnaCs;lnaCs;lnaA
AGAGGCTGCGTGTTT 100 lnaAs;lnaGs;lnaAs;dGs;dGs;dCs;dTs;d
Gs;dCs;dGs;dTs;dGs;lnaTs;lnaTs;lnaT
TTAAAGCCCGAAGAA 101 lnaTs;lnaTs;lnaAs;dAs;dAs;dGs;dCs;dC s;dCs;dGs;dAs;dAs;lnaGs;lnaAs;lnaA
A ACTTTG CTTTTCTG 102 lnaAs;lnaAs;lnaCs;dTs;dTs;dTs;dGs;dC s;dTs;dTs;dTs;dTs;lnaCs;lnaTs;lnaG
GAAAGAATACACACA 103 lnaGs;lnaAs;lnaAs;dAs;dGs;dAs;dAs;d
Ts;dAs;dCs;dAs;dCs;lnaAs;lnaCs;lnaA
ACACACACGCATATG 104 lnaAs;lnaCs;lnaAs;dCs;dAs;dCs;dAs;d
Cs;dGs;dCs;dAs;dTs;lnaAs;lnaTs;lnaG
GTAAATATTCATACA 105 lnaGs;lnaTs;lnaAs;dAs;dAs;dTs;dAs;dT s;dTs;dCs;dAs;dTs;lnaAs;lnaCs;lnaA CATTTATGTATATAT 106 lnaCs;lnaAs;lnaTs;dTs;dTs;dAs;dTs;dG s;dTs;dAs;dTs;dAs;lnaTs;lnaAs;lnaT
ACATATTATAATACC 107 lnaAs;lnaCs;lnaAs;dTs;dAs;dTs;dTs;dA s;dTs;dAs;dAs;dTs;lnaAs;lnaCs;lnaC
CTATA AGTTAG GTAT 108 lnaCs;lnaTs;lnaAs;dTs;dAs;dAs;dGs;dT s;dTs;dAs;dGs;dGs;lnaTs;lnaAs;lnaT
AACTTATATTTGTAT 109 lnaAs;lnaAs;lnaCs;dTs;dTs;dAs;dTs;dA s;dTs;dTs;dTs;dGs;lnaTs;lnaAs;lnaT
ATGATATATGGCCTA 110 lnaAs;lnaTs;lnaGs;dAs;dTs;dAs;dTs;dA s;dTs;dGs;dGs;dCs;lnaCs;lnaTs;lnaA
CTAGGAAATTAAGGC 111 lnaCs;lnaTs;lnaAs;dGs;dGs;dAs;dAs;d
As;dTs;dTs;dAs;dAs;lnaGs;lnaGs;lnaC
G G CTT ATT A A AT A A A 112 lnaGs;lnaGs;lnaCs;dTs;dTs;dAs;dTs;dT s;dAs;dAs;dAs;dTs;lnaAs;lnaAs;lnaAs
ATTT AT A A ATG C AG A 113 lnaAs;lnaTs;lnaTs;dTs;dAs;dTs;dAs;dA s;dAs;dTs;dGs;dCs;lnaAs;lnaGs;lnaA
GATGAGTCAAATACA 114 lnaGs;lnaAs;lnaTs;dGs;dAs;dGs;dTs;d
Cs;dAs;dAs;dAs;dTs;lnaAs;lnaCs;lnaA
ACAAAGATCAGACAT 115 lnaAs;lnaCs;lnaAs;dAs;dAs;dGs;dAs;d
Ts;dCs;dAs;dGs;dAs;lnaCs;lnaAs;lnaT
CATAACTCTATCACC 116 lnaCs;lnaAs;lnaTs;dAs;dAs;dCs;dTs;dC s;dTs;dAs;dTs;dCs;lnaAs;lnaCs;lnaC
TCACCTAAGTAATCA 117 lnaTs;lnaCs;lnaAs;dCs;dCs;dTs;dAs;dA s;dGs;dTs;dAs;dAs;lnaTs;lnaCs;lnaA
1 1 G 1 1 1 AC 1 1 1 1 C 118 lnaTs;lnaTs;lnaGs;dTs;dTs;dTs;dAs;dC s;dGs;dTs;dTs;dTs;lnaTs;lnaGs;lnaC
TGCAGTTTATCTTCC 119 lnaTs;lnaGs;lnaCs;dAs;dGs;dTs;dTs;dT s;dAs;dTs;dCs;dTs;lnaTs;lnaCs;lnaC
CCATTTCTCCCCTCT 120 lnaCs;lnaCs;lnaAs;dTs;dTs;dTs;dCs;dT s;dCs;dCs;dCs;dCs;lnaTs;lnaCs;lnaT
CCCTCTAATATGACT 121 lnaCs;lnaCs;lnaCs;dTs;dCs;dTs;dAs;dA s;dTs;dAs;dTs;dGs;lnaAs;lnaCs;lnaT
I GAU U 1 1 I AAA I 1 122 lnaTs;lnaGs;lnaAs;dCs;dTs;dCs;dTs;dT s;dTs;dTs;dAs;dAs;lnaAs;lnaTs;lnaT
TTAGTTACACAGAAA 123 lnaTs;lnaTs;lnaAs;dGs;dTs;dTs;dAs;dC s;dAs;dCs;dAs;dGs;lnaAs;lnaAs;lnaA
1 I GG I 1 1 I CGU CA 124 lnaTs;lnaTs;lnaGs;dGs;dTs;dTs;dTs;dT s;dGs;dCs;dGs;dCs;lnaTs;lnaCs;lnaA
ACGACATGCCTAACA 125 lnaAs;lnaCs;lnaGs;dAs;dCs;dAs;dTs;d
Gs;dCs;dCs;dTs;dAs;lnaAs;lnaCs;lnaA
CATCAGGCCTTTATC 126 lnaCs;lnaAs;lnaTs;dCs;dAs;dG;dGs;dC s;dCs;dTs;dTs;dTs;lnaAs;lnaTs;lnaC
CCTTAAAGAAGTTTC 127 lnaCs;lnaCs;lnaTs;dTs;dAs;dAs;dAs;dG s;dAs;dAs;dGs;dTs;lnaTs;lnaTs;lnaC
AGTTTCAACACTCTT 128 lnaAs;lnaGs;lnaTs;dTs;dTs;dCs;dAs;dA s;dCs;dAs;dCs;dTs;lnaCs;lnaTs;lnaT
TTGTGTCACCTCATG 129 lnaTs;lnaTs;lnaGs;dTs;dGs;dTs;dCs;dA s;dCs;dCs;dTs;dCs;lnaAs;lnaTs;lnaG GTGTCTACAGAAAAT 130 lnaGs;lnaTs;lnaGs;dTs;dCs;dTs;dAs;dC
s;dAs;dGs;dAs;dAs;lnaAs;lnaAs;lnaT
I G I 1 I GU 1 1 I A I GC 131 lnaTs;lnaGs;lnaTs;dTs;dTs;dGs;dCs;dT
s;dTs;dTs;dTs;dAs;lnaTs;lnaGs;lnaC
CTTC ACG GTTATGTT 132 lnaCs;lnaTs;lnaTs;dCs;dAs;dCs;dGs;dG
s;dTs;dTs;dAs;dTs;lnaGs;lnaTs;lnaT
TTCTTAGTAGTAGCA 133 lnaTs;lnaTs;lnaCs;dTs;dTs;dAs;dGs;dT
s;dAs;dGs;dTs;dAs;lnaGs;lnaCs;lnaA
GT AGT AG C AATA AAT 134 lnaGs;lnaTs;lnaAs;dGs;dTs;dAs;dGs;d
Cs;dAs;dAs;dTs;dAs;lnaAs;lnaAs;lnaT
AAAATAGATGCAAAG 135 lnaAs;lnaAs;lnaAs;dAs;dTs;dAs;dGs;d
As;dTs;dGs;dCs;dAs;lnaAs;lnaAs;lnaG
C AA AGTG CTA ATTAC 136 lnaCs;lnaAs;lnaAs;dAs;dGs;dTs;dGs;d
Cs;dTs;dAs;dAs;dTs;lnaTs;lnaAs;lnaC
GCTAATTACTTGGAA 137 lnaGs;lnaCs;lnaTs;dAs;dAs;dTs;dTs;dA
s;dCs;dTs;dTs;dGs;lnaGs;lnaAs;lnaA
Table 2: A listing of nucleotides and nucleotide modifications
Figure imgf000054_0001
Further gapmer oligonucleotides are provided in Table 3. The gapmer
oligonucleotides in Table 3 each have the structural motif of
lnaNs;lnaNs;lnaNs;dNs;dNs;dNs;dNs;dNs;dNs;dNs;dNs;dNs;lnaNs;lnaNs;lnaN, where InaNs are LNA nucleotides and dNs are DNA nucleotides and the nucleotides are all linked by phosphorothioate linkages as indicated by the "s" after each nucleotide. InaCs are 5- methylcytosine LNAs. Table 3: Further gapmer oligonucleotides
GAPMER BASE SEQUENCE SEQ ID NO
TTTTCTG G C AGTTCT 138
TTTCTG G C AGTTCTC 139
TTCTGGCAGTTCTCA 140
TCTG G C AGTTCTC AG 141
CTGGCAGTTCTCAGA 142
TGGCAGTTCTCAGAC 143
GGCAGTTCTCAGACG 144
GCAGTTCTCAGACGT 145
CAGTTCTCAGACGTA 146
AGTTCTCAGACGTAG 147
GTTCTCAGACGTAGG 148
TTCTCAGACGTAGGA 149
TCTCAGACGTAGGAA 150
CTCAGACGTAGGAAA 151
TCAGACGTAGGAAAT 152
CAGACGTAGGAAATA 153
AGACGTAGGAAATAA 154
GACGTAGGAAATAAG 155
ACGTAGGAAATAAGT 156
CGTAGGAAATAAGTC 157
GTAGGAAATAAGTCA 158
TAGGAAATAAGTCAG 159
AGGAAATAAGTCAGC 160
GGAAATAAGTCAGCA 161
GAAATAAGTCAGCAC 162
A AATA AGTC AG C ACC 163
AATAAGTCAGCACCG 164
ATAAGTCAGCACCGA 165
TAAGTCAGCACCGAA 166
AAGTCAGCACCGAAG 167
AGTCAGCACCGAAGC 168
GTCAGCACCGAAGCA 169
TCAGCACCGAAGCAG 170
CAGCACCGAAGCAGT 171
AGCACCGAAGCAGTG 172
GCACCGAAGCAGTGG 173
CACCGAAGCAGTGGT 174
ACCGAAGCAGTGGTT 175
CCGAAGCAGTGGTTA 176 CGAAGCAGTGGTTAA 177
GAAGCAGTGGTTAAG 178
A AG C AGTG GTTA AG C 179
AG C AGTG GTT AAG CC 180
GCAGTGGTTAAGCCG 181
CAGTGGTTAAGCCGG 182
AGTGGTTAAGCCGGA 183
GTGGTTAAGCCGGAG 184
TGGTTAAGCCGGAGG 185
GGTTAAGCCGGAGGG 186
GTTAAGCCGGAGGGC 187
TTAAGCCGGAGGGCT 188
TAAGCCGGAGGGCTC 189
AAGCCGGAGGGCTCG 190
AGCCGGAGGGCTCGG 191
GCCGGAGGGCTCGGA 192
CCGGAGGGCTCGGAA 193
CGGAGGGCTCGGAAG 194
GGAGGGCTCGGAAGA 195
GAGGGCTCGGAAGAA 196
AGGGCTCGGAAGAAC 197
GGGCTCGGAAGAACG 198
GGCTCGGAAGAACGG 199
GCTCGGAAGAACGGC 200
CTCGGAAGAACGGCA 201
TCGGAAGAACGGCAC 202
CGGAAGAACGGCACC 203
GGAAGAACGGCACCT 204
GAAGAACGGCACCTT 205
AAGAACGGCACCTTT 206
AGAACGGCACU 111 207
GAACGGCACC 1111 C 208
AACGGCACU 11 IU 209
ACGGCACCTTTTCTT 210
CGGCACCI 11 IU 11 211
GGCACU 11 ICI 1 IC 212
GCACU 11 ICI 1 ICI 213
CACCI 11 ICI 1 ICIC 214
ACCTTTTCTTTCTCG 215
CCI 11 ICI 1 ICICGA 216
CTTTTCTTTCTCGAA 217
TTTTCTTTCTCGAAA 218
TTTCTTTCTCGAAAA 219 TTCTTTCTCGAAAAA 220
TCTTTCTCGAAAAAG 221
CTTTCTCGAAAAAGT 222
TTTCTCGAAAAAGTT 223
TTCTCGAAAAAGTTA 224
TCTCGAAAAAGTTAT 225
CTCG AA AA AGTTATA 226
TCGAAAAAGTTATAT 227
CGAAAAAGTTATATG 228
GAAAAAGTTATATGG 229
A AA AAGTT AT ATG G G 230
A AA AGTTATATG G G G 231
A AAGTT AT ATG G G G G 232
A AGTTATATG G G G G C 233
AGTTATATG G G G G CT 234
GTTATATG G G G G CTG 235
TTATATGGGGGCTGA 236
TATATGGGGGCTGAA 237
ATATGGGGGCTGAAT 238
TATGGGGGCTGAATG 239
ATGGGGGCTGAATGA 240
TGGGGGCTGAATGAG 241
GGGGGCTGAATGAGC 242
GGGGCTGAATGAGCT 243
GGGCTGAATGAGCTT 244
GGCTGAATGAGCTTC 245
GCTGAATGAGCTTCT 246
CTGAATGAGCTTCTG 247
TGAATGAGCTTCTGG 248
GAATGAGCTTCTGGA 249
AATGAGCTTCTGGAG 250
ATGAGCTTCTGGAGG 251
TGAGCTTCTGGAGGC 252
GAGCTTCTGGAGGCT 253
AGCTTCTGGAGGCTT 254
GCTTCTGGAGGCTTG 255
CTTCTGGAGGCTTGT 256
TTCTGGAGGCTTGTT 257
TCTGGAGGCTTGTTT 258
CTGGAGGCTTGTTTA 259
TGGAGGCTTGTTTAC 260
GGAGGCTTGTTTACC 261
GAGGCTTGTTTACCG 262 AG G CTTGTTT ACCGT 263
GGCTTGTTTACCGTT 264
GCTTGTTTACCGTTT 265
CI IGI 1 IALLGI 111 266
11 G 111 ALLG 11111 267
1 111 ALLG 111111 268
Gl 1 IALLGI M I MA 269
11 IALLGI 1111 IAI 270
1 IALLGI 1111 IAI 1 271
IALLGI 1111 IAI IG 272
ACCGTTTTTTATTGT 273
LLGI 1111 IAI IC_.IL 274
LGI 1111 IAI IGILA 275
Gl 1111 IAI IGILAL 276
11111 IAI IGILALA 277
1111 IAI IGILALAL 278
TTTTATTGTCACACA 279
TTTATTGTCACACAG 280
TTATTGTCACACAGA 281
TATTGTCACACAGAA 282
ATTGTCACACAGAAA 283
TTGTCACACAGAAAA 284
TGTCACACAGAAAAA 285
GTCACACAGAAAAAG 286
TCACACAGAAAAAGA 287
CACACAGAAAAAGAA 288
ACACAGAAAAAGAAA 289
CACAGAAAAAGAAAC 290
ACAGAAAAAGAAACT 291
CAGAAAAAGAAACTG 292
AGAAAAAGAAACTGC 293
GAAAAAGAAACTGCC 294
AAAAAGAAACTGCCT 295
AAAAGAAACTGCCTT 296
AAAGAAACTGCCTTG 297
AAGAAACTGCCTTGT 298
AGAAACTGCCTTGTC 299
GAAACTGCCTTGTCT 300
A AACTG CCTTGTCTC 301
AACTGCCTTGTCTCC 302
ACTGCCTTGTCTCCC 303
CTGCCTTGTCTCCCT 304
TGCCTTGTCTCCCTT 305 GCCTTGTCTCCCTTC 306
CCTTGTCTCCCTTCC 307
CTTGTCTCCCTTCCG 308
TTGTCTCCCTTCCGG 309
TGTCTCCCTTCCGGG 310
GTCTCCCTTCCGGGA 311
TCTCCCTTCCGGGAA 312
CTCCCTTCCGGGAAT 313
TCCCTTCCGGGAATT 314
CCCTTCCGGGAATTC 315
CCTTCCGGGAATTCT 316
CTTCCGGGAATTCTC 317
TTCCGGGAATTCTCT 318
TCCGGGAATTCTCTC 319
CCGGGAATTCTCTCT 320
CGGGAATTCTCTCTT 321
GGGAATTCTCTCTTT 322
GGAATTCTCTCTTTA 323
GAATTCTCTCTTTAA 324
AATTCTCTCTTTAAG 325
ATTCTCTCTTTAAGA 326
TTCTCTCTTTAAGAC 327
TCTCTCTTTAAGACT 328
CTCTCTTTAAGACTG 329
TCTCTTTAAGACTGT 330
CTCTTTAAGACTGTA 331
TCTTTAAGACTGTAA 332
CTTTAAGACTGTAAG 333
TTTAAGACTGTAAGT 334
TTAAGACTGTAAGTC 335
TAAGACTGTAAGTCG 336
AAGACTGTAAGTCGC 337
AGACTGTAAGTCGCT 338
GACTGTAAGTCGCTG 339
ACTGTAAGTCGCTGC 340
CTGTA AGTCG CTG CC 341
TGTAAGTCGCTGCCT 342
GTAAGTCGCTGCCTG 343
TAAGTCGCTGCCTGA 344
AAGTCGCTGCCTGAG 345
AGTCGCTGCCTGAGT 346
GTCGCTGCCTGAGTG 347
TCGCTGCCTGAGTGG 348 CGCTGCCTGAGTGGT 349
GCTGCCTGAGTGGTT 350
CTGCCTGAGTGGTTT 351
TGCCTGAGTGGTTTC 352
GCCTGAGTGGTTTCA 353
CCTGAGTGGTTTCAT 354
CTGAGTGGTTTCATT 355
TG AGTG GTTTC ATTT 356
GAGIGGI 11 <_ A 1111 357
AGIGGI 1 ICAI 11 lb 358
GIGGI 1 ICAI 11 IGI 359
IGGI 1 ICAI 11 IGI 1 360
G GTTTC ATTTTGTTT 361
Gl 1 ICAI 11 IGI 111 362
11 ICAI 11 IGI 11 IG 363
1 ICAI 11 IGI 11 IGI 364
ICAI 11 IGI 11 IGI 1 365
CAI 11 IGI 11 IGI 11 366
ATTTTGTTTTGTTTT 367
111 IGI 11 IGI 1111 368
11 IGI 11 IGI 111 IC 369
1 IGI 11 IGI 111 ICI 370
IGI 11 IGI 111 ICIG 371
Gl 11 IGI 111 ICIGC 372
111 IGI 111 ICIGCC 373
11 IGI 111 ICIGCCC 374
1 IGI 111 ICIGCCCI 375
IGI 111 ICIGCCCI 1 376
Gl 111 ICIGCCCI IC 377
1111 ICIGCCCI ICI 378
TTTTCTGCCCTTCTC 379
TTTCTGCCCTTCTCT 380
TTCTG CCCTTCTCTT 381
TCTGCCCTTCTCTTT 382
CTGCCCTTCTCTTTC 383
TG CCCTTCTCTTTCT 384
GCCCTTCTCTTTCTT 385
CCCTTCTCTTTCTTC 386
CCTTCTCTTTCTTCT 387
CTTCTCTTTCTTCTT 388
TTCTCTTTCTTCTTT 389
TCTCTTTCTTCTTTT 390
CTCTTTCTTCTTTTG 391
Figure imgf000061_0001
TTTCTG CTTG GTCTC 435
TTCTG CTTG GTCTCC 436
TCTGCTTGGTCTCCT 437
CTG CTTG GTCTCCTG 438
TGCTTGGTCTCCTGC 439
GCTTGGTCTCCTGCT 440
CTTGGTCTCCTGCTG 441
TTG GTCTCCTG CTG G 442
TGGTCTCCTGCTGGG 443
GGTCTCCTGCTGGGG 444
GTCTCCTGCTGGGGT 445
TCTCCTG CTG G G GTT 446
CTCCTG CTG G G GTTG 447
TCCTGCTGGGGTTGG 448
CCTGCTGGGGTTGGT 449
CTG CTG G G GTTG GTG 450
TGCTGGGGTTGGTGG 451
GCTGGGGTTGGTGGT 452
CTGGGGTTGGTGGTA 453
TGGGGTTGGTGGTAC 454
GGGGTTGGTGGTACT 455
G G GTTG GTG GT ACTC 456
GGTTGGTGGTACTCG 457
GTTGGTGGTACTCGT 458
TTG GTG GTACTCGTT 459
TGGTGGTACTCGTTC 460
GGTGGTACTCGTTCC 461
GTGGTACTCGTTCCC 462
TGGTACTCGTTCCCA 463
GGTACTCGTTCCCAC 464
GTACTCGTTCCCACC 465
TACTCGTTCCCACCG 466
ACTCGTTCCCACCGC 467
CTCGTTCCCACCGCA 468
TCGTTCCCACCGCAC 469
CGTTCCCACCGCACA 470
GTTCCC ACCG CAC AG 471
TTCCCACCGCACAGA 472
TCCCACCGCACAGAA 473
CCCACCGCACAGAAC 474
CCACCGCACAGAACC 475
CACCGCACAGAACCC 476
ACCGCACAGAACCCG 477 CCGCACAGAACCCGG 478
CGCACAGAACCCGGC 479
GCACAGAACCCGGCG 480
CACAGAACCCGGCGC 481
ACAGAACCCGGCGCC 482
CAGAACCCGGCGCCT 483
AGAACCCGGCGCCTA 484
GAACCCGGCGCCTAT 485
AACCCGGCGCCTATT 486
ACCCGGCGCCTATTA 487
CCCGGCGCCTATTAT 488
CCGGCGCCTATTATT 489
CGGCGCCTATTATTG 490
GGCGCCTATTATTGG 491
GCGCCTATTATTGGC 492
CGCCTATTATTGGCC 493
GCCTATTATTGGCCA 494
CCTATTATTGGCCAA 495
CTATTATTGGCCAAG 496
TATTATTGGCCAAGA 497
ATTATTGGCCAAGAA 498
TTATTGGCCAAGAAA 499
TATTGGCCAAGAAAC 500
ATTGGCCAAGAAACT 501
TTGGCCAAGAAACTT 502
TGGCCAAGAAACTTG 503
GGCCAAGAAACTTGA 504
GCCAAGAAACTTGAG 505
CCAAGAAACTTGAGC 506
CAAGAAACTTGAGCA 507
AAGAAACTTGAGCAG 508
AGAAACTTGAGCAGC 509
GAAACTTGAGCAGCC 510
AAACTTGAGCAGCCT 511
AACTTGAGCAGCCTG 512
ACTTGAGCAGCCTGT 513
CTTGAGCAGCCTGTT 514
TTGAGCAGCCTGTTT 515
I GAGCAGCC I G I 1 1 1 516
GAGCAGCCTGTTTTG 517
AGCAGCC 1 G 1 1 1 1 GA 518
GCAGCC I I 1 1 I AA 519
CAGCU I 1 1 I AAA 520 AGO. 1 b 1 1 1 1 bAAAA 521 bCU b l 1 1 1 bAAAAb 522
CU b l 1 1 I bAAAAb l 523 1 b 1 1 1 1 bAAAAb 1 C 524
1 b 1 1 1 1 bAAAAb 1 525 b 1 1 1 1 bAAAAb 1 LLC 526
TTTTGAAAAGTCCCT 527
TTTGAAAAGTCCCTC 528
TTGAAAAGTCCCTCG 529
TGAAAAGTCCCTCGC 530
GAAAAGTCCCTCGCT 531
AAAAGTCCCTCGCTC 532
AAAGTCCCTCGCTCA 533
AAGTCCCTCGCTCAG 534
AGTCCCTCGCTCAGA 535
GTCCCTCGCTCAGAA 536
TCCCTCGCTCAGAAA 537
CCCTCGCTCAGAAAT 538
CCTCGCTCAGAAATG 539
CTCGCTCAGAAATGC 540
TCGCTCAGAAATGCC 541
CGCTCAGAAATGCCA 542
GCTCAGAAATGCCAG 543
CTCAGAAATGCCAGC 544
TCAGAAATGCCAGCT 545
CAGAAATGCCAGCTT 546
AGAAATGCCAGCTTG 547
GAAATGCCAGCTTGC 548
AAATGCCAGCTTGCA 549
A ATG CC AG CTTG C AG 550
ATGCCAGCTTGCAGA 551
TGCCAGCTTGCAGAT 552
GCCAGCTTGCAGATG 553
CCAGCTTGCAGATGG 554
CAGCTTGCAGATGGC 555
AGCTTGCAGATGGCT 556
GCTTGCAGATGGCTA 557
CTTGCAGATGGCTAA 558
TTGCAGATGGCTAAT 559
TGCAGATGGCTAATC 560
GCAGATGGCTAATCA 561
CAGATGGCTAATCAA 562
AGATGGCTAATCAAA 563 GATGGCTAATCAAAG 564
ATGGCTAATCAAAGA 565
TGGCTAATCAAAGAG 566
GGCTAATCAAAGAGA 567
GCTAATCAAAGAGAC 568
CTAATCAAAGAGACG 569
TAATCAAAGAGACGT 570
AATCAAAGAGACGTG 571
ATCAAAGAGACGTGG 572
TCAAAGAGACGTGGA 573
CAAAGAGACGTGGAT 574
AAAGAGACGTGGATC 575
AAGAGACGTGGATCC 576
AGAGACGTGGATCCA 577
GAGACGTGGATCCAG 578
AGACGTGGATCCAGG 579
GACGTGGATCCAGGA 580
ACGTGGATCCAGGAG 581
CGTGGATCCAGGAGG 582
GTGGATCCAGGAGGC 583
TGGATCCAGGAGGCT 584
GGATCCAGGAGGCTC 585
GATCCAGGAGGCTCA 586
ATCCAGGAGGCTCAT 587
TCCAGGAGGCTCATT 588
CCAGGAGGCTCATTT 589
CAGGAGGCTCATTTG 590
AGGAGGCTCATTTGA 591
GGAGGCTCATTTGAG 592
GAGGCTCATTTGAGT 593
AGGCTCATTTGAGTA 594
GGCTCATTTGAGTAC 595
GCTCATTTGAGTACC 596
CTCATTTGAGTACCG 597
TCATTTGAGTACCGG 598
CATTTGAGTACCGGA 599
ATTTGAGTACCGGAG 600
TTTGAGTACCGGAGC 601
TTGAGTACCGGAGCC 602
TGAGTACCGGAGCCT 603
GAGTACCGGAGCCTC 604
AGTACCGGAGCCTCT 605
GTACCGGAGCCTCTG 606 TACCGGAGCCTCTGA 607
ACCGGAGCCTCTGAG 608
CCGGAGCCTCTGAGA 609
CGGAGCCTCTGAGAG 610
GGAGCCTCTGAGAGT 611
GAGCCTCTGAGAGTC 612
AGCCTCTGAGAGTCC 613
GCCTCTGAGAGTCCA 614
CCTCTGAGAGTCCAT 615
CTCTGAGAGTCCATG 616
TCTGAGAGTCCATGG 617
CTGAGAGTCCATGGA 618
TGAGAGTCCATGGAT 619
GAGAGTCCATGGATT 620
AGAGTCCATGGATTT 621
GAGICCAIGGAI 111 622
AGICCAIGGAI 1111 623
GICCAIGGAI U NA 624
ICCAIGGAI 111 IAI 625
CCAIGGAI 111 IAI 1 626
CAIGGAI 111 IAI 11 627
AIGGAI 111 IAI 111 628
IGGAI 111 IAI 11 IA 629
GGAI 111 IAI 11 IAI 630
GAI 111 IAI 11 IAI 1 631
Al 111 IAI 11 IAI lb 632
1111 IAI 11 IAI IGG 633
TTTTATTTTATTGGG 634
11 IAI 11 IAI IGGGC 635
1 IAI 11 IAI IGGGCC 636
IAI 11 IAI IGGGCCC 637
ATTTTATTGGGCCCC 638
TTTTATTGGGCCCCA 639
TTTATTGGGCCCCAA 640
TTATTGGGCCCCAAC 641
TATTGGGCCCCAACA 642
ATTGGGCCCCAACAA 643
TTGGGCCCCAACAAA 644
TGGGCCCCAACAAAG 645
GGGCCCCAACAAAGT 646
GGCCCCAACAAAGTG 647
GCCCCAACAAAGTGC 648
CCCCAACAAAGTGCC 649 CCCAACAAAGTGCCA 650
CCAACAAAGTGCCAG 651
CAACAAAGTGCCAGG 652
A AC AA AGTG CC AG G C 653
ACAAAGTGCCAGGCA 654
C AA AGTG CC AG G C AT 655
AAAGTGCCAGGCATA 656
A AGTG CC AG G C ATAG 657
AGTGCCAGGCATAGC 658
GTGCCAGGCATAGCG 659
TGCCAGGCATAGCGA 660
GCCAGGCATAGCGAG 661
CCAGGCATAGCGAGA 662
CAGGCATAGCGAGAG 663
AGGCATAGCGAGAGA 664
GGCATAGCGAGAGAA 665
GCATAGCGAGAGAAG 666
CATAGCGAGAGAAGT 667
ATAGCGAGAGAAGTG 668
TAGCGAGAGAAGTGT 669
AGCGAGAGAAGTGTT 670
GCGAGAGAAGTGTTT 671
CGAGAGAAGTGTTTA 672
GAGAGAAGTGTTTAC 673
AGAGAAGTGTTTACA 674
GAGAAGTGTTTACAA 675
AGAAGTGTTTACAAA 676
GAAGTGTTTACAAAT 677
AAGTGTTTACAAATG 678
AGTGTTTACAAATGT 679
GTGTTTACAAATGTG 680
TGTTTACAAATGTGA 681
GTTTACAAATGTGAT 682
TTTACAAATGTGATC 683
TTACAAATGTGATCT 684
TACAAATGTGATCTC 685
ACAAATGTGATCTCC 686
CAAATGTGATCTCCG 687
AAATGTGATCTCCGC 688
AATGTGATCTCCGCG 689
ATGTGATCTCCGCGG 690
TGTGATCTCCGCGGA 691
GTGATCTCCGCGGAT 692 TGATCTCCGCGGATT 693
GATCTCCGCGGATTC 694
ATCTCCGCGGATTCT 695
TCTCCGCGGATTCTC 696
CTCCGCGGATTCTCA 697
TCCGCGGATTCTCAC 698
CCGCGGATTCTCACG 699
CGCGGATTCTCACGA 700
GCGGATTCTCACGAG 701
CGGATTCTCACGAGA 702
GGATTCTCACGAGAG 703
GATTCTCACGAGAGC 704
ATTCTCACGAGAGCC 705
TTCTCACGAGAGCCA 706
TCTCACGAGAGCCAT 707
CTCACGAGAGCCATG 708
TCACGAGAGCCATGT 709
CACGAGAGCCATGTA 710
ACGAGAGCCATGTAG 711
CGAGAGCCATGTAGT 712
GAGAGCCATGTAGTG 713
AGAGCCATGTAGTGG 714
GAGCCATGTAGTGGG 715
AGCCATGTAGTGGGG 716
GCCATGTAGTGGGGA 717
CCATGTAGTGGGGAA 718
CATGTAGTGGGGAAG 719
ATGTAGTGGGGAAGA 720
TGTAGTGGGGAAGAT 721
GTAGTGGGGAAGATA 722
TAGTGGGGAAGATAA 723
AGTGGGGAAGATAAC 724
GTGGGGAAGATAACC 725
TGGGGAAGATAACCA 726
GGGGAAGATAACCAC 727
GGGAAGATAACCACA 728
GGAAGATAACCACAT 729
GAAGATAACCACATC 730
AAGATAACCACATCC 731
AGATAACCACATCCC 732
GATAACCACATCCCC 733
ATAACCACATCCCCA 734
TAACCACATCCCCAT 735 AACCACATCCCCATT 736
ACCACATCCCCATTT 737
CCACAI CCCAI 111 738
CACAI CCCAI I MA 739
ACAI CCCAI 111 AC 740
CAI CCCAI 11 IACA 741
AI CCCAI 11 IACAG 742
I CCCAI 11 IACAG A 743
CCCCAI 11 IACA AI 744
CCCATTTTACAGATG 745
CCAI 11 IACAGAI G 746
LAI 11 IACAGAI GC 747
ATTTTACAGATGGCG 748
TTTTACAGATGGCGA 749
TTTACAGATGGCGAA 750
TTACAGATGGCGAAA 751
TACAGATGGCGAAAT 752
ACAGATGGCGAAATG 753
CAGATGGCGAAATGA 754
AGATGGCGAAATGAG 755
GATGGCGAAATGAGG 756
ATGGCGAAATGAGGT 757
TGGCGAAATGAGGTT 758
GGCGAAATGAGGTTC 759
GCGAAATGAGGTTCA 760
CGAAATGAGGTTCAG 761
GAAATGAGGTTCAGA 762
AAATGAGGTTCAGAG 763
AATGAGGTTCAGAGA 764
ATGAGGTTCAGAGAG 765
TGAGGTTCAGAGAGC 766
GAGGTTCAGAGAGCT 767
AGGTTCAGAGAGCTT 768
GGTTCAGAGAGCTTC 769
GTTCAGAGAGCTTCA 770
TTCAGAGAGCTTCAG 771
TCAGAGAGCTTCAGT 772
CAGAGAGCTTCAGTA 773
AGAGAGCTTCAGTAA 774
GAGAGCTTCAGTAAT 775
AGAGCTTCAGTAATT 776
GAGCTTCAGTAATTC 777
AGCTTCAGTAATTCA 778 G CTTC AGTA ATTC AG 779
CTTC AGTA ATTC AG C 780
TTCAGTAATTCAGCC 781
TC AGTA ATTC AG CCA 782
C AGT AATTC AG CCA A 783
AGTAATTCAGCCAAG 784
GTAATTCAGCCAAGA 785
TAATTCAGCCAAGAT 786
AATTCAGCCAAGATC 787
ATTCAGCCAAGATCA 788
TTCAGCCAAGATCAC 789
TCAGCCAAGATCACA 790
CAGCCAAGATCACAC 791
AGCCAAGATCACACA 792
GCCAAGATCACACAG 793
CCAAGATCACACAGC 794
CAAGATCACACAGCT 795
AAGATCACACAGCTA 796
AGATCACACAGCTAG 797
GATCACACAGCTAGT 798
ATC AC AC AG CTAGT A 799
TC AC AC AG CT AGTA A 800
C AC AC AG CTAGTA AA 801
AC AC AG CT AGT AA AT 802
C AC AG CTAGTA AATG 803
ACAGCTAGTAAATGG 804
C AG CTAGTA AATG G G 805
AGCTAGTAAATGGGA 806
GCTAGTAAATGGGAG 807
CTAGTAAATGGGAGA 808
TAGTAAATGGGAGAT 809
AGTAAATGGGAGATA 810
GTAAATGGGAGATAT 811
TAAATGGGAGATATG 812
AAATGGGAGATATGG 813
AATGGGAGATATGGG 814
ATGGGAGATATGGGA 815
TGGGAGATATGGGAT 816
GGGAGATATGGGATT 817
GGAGATATGGGATTG 818
GAGATATGGGATTGA 819
AGATATGGGATTGAA 820
GATATGGGATTGAAC 821 ATATGGGATTGAACT 822
TATGGGATTGAACTC 823
ATGGGATTGAACTCA 824
TGGGATTGAACTCAG 825
GGGATTGAACTCAGA 826
GGATTGAACTCAGAT 827
GATTGAACTCAGATC 828
ATTGAACTCAGATCT 829
TTGAACTCAGATCTA 830
TGAACTCAGATCTAA 831
GAACTCAGATCTAAT 832
AACTCAGATCTAATT 833
ACTCAGATCTAATTC 834
CTCAGATCTAATTCC 835
TCAGATCTAATTCCA 836
CAGATCTAATTCCAA 837
AGATCTAATTCCAAA 838
GATCTAATTCCAAAA 839
ATCTAATTCCAAAAC 840
TCTAATTCCAAAACT 841
CTAATTCCAAAACTC 842
TAATTCCAAAACTCA 843
AATTCCAAAACTCAG 844
ATTCCAAAACTCAGG 845
TTCCAAAACTCAGGC 846
TCCAAAACTCAGGCT 847
CCAAAACTCAGGCTG 848
C AA AACTC AG G CTGT 849
A AA ACTC AG G CTGTT 850
A AACTC AG G CTGTTC 851
A ACTC AG G CTGTTCG 852
ACTCAGGCTGTTCGC 853
CTCAGGCTGTTCGCT 854
TCAGG CTGTTCG CTG 855
CAGGCTGTTCGCTGA 856
AGGCTGTTCGCTGAT 857
GGCTGTTCGCTGATT 858
GCTGTTCGCTGATTG 859
CTGTTCGCTGATTGC 860
TGTTCGCTGATTGCA 861
GTTCGCTGATTGCAT 862
TTCGCTGATTGCATC 863
TCGCTGATTGCATCT 864 CGCTGATTGCATCTG 865
GCTGATTGCATCTGA 866
CTGATTGCATCTGAT 867
TGATTGCATCTGATA 868
GATTGCATCTGATAT 869
ATTGCATCTGATATC 870
TTGCATCTGATATCC 871
TG CATCTG ATATCC A 872
GCATCTGATATCCAG 873
CATCTGATATCCAGA 874
ATCTGATATCCAGAT 875
TCTGATATCCAGATT 876
CTGATATCCAGATTA 877
TGATATCCAGATTAC 878
GATATCCAGATTACA 879
ATATCCAGATTACAC 880
TATCCAGATTACACC 881
ATCCAGATTACACCA 882
TCCAGATTACACCAA 883
CCAGATTACACCAAG 884
CAGATTACACCAAGA 885
AGATTACACCAAGAG 886
GATTACACCAAGAGG 887
ATTACACCAAGAGGC 888
TTACACCAAGAGGCT 889
TACACCAAGAGGCTG 890
ACACCAAGAGGCTGA 891
CACCAAGAGGCTGAG 892
ACCAAGAGGCTGAGA 893
CCAAGAGGCTGAGAT 894
CAAGAGGCTGAGATG 895
AAGAGGCTGAGATGT 896
AGAGGCTGAGATGTC 897
GAGGCTGAGATGTCT 898
AGGCTGAGATGTCTG 899
GGCTGAGATGTCTGG 900
GCTGAGATGTCTGGG 901
CTGAGATGTCTGGGT 902
TGAGATGTCTGGGTC 903
GAGATGTCTGGGTCC 904
AGATGTCTGGGTCCA 905
GATGTCTGGGTCCAT 906
ATGTCTGGGTCCATT 907 TGTCTGGGTCCATTT 908
GIUGGGIO.AI 111 909
IUGGGIO.AI 11 IA 910
UGGGIO.AI 11 IAI 911
TG G GTCC ATTTTATG 912
GGGIO.AI 11 IAIGA 913
GGIO.AI 11 IAIGAI 914
GIO.AI 11 IAIGAI 1 915
IO.AI 11 IAIGAI IA 916
O.AI 11 IAIGAI 1 AG 917
LAI 11 IAIGAI IAGA 918
ATTTTATGATTAGAA 919
TTTTATGATTAGAAA 920
TTTATGATTAGAAAA 921
TTATGATTAGAAAAA 922
TATGATTAGAAAAAA 923
ATGATTAGAAAAAAA 924
TGATTAGAAAAAAAA 925
GATTAGAAAAAAAAA 926
ATTAGAAAAAAAAAT 927
TTAGAAAAAAAAATC 928
TAGAAAAAAAAATCC 929
AGAAAAAAAAATCCT 930
GAAAAAAAAATCCTG 931
AAAAAAAAATCCTGT 932
AAAAAAAATCCTGTT 933
AAAAAAATCCTGTTC 934
AAAAAATCCTGTTCC 935
AAAAATCCTGTTCCT 936
AAAATCCTGTTCCTT 937
AAATCCTGTTCCTTT 938
AATCCTGTTCCTTTC 939
ATCCTGTTCCTTTCA 940
TCCTGTTCCTTTCAG 941
CCTGTTCCTTTCAGA 942
CTGTTCCTTTCAGAA 943
TGTTCCTTTCAGAAA 944
GTTCCTTTCAGAAAT 945
TTCCTTTCAGAAATA 946
TCCTTTCAGAAATAG 947
CCTTTCAGAAATAGT 948
CTTTCAGAAATAGTG 949
TTTCAGAAATAGTGT 950 TTCAGAAATAGTGTG 951
TCAGAAATAGTGTGC 952
CAGAAATAGTGTGCA 953
AGAAATAGTGTGCAC 954
GAAATAGTGTGCACT 955
A AATAGTGTG C ACTA 956
A AT AGTGTG C ACT AT 957
ATAGTGTGCACTATT 958
TAGTGTG C ACTATTT 959
AGTGTG C ACTATTTG 960
GTGTG C ACTATTTG G 961
TGTGCACTATTTGGG 962
GTGCACTATTTGGGA 963
TGCACTATTTGGGAT 964
G C ACTATTTG G G ATT 965
CACTATTTGGGATTC 966
ACTATTTGGGATTCT 967
CTATTTGGGATTCTT 968
I A I 1 I GGGA I I C I 1 1 969
ATTTGGGATTCTTTA 970
TTTGGGATTCTTTAG 971
TTGGGATTCTTTAGA 972
TGGGATTCTTTAGAA 973
GGGA I I C I 1 I AGAAA 974
GGA I I I 1 I AGAAAC 975
GA I I C I 1 I AGAAAC I 976
ATTCTTTAGAAACTT 977
TTCTTTAGAAACTTC 978
TCTTTAGAAACTTCC 979
CTTTAGAAACTTCCT 980
TTTAGAAACTTCCTT 981
TTAGAAACTTCCTTG 982
TAGAAACTTCCTTGG 983
AGAAACTTCCTTGGA 984
GAAACTTCCTTGGAG 985
AAACTTCCTTGGAGA 986
AACTTCCTTGGAGAA 987
ACTTCCTTGGAGAAA 988
CTTCCTTGGAGAAAA 989
TTCCTTGGAGAAAAT 990
TCCTTGGAGAAAATT 991
CCTTGGAGAAAATTA 992
CTTGGAGAAAATTAA 993 TTGGAGAAAATTAAT 994
TG G AG AAAATTAATG 995
GGAGAAAATTAATGA 996
GAGAAAATTAATGAT 997
AGAAAATTAATGATT 998
GAAAATTAATGATTC 999
AAAATTAATGATTCA 1000
AAATTAATGATTCAA 1001
AATTAATGATTCAAG 1002
ATTAATGATTCAAGA 1003
TTAATGATTCAAGAT 1004
TAATGATTCAAGATT 1005
AATGATTCAAGATTG 1006
ATGATTCAAGATTGA 1007
TGATTCAAGATTGAG 1008
GATTCAAGATTGAGA 1009
ATTCAAGATTGAGAA 1010
TTCAAGATTGAGAAA 1011
TCAAGATTGAGAAAA 1012
CAAGATTGAGAAAAC 1013
AAGATTGAGAAAACA 1014
AGATTGAGAAAACAT 1015
GATTGAGAAAACATT 1016
ATTGAGAAAACATTT 1017
TTGAGAAAACATTTA 1018
TGAGAAAACATTTAT 1019
GAGAAAACATTTATA 1020
AGAAAACATTTATAC 1021
GAAAACATTTATACA 1022
AAAACATTTATACAA 1023
AAACATTTATACAAC 1024
AACATTTATACAACC 1025
ACATTTATACAACCT 1026
CATTTATACAACCTC 1027
ATTTATACAACCTCA 1028
TTTATACAACCTCAG 1029
TTATACAACCTCAGG 1030
TAT AC AACCTC AG G C 1031
ATACAACCTCAGGCC 1032
TAC AACCTC AG G CC A 1033
ACAACCTCAGGCCAC 1034
CAACCTCAGGCCACA 1035
AACCTCAGGCCACAC 1036 ACCTCAGGCCACACT 1037
CCTCAGGCCACACTC 1038
CTCAGGCCACACTCT 1039
TC AG G CC AC ACTCTT 1040
CAGGCCACACTCTTC 1041
AG G CC AC ACTCTTCT 1042
GGCCACACTCTTCTC 1043
GCCACACTCTTCTCT 1044
CCACACTCTTCTCTC 1045
CACACTCTTCTCTCT 1046
ACACTCTTCTCTCTC 1047
CACTCTTCTCTCTCT 1048
ACTCTTCTCTCTCTT 1049
CTCTTCTCTCTCTTG 1050
TCTTCTCTCTCTTGT 1051
CTTCTCTCTCTTGTT 1052
TTCTCTCTCTTGTTA 1053
TCTCTCTCTTGTTAT 1054
CTCTCTCTTGTTATT 1055
TCTCTCTTGTTATTA 1056
CTCTCTTGTTATTAA 1057
TCTCTTGTTATTAAC 1058
CTCTTGTTATTAACC 1059
TCTTGTTATTAACCA 1060
CTTGTTATTAACCAT 1061
TTGTTATTAACCATT 1062
TGTTATTAACCATTG 1063
GTTATTA ACC ATTG C 1064
TTATTAACCATTGCA 1065
TATTAACCATTGCAC 1066
ATT AACC ATTG C ACC 1067
TTAACCATTGCACCA 1068
TA ACC ATTG C ACC AC 1069
AACCATTGCACCACT 1070
ACCATTGCACCACTG 1071
CCATTGCACCACTGC 1072
CATTGCACCACTGCA 1073
ATTGCACCACTGCAA 1074
TTGCACCACTGCAAT 1075
TGCACCACTGCAATG 1076
GCACCACTGCAATGT 1077
C ACCACTG CAATGTT 1078
ACC ACTG C AATGTTG 1079 CCACTGCAATGTTGG 1080
C ACTG C AATGTTG G C 1081
ACTGCAATGTTGGCT 1082
CTG C AATGTTG G CTG 1083
TGCAATGTTGGCTGC 1084
G C AATGTTG G CTG CT 1085
C AATGTTG G CTG CTT 1086
AATGTTGGCTGCTTT 1087
ATGTTG G CTG CTTTG 1088
TGTTGGCTGCTTTGA 1089
GTTGGCTGCTTTGAA 1090
TTGGCTGCTTTGAAA 1091
TGGCTGCTTTGAAAA 1092
GGCTGCTTTGAAAAT 1093
GCTGCTTTGAAAATC 1094
CTGCTTTGAAAATCT 1095
TG CTTTG AAAATCTC 1096
GCTTTGAAAATCTCA 1097
CTTTGAAAATCTCAT 1098
TTTGAAAATCTCATA 1099
TTGAAAATCTCATAG 1100
TG AAAATCTC ATAGT 1101
GAAAATCTCATAGTT 1102
AAAATCTCATAGTTT 1103
AAAICICAIAGI 111 1104
AAIUCAIAGI 1111 1105
ATCTCATAGTTTTTG 1106
ICICAIAGI M M GG 1107
CICAIAGI 111 IGGA 1108
ICAIAGI 111 IGGAC 1109
CAIA M M I GACA 1110
AIAGI 111 IGGACAG 1111
1 AG M M 1 GACAGC 1112
AG M M 1 GACAGCA 1113
G M M 1 GACAGCAG 1114
M M 1 GGACAGCAG 1 1115
TTTTGGACAGCAGTA 1116
TTTGGACAGCAGTAT 1117
TTGGACAGCAGTATT 1118
TGGACAGCAGTATTA 1119
GGACAGCAGTATTAG 1120
GACAGCAGTATTAGT 1121
AC AG C AGTATTAGTA 1122 C AG C AGT ATTAGT AA 1123
AG C AGTATTAGTA AA 1124
G C AGT ATTAGT A A AG 1125
CAGTATTAGTAAAGT 1126
AGTATTAGTAAAGTG 1127
GTATTAGTAAAGTGT 1128
TATTAGTAAAGTGTA 1129
ATTAGTAAAGTGTAA 1130
TTAGTAAAGTGTAAG 1131
TAGTAAAGTGTAAGA 1132
AGTAAAGTGTAAGAA 1133
GTAAAGTGTAAGAAA 1134
TAAAGTGTAAGAAAA 1135
AAAGTGTAAGAAAAT 1136
AAGTGTAAGAAAATT 1137
AGTGTAAGAAAATTG 1138
GTGTAAGAAAATTGT 1139
TGTAAGAAAATTGTC 1140
GTAAGAAAATTGTCA 1141
TAAGAAAATTGTCAA 1142
AAGAAAATTGTCAAA 1143
AGAAAATTGTCAAAG 1144
GAAAATTGTCAAAGC 1145
A AA ATTGTC A AAG CT 1146
A AATTGTC A A AG CTT 1147
A ATTGTC A AAG CTTG 1148
ATTGTC A A AG CTTG G 1149
TTGTC A AAG CTTG G A 1150
TGTCAAAGCTTGGAG 1151
GTCAAAGCTTGGAGC 1152
TCAAAGCTTGGAGCT 1153
CAAAGCTTGGAGCTA 1154
AAAGCTTGGAGCTAT 1155
AAGCTTGGAGCTATG 1156
AGCTTGGAGCTATGC 1157
GCTTGGAGCTATGCT 1158
CTTGGAGCTATGCTT 1159
TTGGAGCTATGCTTG 1160
TGGAGCTATGCTTGT 1161
GGAGCTATGCTTGTT 1162
GAGCTATGCTTGTTG 1163
AGCTATGCTTGTTGA 1164
GCTATGCTTGTTGAA 1165 CTATGCTTGTTGAAC 1166
TATGCTTGTTGAACT 1167
ATGCTTGTTGAACTT 1168
TG CTTGTTG AACTTT 1169
G 1 1 1 1 GAA 1 1 1 1 1170 1 1 1 1 GAA 1 1 1 1 1171
1 I G I I GAAU 1 1 I G I 1172
I G I I GAAU 1 1 I G I A 1173 1 1 GAA 1 1 1 1 1 AC 1174
1 1 GAA 1 1 1 1 1 ACC 1175
I GAAU 1 1 I I ACCA 1176
GAACTTTTGTACCAA 1177
AACTTTTGTACCAAT 1178
AU 1 1 I I ACCAA I A 1179
CTTTTGTACCAATAG 1180
TTTTGTACCAATAGC 1181
TTTGT ACC AATAG C A 1182
TTGTACCAATAGCAC 1183
TGTACCAATAGCACC 1184
GTACCAATAGCACCT 1185
TACCAATAGCACCTT 1186
ACC AATAG C ACCTTT 1187
CCAATAGCACCTTTA 1188
C AATAG C ACCTTTAC 1189
AATAGCACCTTTACC 1190
ATAGCACCTTTACCA 1191
TAGCACCTTTACCAG 1192
AGCACCTTTACCAGA 1193
GCACCTTTACCAGAG 1194
CACCTTTACCAGAGG 1195
ACCTTTACCAGAGGC 1196
CCTTTACCAGAGGCT 1197
CTTTACCAGAGGCTG 1198
TTTACCAGAGGCTGC 1199
TTACCAGAGGCTGCG 1200
TACCAGAGGCTGCGT 1201
ACCAGAGGCTGCGTG 1202
CCAGAGGCTGCGTGT 1203
CAGAGGCTGCGTGTT 1204
AGAGGCTGCGTGTTT 1205
GAGGCTGCGTGTTTA 1206
AG G CTG CGTGTTTA A 1207
GGCTGCGTGTTTAAA 1208 GCTGCGTGTTTAAAG 1209
CTGCGTGTTTAAAGC 1210
TG CGTGTTTA AAG CC 1211
GCGTGTTTAAAGCCC 1212
CGTGTTTAAAGCCCG 1213
GTGTTTAAAGCCCGA 1214
TGTTTAAAGCCCGAA 1215
GTTTAAAGCCCGAAG 1216
TTTAAAGCCCGAAGA 1217
TTAAAGCCCGAAGAA 1218
TAAAGCCCGAAGAAA 1219
AAAGCCCGAAGAAAA 1220
AAGCCCGAAGAAAAC 1221
AGCCCGAAGAAAACT 1222
GCCCGAAGAAAACTT 1223
CCCGAAGAAAACTTT 1224
CCGAAGAAAACTTTG 1225
CGAAGAAAACTTTGC 1226
GAAGAAAACTTTGCT 1227
AAGAAAACTTTGCTT 1228
AG AAAACTTTG CTTT 1229
GAAAACTTTGCTTTT 1230
AAAACTTTG CTTTTC 1231
AAAC I 1 I GC I 1 1 I U 1232
A ACTTTG CTTTTCTG 1233
ACTTTGCTTTTCTGA 1234
CTTTGCTTTTCTGAA 1235
TTTGCTTTTCTGAAA 1236
TTGCTTTTCTGAAAG 1237
TG CTTTTCTG AAAG A 1238
GC I 1 1 I C I GAAAGAA 1239
CTTTTCTGAAAGAAT 1240
TTTTCTGAAAGAATA 1241
TTTCTGAAAGAATAC 1242
TTCTGAAAGAATACA 1243
TCTGAAAGAATACAC 1244
CTGAAAGAATACACA 1245
TGAAAGAATACACAC 1246
GAAAGAATACACACA 1247
AAAGAATACACACAC 1248
AAGAATACACACACA 1249
AGAATACACACACAC 1250
GAATACACACACACA 1251 AATACACACACACAC 1252
ATACACACACACACG 1253
TACACACACACACGC 1254
ACACACACACACGCA 1255
CACACACACACGCAT 1256
ACACACACACGCATA 1257
CACACACACGCATAT 1258
ACACACACGCATATG 1259
CACACACGCATATGT 1260
ACACACGCATATGTA 1261
CACACGCATATGTAA 1262
ACACGCATATGTAAA 1263
CACGCATATGTAAAT 1264
ACGCATATGTAAATA 1265
CGCATATGTAAATAT 1266
G C ATATGTA AATATT 1267
CATATGTAAATATTC 1268
ATATGTAAATATTCA 1269
TATGTAAATATTCAT 1270
ATGTAAATATTCATA 1271
TGTAAATATTCATAC 1272
GTAAATATTCATACA 1273
TAAATATTCATACAT 1274
AAATATTCATACATT 1275
AATATTCATACATTT 1276
ATATTCATACATTTA 1277
TATTCATACATTTAT 1278
ATTCATACATTTATG 1279
TTCATACATTTATGT 1280
TCATACATTTATGTA 1281
CATACATTTATGTAT 1282
ATACATTTATGTATA 1283
TACATTTATGTATAT 1284
ACATTTATGTATATA 1285
CATTTATGTATATAT 1286
ATTTATGTATATATA 1287
TTTATGTATATATAC 1288
TTATGTATATATACA 1289
TATGTATATATACAT 1290
ATGTATATATACATA 1291
TGTATATATACATAT 1292
GTATATATACATATT 1293
TATATATACATATTA 1294 ATATATACATATTAT 1295
TATATACATATTATA 1296
ATATACATATTATAA 1297
TATACATATTATAAT 1298
ATACATATTATAATA 1299
TACATATTATAATAC 1300
ACATATTATAATACC 1301
CATATTATAATACCT 1302
ATATTATAATACCTA 1303
TATTATAATACCTAT 1304
ATTATAATACCTATA 1305
TTATAATACCTATAA 1306
TATAATACCTATAAG 1307
ATAATACCTATAAGT 1308
TAATACCTATAAGTT 1309
AATACCTATAAGTTA 1310
ATACCTATAAGTTAG 1311
TACCTATAAGTTAGG 1312
ACCTATA AGTT AG GT 1313
CCTATAAGTTAGGTA 1314
CT AT A AGTT AG GTAT 1315
TATAAGTTAGGTATA 1316
ATAAGTTAGGTATAA 1317
TAAGTTAGGTATAAC 1318
AAGTTAGGTATAACT 1319
AGTT AG GT ATA ACTT 1320
GTTAGGTATAACTTA 1321
TT AG GTATA ACTT AT 1322
TAGGTATAACTTATA 1323
AGGTATAACTTATAT 1324
G GT AT AACTTAT ATT 1325
GTATAACTTATATTT 1326
TATAACTTATATTTG 1327
ATAACTTATATTTGT 1328
TAACTTATATTTGTA 1329
AACTTATATTTGTAT 1330
ACTTATATTTGTATA 1331
CTTATATTTGTATAT 1332
TTATATTTGTATATG 1333
TATATTTGTATATGA 1334
ATATTTGTATATGAT 1335
TATTTGTATATGATA 1336
ATTTGTATATGATAT 1337 TTTGTATATGATATA 1338
TTGTATATGATATAT 1339
TGTATATGATATATG 1340
GTATATGATATATGG 1341
TATATGATATATGGC 1342
ATATGATATATGGCC 1343
TATGATATATGGCCT 1344
ATGATATATGGCCTA 1345
TGATATATGGCCTAG 1346
GATATATGGCCTAGG 1347
ATATATGGCCTAGGA 1348
TATATGGCCTAGGAA 1349
ATATGGCCTAGGAAA 1350
TATGGCCTAGGAAAT 1351
ATGGCCTAGGAAATT 1352
TGGCCTAGGAAATTA 1353
GGCCTAGGAAATTAA 1354
GCCTAGGAAATTAAG 1355
CCTAGGAAATTAAGG 1356
CTAGGAAATTAAGGC 1357
TAGGAAATTAAGGCT 1358
AGGAAATTAAGGCTT 1359
GGAAATTAAGGCTTA 1360
GAAATTAAGGCTTAT 1361
A AATT AAG G CTT ATT 1362
A ATT A AG G CTT ATT A 1363
ATT AAG G CTT ATT A A 1364
TT A AG G CTT ATT A A A 1365
TA AG G CTTATT A AAT 1366
A AG G CTT ATT A A AT A 1367
AG G CTTATTA AATA A 1368
G G CTT ATT A A AT A A A 1369
G CTT ATT A A AT A A A A 1370
CTTATTAAATAAAAT 1371
TT ATT AAATAA AATT 1372
TATTAAATAAAATTT 1373
ATTAAATAAAATTTA 1374
TTAAATAAAATTTAT 1375
TAAATAAAATTTATA 1376
AAATAAAATTTATAA 1377
AATAAAATTTATAAA 1378
ATAAAATTTATAAAT 1379
TAAAATTTATAAATG 1380 AAAATTTATAAATGC 1381
A A ATTT AT A A ATG C A 1382
A ATTTATA A ATG C AG 1383
ATTT AT A A ATG C AG A 1384
TTTATAAATGCAGAT 1385
TTATAAATGCAGATG 1386
TATAAATGCAGATGA 1387
ATAAATGCAGATGAG 1388
TAAATGCAGATGAGT 1389
AAATGCAGATGAGTC 1390
AATGCAGATGAGTCA 1391
ATGCAGATGAGTCAA 1392
TGCAGATGAGTCAAA 1393
GCAGATGAGTCAAAT 1394
CAGATGAGTCAAATA 1395
AGATGAGTCAAATAC 1396
GATGAGTCAAATACA 1397
ATGAGTCAAATACAA 1398
TG AGTC A AATAC A AA 1399
GAGTCAAATACAAAG 1400
AGTCAAATACAAAGA 1401
GTCAAATACAAAGAT 1402
TCAAATACAAAGATC 1403
CAAATACAAAGATCA 1404
AAATACAAAGATCAG 1405
AATACAAAGATCAGA 1406
ATACAAAGATCAGAC 1407
TACAAAGATCAGACA 1408
ACAAAGATCAGACAT 1409
CAAAGATCAGACATA 1410
AAAGATCAGACATAA 1411
AAGATCAGACATAAC 1412
AGATCAGACATAACT 1413
GATCAGACATAACTC 1414
ATCAGACATAACTCT 1415
TCAGACATAACTCTA 1416
CAGACATAACTCTAT 1417
AGACATAACTCTATC 1418
GACATAACTCTATCA 1419
ACATAACTCTATCAC 1420
CATAACTCTATCACC 1421
ATAACTCTATCACCT 1422
TAACTCTATCACCTA 1423 AACTCTATCACCTAA 1424
ACTCTATCACCTAAG 1425
CTCTATCACCTAAGT 1426
TCTATCACCTAAGTA 1427
CTATCACCTAAGTAA 1428
TATCACCTAAGTAAT 1429
ATCACCTAAGTAATC 1430
TCACCTAAGTAATCA 1431
CACCTAAGTAATCAT 1432
ACCTAAGTAATCATT 1433
CCTAAGTAATCATTG 1434
CTAAGTAATCATTGT 1435
TAAGTAATCATTGTT 1436
AAGTAATCATTGTTT 1437
AGTAATCATTGTTTA 1438
GTAATCATTGTTTAC 1439
TAATCATTGTTTACG 1440
AATCATTGTTTACGT 1441
ATCATTGTTTACGTT 1442
TCATTGTTTACGTTT 1443
CAI IGI 1 IACGI 111 1444
Al IGI 1 IACGI I MG 1445
11 111 AC 1111 C 1446
1 G 111 ACG 1111 GCA 1447
GTTTACGTTTTGCAG 1448
TTTACGTTTTGCAGT 1449
11 ACG 1111 GCAG 11 1450
IACGI 11 IGCAGI 11 1451
ACGI 11 IGCAGI 1 IA 1452
CGI 11 IGCAGI 1 IAI 1453
GI N IGCAGI 1 IAIC 1454
TTTTGCAGTTTATCT 1455
TTTG C AGTTTATCTT 1456
TTG C AGTTT ATCTTC 1457
TGCAGTTTATCTTCC 1458
G C AGTTTATCTTCC A 1459
CAGTTTATCTTCCAT 1460
AGTTTATCTTCCATT 1461
GTTTATCTTCCATTT 1462
TTTATCTTCCATTTC 1463
TTATCTTCCATTTCT 1464
TATCTTCCATTTCTC 1465
ATCTTCCATTTCTCC 1466 TCTTCCATTTCTCCC 1467
CTTCCATTTCTCCCC 1468
TTCCATTTCTCCCCT 1469
TCCATTTCTCCCCTC 1470
CCATTTCTCCCCTCT 1471
CATTTCTCCCCTCTA 1472
ATTTCTCCCCTCTAA 1473
TTTCTCCCCTCTAAT 1474
TTCTCCCCTCTAATA 1475
TCTCCCCTCTAATAT 1476
CTCCCCTCTAATATG 1477
TCCCCTCTAATATGA 1478
CCCCTCTAATATGAC 1479
CCCTCTAATATGACT 1480
CCTCTAATATGACTC 1481
CTCTAATATGACTCT 1482
TCTAATATGACTCTT 1483
CTAATATGACTCTTT 1484
I AA I A I GAU U 1 1 1 1485
AATATGACTCTTTTA 1486
ATATGACTCTTTTAA 1487
TATGACTCTTTTAAA 1488
ATGACTCTTTTAAAT 1489
I GAU U 1 1 I AAA I 1 1490
GACTCTTTTAAATTT 1491
ACTCTTTTAAATTTA 1492
CTCTTTTAAATTTAG 1493
I U 1 1 I AAA I 1 I AG I 1494
CTTTTAAATTTAGTT 1495
TTTTAAATTTAGTTA 1496
TTTAAATTTAGTTAC 1497
TTAAATTTAGTTACA 1498
TAAATTTAGTTACAC 1499
AAATTTAGTTACACA 1500
AATTTAGTTACACAG 1501
ATTTAGTTACACAGA 1502
TTTAGTTACACAGAA 1503
TTAGTTACACAGAAA 1504
TAGTTACACAGAAAT 1505
AGTTACACAGAAATT 1506
GTTACACAGAAATTG 1507
TTACACAGAAATTGG 1508
TACACAGAAATTGGT 1509 ACACAGAAATTGGTT 1510
CACAGAAATTGGTTT 1511
ACAGAAATTGGTTTT 1512
CAGAAAI IGGI 11 lb 1513
AGAAATTGGTTTTGC 1514
GAAAI IGGI 11 I CG 1515
AAAI IGGI 11 IGCGC 1516
AAI IGGI 11 IGCGC 1 1517
Al IGGI 11 IGCGCIC 1518
1 IGGI 11 IGCGC ICA 1519
IGGI 11 IGCGCICAC 1520
GGI 11 IGCGC ICACG 1521
GI N IGCGC ICACGA 1522
TTTTGCGCTCACGAC 1523
TTTGCGCTCACGACA 1524
TTGCGCTCACGACAT 1525
TGCGCTCACGACATG 1526
GCGCTCACGACATGC 1527
CGCTCACGACATGCC 1528
GCTCACGACATGCCT 1529
CTCACGACATGCCTA 1530
TCACGACATGCCTAA 1531
CACGACATGCCTAAC 1532
ACGACATGCCTAACA 1533
CGACATGCCTAACAT 1534
GACATGCCTAACATC 1535
ACATGCCTAACATCA 1536
CATGCCTAACATCAG 1537
ATGCCTAACATCAGG 1538
TG CCTA AC ATC AG G C 1539
GCCTAACATCAGGCC 1540
CCTAACATCAGGCCT 1541
CTA AC ATC AG G CCTT 1542
TAACATCAGGCCTTT 1543
A AC ATC AG G CCTTTA 1544
ACATCAGGCCTTTAT 1545
CATCAGGCCTTTATC 1546
ATCAGGCCTTTATCC 1547
TCAGGCCTTTATCCT 1548
CAGGCCTTTATCCTT 1549
AG G CCTTTATCCTTA 1550
GGCCTTTATCCTTAA 1551
GCCTTTATCCTTAAA 1552 CCTTTATCCTTAAAG 1553
CTTTATCCTTAAAGA 1554
TTTATCCTTAAAGAA 1555
TTATCCTTAAAGAAG 1556
TATCCTTAAAGAAGT 1557
ATCCTTAAAGAAGTT 1558
TCCTTAAAGAAGTTT 1559
CCTTAAAGAAGTTTC 1560
CTTAAAGAAGTTTCA 1561
TTAAAGAAGTTTCAA 1562
TAAAGAAGTTTCAAC 1563
AAAGAAGTTTCAACA 1564
AAGAAGTTTCAACAC 1565
AGAAGTTTCAACACT 1566
GAAGTTTCAACACTC 1567
AAGTTTCAACACTCT 1568
AGTTTCAACACTCTT 1569
GTTTCAACACTCTTG 1570
TTTCAACACTCTTGT 1571
TTCAACACTCTTGTG 1572
TCAACACTCTTGTGT 1573
CAACACTCTTGTGTC 1574
AACACTCTTGTGTCA 1575
ACACTCTTGTGTCAC 1576
CACTCTTGTGTCACC 1577
ACTCTTGTGTCACCT 1578
CTCTTGTGTCACCTC 1579
TCTTGTGTCACCTCA 1580
CTTGTGTCACCTCAT 1581
TTGTGTCACCTCATG 1582
TGTGTCACCTCATGT 1583
GTGTCACCTCATGTG 1584
TGTCACCTCATGTGT 1585
GTCACCTCATGTGTC 1586
TCACCTCATGTGTCT 1587
CACCTCATGTGTCTA 1588
ACCTCATGTGTCTAC 1589
CCTCATGTGTCTACA 1590
CTCATGTGTCTACAG 1591
TCATGTGTCTACAGA 1592
CATGTGTCTACAGAA 1593
ATGTGTCTACAGAAA 1594
TGTGTCTACAGAAAA 1595 GTGTCTACAGAAAAT 1596
TGTCTACAGAAAATG 1597
GTCTACAGAAAATGT 1598
TCTACAGAAAATGTT 1599
CTACAGAAAATGTTT 1600
TACAGAAAATGTTTG 1601
ACAGAAAATGTTTGC 1602
CAGAAAATGTTTGCT 1603
AGAAAATGTTTGCTT 1604
GAAAATGTTTGCTTT 1605
AAAAIGI 1 IGU 111 1606
AAA 1 11 IGU 11 IA 1607
AAIGI 1 IGU 11 IAI 1608
AIGI 1 IGU 11 IAIG 1609
IGI 1 IGU 11 IAIGC 1610
Gl 1 IGU 11 IAIGCC 1611
11 IGU 11 IAIGCU 1612
TTGCTTTTATGCCTT 1613
IGU 11 IAIGCU IC 1614
GU 11 IAIGCU ICA 1615
CTTTTATG CCTTC AC 1616
TTTTATGCCTTCACG 1617
TTTATGCCTTCACGG 1618
TTATGCCTTCACGGT 1619
TATGCCTTCACGGTT 1620
ATGCCTTCACGGTTA 1621
TGCCTTCACGGTTAT 1622
GCCTTCACGGTTATG 1623
CCTTCACGGTTATGT 1624
CTTC ACG GTTATGTT 1625
TTCACGGTTATGTTT 1626
ICACGGI IAIGI 111 1627
CACGGTTATGTTTTC 1628
ACGGI IAIGI 11 Id 1629
CGGTTATGTTTTCTT 1630
GGI IAIGI 11 Id IA 1631
Gl IAIGI 11 Id 1 AG 1632
1 IAIGI 11 Id IAGI 1633
IAIGI 11 Id 1 AG 1 A 1634
AIGI 11 Id 1 AG 1 AG 1635
IGI 11 Id 1 AG 1 AG 1 1636
Gl 11 Id 1 AG 1 AG 1 A 1637
TTTTCTTAGTAGTAG 1638 TTTCTTAGTAGT AG C 1639
TTCTTAGTAGTAGCA 1640
TCTTAGTAGTAGCAA 1641
CTT AGTAGTAG C A AT 1642
TT AGTAGTAG C AATA 1643
TAGT AGT AG C AAT AA 1644
AGTAGTAGCAATAAA 1645
GT AGT AG C AATA AAT 1646
TAGTAGCAATAAATA 1647
AGTAG C A AT AA AT AA 1648
GT AG C AATA AATA AA 1649
TAG C AATA AATA AAA 1650
AG C A AT AA AT AA AAT 1651
G C AATA AATA AA AT A 1652
CAATAAATAAAATAG 1653
AATAAATAAAATAGA 1654
ATAAATAAAATAGAT 1655
TAAATAAAATAGATG 1656
AAATAAAATAGATGC 1657
AATAAAATAGATGCA 1658
ATAAAATAGATGCAA 1659
TAAAATAGATGCAAA 1660
AAAATAGATGCAAAG 1661
AAATAGATGCAAAGT 1662
AATAGATGCAAAGTG 1663
ATAGATGCAAAGTGC 1664
TAGATGCAAAGTGCT 1665
AGATGCAAAGTGCTA 1666
GATGCAAAGTGCTAA 1667
ATG C AA AGTG CTA AT 1668
TG C A AAGTG CT AATT 1669
G C AA AGTG CTA ATTA 1670
C AA AGTG CTA ATTAC 1671
A AAGTG CTA ATTACT 1672
AAGTG CTA ATTACTT 1673
AGTG CTA ATTACTTG 1674
GTG CTA ATTACTTG G 1675
TGCTAATTACTTGGA 1676
GCTAATTACTTGGAA 1677
C AATGTTG G CTG CTT 1678
A ATGTTG G CTG CTTT 1679
ATGTTG G CTG CTTTG 1680
TGTTGGCTGCTTTGA 1681 GTTGGCTGCTTTGAA 1682
TTGGCTGCTTTGAAA 1683
TGGCTGCTTTGAAAA 1684
GGCTGCTTTGAAAAT 1685
GCTGCTTTGAAAATC 1686
CTGCTTTGAAAATCT 1687
TG CTTTG AAAATCTC 1688
GCTTTGAAAATCTCA 1689
CTTTGAAAATCTCAT 1690
TTTGAAAATCTCATA 1691
TTGAAAATCTCATAG 1692
TG AAAATCTC ATAGT 1693
GAAAATCTCATAGTT 1694
AAAATCTCATAGTTT 1695
AAAICICAIAGI 111 1696
AAICICAIAGI 1111 1697
ATCTCATAGTTTTTG 1698
ICICAIAGI M M GG 1699
CICAIAGI 111 IGGA 1700
ICAIAGI 111 IGGAC 1701
CAIA M M I GACA 1702
AIAGI 111 IGGACAG 1703
1 AG M M 1 GACAGC 1704
AG M M 1 GACAGCA 1705
G M M 1 GACAGCAG 1706
M M 1 GGACAGCAG 1 1707
TTTTGGACAGCAGTA 1708
TTTGGACAGCAGTAT 1709
TTGGACAGCAGTATT 1710
TGGACAGCAGTATTA 1711
GGACAGCAGTATTAG 1712
GACAGCAGTATTAGT 1713
AC AG C AGTATT AGTA 1714
C AG C AGT ATTAGT AA 1715
AGCAGTATTAGTAAA 1716
G C AGT ATTAGT A A AG 1717
CAGTATTAGTAAAGT 1718
AGTATT AGTAAAGTG 1719
GTATTAGTAAAGTGT 1720
TATTAGTAAAGTGTA 1721
ATTAGTAAAGTGTAA 1722
TTAGTAAAGTGTAAG 1723
TAGTAAAGTGTAAGA 1724 AGTAAAGTGTAAGAA 1725
GTAAAGTGTAAGAAA 1726
TAAAGTGTAAGAAAA 1727
AAAGTGTAAGAAAAT 1728
AAGTGTAAGAAAATT 1729
AGTGTAAGAAAATTG 1730
GTGTAAGAAAATTGT 1731
TGTAAGAAAATTGTC 1732
GTAAGAAAATTGTCA 1733
TAAGAAAATTGTCAA 1734
AAGAAAATTGTCAAA 1735
AGAAAATTGTCAAAG 1736
GAAAATTGTCAAAGC 1737
A AA ATTGTC A AAG CT 1738
A AATTGTC A A AG CTT 1739
A ATTGTC A AAG CTTG 1740
ATTGTC A A AG CTTG G 1741
TTGTC A AAG CTTG G A 1742
TGTCAAAGCTTGGAG 1743
GTCAAAGCTTGGAGC 1744
TCAAAGCTTGGAGCT 1745
CAAAGCTTGGAGCTA 1746
AAAGCTTGGAGCTAT 1747
AAGCTTGGAGCTATG 1748
AGCTTGGAGCTATGC 1749
GCTTGGAGCTATGCT 1750
CTTGGAGCTATGCTT 1751
TTGGAGCTATGCTTG 1752
TGGAGCTATGCTTGT 1753
GGAGCTATGCTTGTT 1754
GAGCTATGCTTGTTG 1755
AGCTATGCTTGTTGA 1756
GCTATGCTTGTTGAA 1757
CTATGCTTGTTGAAC 1758
TATGCTTGTTGAACT 1759
ATGCTTGTTGAACTT 1760
TG CTTGTTG AACTTT 1761
GC 1 I G I 1 AAC 1 1 1 1 1762
C I 1 1 1 GAAC 1 1 1 1 1763
1 I G I I GAAC I 1 1 I G I 1764
I G I I GAAC I 1 1 I G I A 1765 1 1 AAC 1 1 1 1 1 AC 1766
1 1 GAAC 1 1 1 1 1 ACC 1767 I GAAU 1 1 I G I AO.A 1768
GAACTTTTGTACCAA 1769
AACTTTTGTACCAAT 1770
AU 1 1 I G I AO.AA I A 1771
CTTTTGTACCAATAG 1772
TTTTGTACCAATAGC 1773
TTTGT ACC AATAG C A 1774
TTGTACCAATAGCAC 1775
TGTACCAATAGCACC 1776
GTACCAATAGCACCT 1777
TACCAATAGCACCTT 1778
ACC AATAG C ACCTTT 1779
CCAATAGCACCTTTA 1780
C AATAG C ACCTTTAC 1781
AATAGCACCTTTACC 1782
ATAGCACCTTTACCA 1783
TAGCACCTTTACCAG 1784
AGCACCTTTACCAGA 1785
GCACCTTTACCAGAG 1786
CACCTTTACCAGAGG 1787
ACCTTTACCAGAGGC 1788
CCTTTACCAGAGGCT 1789
CTTTACCAGAGGCTG 1790
TTTACCAGAGGCTGC 1791
TTACCAGAGGCTGCG 1792
TACCAGAGGCTGCGT 1793
ACCAGAGGCTGCGTG 1794
CCAGAGGCTGCGTGT 1795
CAGAGGCTGCGTGTT 1796
AGAGGCTGCGTGTTT 1797
GAGGCTGCGTGTTTA 1798
AG G CTG CGTGTTTA A 1799
GGCTGCGTGTTTAAA 1800
GCTGCGTGTTTAAAG 1801
CTGCGTGTTTAAAGC 1802
TG CGTGTTTA AAG CC 1803
GCGTGTTTAAAGCCC 1804
CGTGTTTAAAGCCCG 1805
GTGTTTAAAGCCCGA 1806
TGTTTAAAGCCCGAA 1807
GTTTAAAGCCCGAAG 1808
TTTAAAGCCCGAAGA 1809
TTAAAGCCCGAAGAA 1810 TAAAGCCCGAAGAAA 1811
AAAGCCCGAAGAAAA 1812
AAGCCCGAAGAAAAC 1813
AGCCCGAAGAAAACT 1814
GCCCGAAGAAAACTT 1815
CCCGAAGAAAACTTT 1816
CCGAAGAAAACTTTG 1817
CGAAGAAAACTTTGC 1818
GAAGAAAACTTTGCT 1819
AAGAAAACTTTGCTT 1820
AG AAAACTTTG CTTT 1821
GAAAACTTTGCTTTT 1822
AAAACTTTG CTTTTC 1823
AAAU 1 I GU 1 1 I U 1824
A ACTTTG CTTTTCTG 1825
ACTTTGCTTTTCTGA 1826
CTTTGCTTTTCTGAA 1827
TTTGCTTTTCTGAAA 1828
TTGCTTTTCTGAAAG 1829
TG CTTTTCTG AAAG A 1830
GC I 1 1 1 C 1 AAAGAA 1831
CTTTTCTGAAAGAAT 1832
TTTTCTGAAAGAATA 1833
Without further elaboration, it is believed that one skilled in the art can, based on the description provided herein, utilize the present invention to its fullest extent. The specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."
The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another
embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

Claims

CLAIMS What is claimed is:
1. A method of maintaining or increasing the number of naive T cells in a T cell population, the method comprising delivering ex vivo an oligonucleotide that inhibits the interaction of FAS-AS1 with RBM5 to a T cell population comprising naive T cells.
2. The method of claim 1, wherein delivering the oligonucleotide results in an increase in soluble Fas (sFas) expression in the naive T cells in the T cell population compared to sFas expression in control naive T cells in a control T cell population to which the oligonucleotide has not been delivered.
3. The method of claim 1 or 2, wherein the T cell population is a CD4+ T cell population.
4. The method of any one of claims 1 to 3, wherein the method further comprises: a) isolating T cells from a sample obtained from a donor subject; and b) selecting CD4+ T cells from the isolated T cells, thereby producing the T cell population comprising naive T cells to which the oligonucleotide is delivered.
5. The method of claim 4, wherein the method further comprises administering the T cell population to a host subject after the oligonucleotide has been delivered to the T cell population.
6. The method of claim 5, wherein the donor subject and the host subject are the same.
7. The method of claim 5, wherein the donor subject and the host subject are different.
8. The method of any one of claims 1 to 7, wherein the method further comprises transfecting the T cell population with an expression construct encoding a chimeric antigen receptor (CAR).
9. The method of claim 8, wherein the transfection occurs before delivery of the oligonucleotide to the T cell population.
10. The method of claim 8, wherein the transfection occurs after delivery of the oligonucleotide to the T cell population.
11. The method of any one of claims 8 to 10, wherein the CAR is specific for a tumor antigen.
12. The method of claim 11, wherein the host subject has cancer.
13. The method of any one of claims 1 to 12, wherein the T cell population is a human T cell population.
14. The method of any one of claims 1 to 13, wherein the oligonucleotide comprises a region of complementarity that is complementary with at least 8 nucleotides of FAS-AS l.
15. The method of claim 14, wherein the oligonucleotide reduces the level of FAS-AS l.
16. The method of claim 15, wherein the oligonucleotide is a gapmer, an siRNA, a ribozyme or an aptamer that causes degradation of FAS-AS l.
17. The method of claim 14, wherein the oligonucleotide sterically interferes with the interaction of FAS-AS l with RBM5.
18. The method of claim 17, wherein the oligonucleotide is a mixmer.
19. The method of any one of claims 1 to 18, wherein the oligonucleotide is single stranded.
20. The method of claim 19, wherein the oligonucleotide is a gapmer comprising a region of complementarity that is complementary with at least 8 nucleotides of FAS-AS l.
21. The method of claim 20, wherein the gapmer comprises the general formula:
wherein each instance of X , X3 is independently a modified or unmodified nucleotide, wherein m and o are independently integers in a range of 1 to 10, reflecting the number of instances of X1 and X3, respectively, linked consecutively together through
internucleotide linkages, wherein each instance of X2 is a deoxyribonucleotide, wherein n is an integer in a range of 6 to 20, reflecting the number of instances of X2 linked consecutively together through internucleotide linkages.
22. The method of claim 21, wherein at least one of X1, X3 is a 2'-modified nucleotide.
5 23. The method of claim 22, wherein the 2'-modified nucleotide is a 2'-0,4'-C- bridged nucleotide.
24. The method of claim 23, wherein the 2'-modified nucleotide is a 2'-0,4'-C- methylene bridged nucleotide.
25. The method of claim 22, wherein the a 2'-modified nucleotide is a 2'-0-methyl l o nucleotide.
26. The method of any one of claims 1 to 25, wherein the oligonucleotide is 8 to 30 nucleotides in length.
PCT/US2018/047473 2017-08-22 2018-08-22 Modulation of soluble fas expression WO2019040590A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762548483P 2017-08-22 2017-08-22
US62/548,483 2017-08-22

Publications (1)

Publication Number Publication Date
WO2019040590A1 true WO2019040590A1 (en) 2019-02-28

Family

ID=65439873

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/047473 WO2019040590A1 (en) 2017-08-22 2018-08-22 Modulation of soluble fas expression

Country Status (1)

Country Link
WO (1) WO2019040590A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
DE202023106198U1 (en) 2022-10-28 2024-03-21 CureVac SE Nucleic acid-based vaccine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014039044A1 (en) * 2012-09-06 2014-03-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Methods of producing t memory stem cell populations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014039044A1 (en) * 2012-09-06 2014-03-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Methods of producing t memory stem cell populations

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AUNE, THOMAS M. ET AL.: "Long noncoding RNAs in T lymphocytes", JOURNAL OF LEUKOCYTE BIOLOGY, vol. 99, January 2016 (2016-01-01), pages 31 - 44, XP055580547 *
DESBARATS, J. ET AL.: "Dichotomy between naive and memory CD 4+ T cell responses to Fas engagement", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 96, July 1999 (1999-07-01), pages 8104 - 8109, XP055580499 *
KLEBANOFF, CHRISTOPHER A. ET AL.: "Memory T cell -driven differentiation of naive cells impairs adoptive immunotherapy", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 126, no. 1, January 2016 (2016-01-01), pages 318 - 334, XP055580492 *
SEHGAL, L. ET AL.: "FAS-antisense 1 IncRNA and production of soluble versus membrane Fas in B- cell lymphoma", LEUKEMIA, vol. 28, 2014, pages 2376 - 2387, XP055580494 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
DE202023106198U1 (en) 2022-10-28 2024-03-21 CureVac SE Nucleic acid-based vaccine

Similar Documents

Publication Publication Date Title
AU2018202634B2 (en) Multimeric oligonucleotide compounds
AU2019200789B2 (en) Multimeric oligonucleotide compounds
US10041074B2 (en) Euchromatic region targeting methods for modulating gene expression
WO2019040590A1 (en) Modulation of soluble fas expression
WO2015023938A1 (en) Epigenetic regulators of frataxin
US20150225722A1 (en) Methods for selective targeting of heterochromatin forming non-coding rna
EP3033425A1 (en) Compositions and methods for modulating expression of frataxin
KR20160074368A (en) Compositions and methods for modulating utrn expression
JP2016521556A (en) Compositions and methods for modulating FOXP3 expression
WO2016130943A1 (en) Hybrid oligonucleotides and uses thereof
WO2016130929A1 (en) Targeting oligonucleotides and uses thereof to modulate gene expression
WO2018031871A1 (en) Ex vivo modulation of foxp3 expression

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18847999

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18847999

Country of ref document: EP

Kind code of ref document: A1