US20200332298A1

US20200332298A1 - Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins

Info

Publication number: US20200332298A1
Application number: US16/439,390
Authority: US
Inventors: Wilhelmina M.C. van Roon-Mom; Melvin Maurice Evers; Barry Antonius Pepers; Annemieke Aartsma-Rus; Garrit-Jan Boudewijn van Ommen
Original assignee: Leids Universitair Medisch Centrum LUMC
Current assignee: Leids Universitair Medisch Centrum LUMC
Priority date: 2010-08-05
Filing date: 2019-06-12
Publication date: 2020-10-22
Also published as: US20140039037A1; AU2011286539B2; CA2807187A1; EP2601294A1; NZ607297A; AU2011286539A1; US20130198877A1; US10364432B2; CA2807187C; US9611471B2; EP2601294B1; WO2012018257A1; US20200224203A1; DK2601294T3; US20170226521A1; US10590421B2; US20170175117A1

Abstract

The invention relates to means and methods for removing a proteolytic cleavage site from a protein comprising providing a cell that expresses pre-mRNA encoding the protein with an anti-sense oligonucleotide that induces skipping of the exonic sequence that encodes the proteolytic cleavage site, the method further comprising allowing translation of mRNA produced from the pre-mRNA.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/439,776, filed Feb. 22, 2017, pending, which is a continuation of U.S. patent application Ser. No. 13/814,203, filed Apr. 12, 2013, now U.S. Pat. No. 9,611,471, issued Apr. 4, 2017, which is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/NL2011/050549, filed Aug. 4, 2011, designating the United States of America and published in English as International Patent Publication WO 2012/018257 A1 on Feb. 9, 2012, which claims the benefit under Article 8 of the Patent Cooperation Treaty and under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No. 61/370,855, filed Aug. 5, 2010, and to European Patent Application Serial No. 10172076.1, filed Aug. 5, 2010, the contents of the entirety of each of which are hereby incorporated herein by this reference.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTING SUBMITTED AS A TXT AND PDF FILES

Pursuant to 37 C.F.R. § 1.821(c) or (e), files containing a TXT version and a PDF version of the Sequence Listing have been submitted concomitant with this application, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

This application relates to the field of biotechnology and genetic and acquired diseases. In particular, it relates to the alteration of mRNA processing of specific pre-mRNA to remove a proteolytic cleavage site from a protein encoded by the pre-mRNA.

BACKGROUND

Proteolytic processing is a major form of post-translational modification that occurs when a protease cleaves one or more bonds in a target protein to modify its activity. This processing may lead to activation, inhibition, alteration or destruction of the protein's activity. Many cellular processes are controlled by proteolytic processing. The attacking protease may remove a peptide segment from either end of the target protein, but it may also cleave internal bonds in the protein that lead to major changes in the structure and function of the protein.
Proteolytic processing is a highly specific process. The mechanism of proteolytic processing varies according to the protein being processed, location of the protein, and the protease.
Proteolytic processing can have various functions. For instance, proteolysis of precursor proteins regulates many cellular processes including gene expression, embryogenesis, the cell cycle, programmed cell death, intracellular protein targeting and endocrine/neural functions. In all of these processes, proteolytic cleavage of precursor proteins is necessary. The proteolysis is often done by serine proteases in the secretory pathways. These proteases are calcium-dependent serine endoproteases and are related to yeast and subtilisin proteases and, therefore, called Subtilisin-like Proprotein Convertases (SPCs) or PCs. Seven members of this family have been identified and characterized and each have conserved signal peptides, pro-regions, catalytic and P-domains but differ in their C-terminal domains in mammals.
Autocatalytic cleavage of an N-terminal propeptide activates these proteases, which is required for folding, and activity also causes the release of prodomain. Other examples of function associated with proteolytic processing are the blood clotting cascades, the metaloendopeptidases, the secretases and the caspases. Yet other examples are the viral proteases that specifically process viral polyproteins.
The prior art describes various strategies to inhibit the various proteases. For instance, gamma-secretase inhibitors are presently being developed for the treatment of T cell acute lymphoblastic leukemia (Nature Medicine 2009, 15:50-58). Caspase inhibitors are being developed for a variety of different applications (The Journal of Biological Chemistry 1998, 273:32608-32613), for instance, in the treatment of sepsis (Nature Immunology 2000, 1:496-501).
A problem with the use of protease inhibitors is that these proteins typically have a range of targets in the human body and, associated therewith, a range of effects. Inhibiting a protease in the human body through the action of a protease inhibitor thus, not only inhibits the desired effect, but typically also has a range of other effects that may or may not affect the utility of the protease inhibitor for the indicated disease. Another problem associated with protease inhibitors is that it is not always easy to produce an inhibitor that is sufficiently specific for the target protease and, therefore, may also inhibit other proteases.

DISCLOSURE

The disclosure provides an alternative approach to interfere with the proteolytic processing of target proteins. Instead of designing inhibitors to the proteases, the target protein itself is modified. In the art, it is known to modify a protease cleavage site in a target protein. This is typically done by introducing point mutations into the coding region of a protein. These mutations typically break up the recognition sequence of the protease. These types of modification are usually introduced into a cDNA copy of the gene and this altered copy is inserted into the DNA of cells by recombinant DNA technology. Although this can be done in the laboratory, it is difficult to implement such strategies in the clinic, if only because gene therapy applications that rely on the introduction of a complete gene are, at present, not very efficient, and the original gene associated with the problem is not removed.
Provided herein is a method for removing a proteolytic cleavage site from a protein comprising providing a cell that expresses a pre-mRNA encoding the protein with an antisense oligonucleotide (AON) that induces skipping of the exon sequence that encodes the proteolytic cleavage site, the method further comprising allowing translation of mRNA produced from the pre-mRNA.
A method hereof is particularly useful for removing proteolytic cleavage sites from proteins. It does not require removal or modification of the gene itself, but rather, prevents the incorporation of the genetic code for the proteolytic cleavage site into the coding region of the protein in the mature mRNA. In this way, the process is reversible. The oligonucleotide has a finite life span in the cell and, therefore, has a finite effect on the removal. Another advantage is that the removal is not absolute. Not all pre-mRNA coding for the target protein that is generated by the cell is typically targeted. It is possible to achieve high levels of skipping. The skipping efficiency depends, for instance, on the particular target, the particular exon sequence to be skipped, the particular AON design, and/or the amount of AON used. Skipping percentages are typically expressed as the ratio of mRNA that does not have the coding part of the proteolytic cleavage site (skipped mRNA) versus the sum of skipped mRNA and unmodified mRNA coding for the unmodified target protein (unmodified mRNA). The possibility of tailoring the percentage of skipping is advantageous; for instance, when the unmodified protein is associated with a toxic phenotype but also has a positive function to perform that is not performed (as well) by the modified protein. By removing the proteolytic cleavage site only from a fraction of the protein formed, it is possible to reduce the toxic property, while leaving the positive or desired function of the unmodified protein at least partially intact.
A method hereof modulates the splicing of a pre-mRNA into an mRNA, such that an exon sequence that codes for a proteolytic cleavage site that is present in the exons encoded by the pre-mRNA is not included in the mature mRNA produced from the pre-mRNA. Protein that is subsequently translated from this mRNA does not contain the proteolytic cleavage site. The invention, thus, does not actually remove a proteolytic cleavage site from a protein that has already been formed. Rather, it promotes the production of a novel protein that does not contain the proteolytic cleavage site. However, when looking at a cell as an entity wherein protein synthesis and degradation are at equilibrium, the result of a method of the invention can be seen as removing a proteolytic cleavage site from a protein. Unmodified target protein is gradually replaced by target protein that does not contain the proteolytic cleavage site. Thus, provided is a method for producing a cell that contains a modified protein that lacks a proteolytic cleavage site, when compared to the unmodified protein encoded in the genome, the method comprising providing a cell that expresses pre-mRNA encoding the protein with an AON that induces skipping of the exon sequence or part of the exon sequence that encodes the proteolytic cleavage site, the method further comprising allowing translation of mRNA produced from the pre-mRNA in the cell. The novel mRNA from which the coding sequence for the proteolytic cleavage site is removed is a shortened or smaller coding sequence that codes for a shorter or smaller version of the unmodified protein. Often, the modified protein is an internally deleted version of the unmodified protein, wherein the internal deletion at least breaks up and, preferably, deletes the proteolytic cleavage site.
Antisense-mediated modulation of splicing (also referred to as exon-skipping) is one of the fields where AONs have been able to live up to their expectations. In this approach, AONs are implemented to facilitate cryptic splicing, to change levels of alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. The antisense-mediated modulation of splicing has been diversified since its first introduction and now many different kinds of manipulations are possible. Apart from classical exon skipping where typically an entire exon is skipped from the mature mRNA, it is, for instance, possible to skip a part of an exon. Exon inclusion is also possible. The latter occurs when AONs targeted toward appropriate intron sequences are coupled to the business end of SR-proteins.
Exon skipping has been used to restore cryptic splicing, to change levels of alternatively spliced genes, and to restore disrupted open reading frames. This approach has been employed with a number of genes including Apolipoprotein B, Bcl-X, Collagen type 7, dystrophin, dysferlin, prostate-specific membrane antigen, IL-5 receptor alpha, MyD88, Tau, TNFalpha2 receptor, Titin, WT1, beta-globulin, and CFTR. Accordingly, in preferred embodiments, methods are provided for removing a proteolytic cleavage site from a protein, wherein the protein is not Apolipoprotein B, Bcl-X, Collagen type 7, dystrophin, dysferlin, prostate-specific membrane antigen, IL-5 receptor alpha, MyD88, Tau, TNFalpha2 receptor, Titin, WT1, beta-globulin, or CFTR; more preferably, the protein is not dystrophin.
In contrast to the previous uses for exon-skipping, provided is a method for removing a proteolytic cleavage site in order to treat an individual, restore function to a protein, or reduce toxicity of a protein. The methods and oligonucleotides described herein are particularly useful for removing proteolytic cleavage sites from a protein, wherein the protein is involved in a neurodegenerative disorder.
Prevention of inclusion of a coding part for a proteolytic cleavage site into mature mRNA is, in the present invention, typically achieved by means of exon-skipping. Antisense oligonucleotides for exon-skipping typically enable skipping of an exon or the 5′ or 3′ part of it. Antisense oligonucleotides can be directed toward the 5′ splice site, the 3′ splice site, to both splice sites, to one or more exon-internal sites and to intron sequences, for instance, specific for the branch point. The latter enables skipping of the upstream exon.
Skipping of the nucleotides that code for the proteolytic cleavage site is typically achieved by skipping the exon that contains the nucleotides that code for the proteolytic cleavage site. The proteolytic cleavage site comprises the recognition sequence for the specific protease and the two amino acids between which the peptide linkage is cleaved by the protease. The proteolytic cleavage site can overlap the boundary of two adjacent exons or, if a part of the exon is skipped, overlap the exon sequence that contains the cryptic splice acceptor/donor sequence. In this embodiment, it is preferred to skip the exon sequence that codes for the peptide linkage that is cleaved by the protease. Whether or not a recognition sequence for a protease is actually used in nature depends, not only on the presence of the recognition sequence itself, but also on the location of the site in the folded protein. An internally located recognition site is typically not used in nature. In the invention, a proteolytic cleavage site is an active proteolytic cleavage site that is actually used in nature.
Skipping of the exon that contains the nucleotides that code for the proteolytic cleavage site is preferably achieved by means of an AON that is directed toward an exon internal sequence. An oligonucleotide is said to be directed toward an exon internal sequence if the complementarity region that contains the sequence identity to the reverse complement of the target pre-mRNA is within the exon boundary. Presently, all exons that have been targeted by means of exon-skipping can be induced to be skipped from the mature mRNA, often with one AON and sometimes with two AONs directed toward the exon. However, not all AONs that can be designed induce detectable amounts of skipping. The most experience with exon-skipping has been gained in the DMD system. Using AON directed toward exon-internal sequences, it has been shown that all exons can be skipped (with the exception, of course, of the first and the last exon). However, not all AON designed against an exon-internal sequence actually induce detectable amounts of skipping of the targeted exon. The frequency of randomly selected exon-internal AON that induce skipping is around 30%, depending on the actual exon that is targeted. Since the first trials, however, the experience gained from AON that successfully induced skipping has resulted in a significant improvement of the success ratio of a designed AON (PMID: 18813282, Aartsma-Rus et al., Mol. Ther. 17(3):548 (2009). The factors that improve the success ratio include, among others, the predicted structure of the exon RNA at the target site, the exact sequence targeted, and the predicted presence or absence of specific SR-protein binding sites in the target site (ibid).
Skipping of an exon sequence encoding a proteolytic cleavage site is preferably such that downstream amino acids of the target protein are present in the newly formed protein. In this way, the proteolytic cleavage site is removed while leaving much of the downstream protein intact. In this embodiment, the functionality of the modified protein is at least part of the functionality of the protein as present in normal individuals. Thus, preferably, the modified protein contains an “in frame” deletion of the proteolytic cleavage site. Preferably, the “in frame” deleted protein has at least 20%, preferably at least 50% of the functionality of the unmodified protein in a normal individual. Thus, in certain embodiments, the number of nucleotides that is skipped is dividable by three. Skipping of an exon sequence that codes for a proteolytic cleavage site is typically achieved by skipping the exon that contains this sequence. Skipping of the target exon is sufficient if this exon contains a number of nucleotides that is dividable by three. If the exon contains another number, it is preferred to also skip an adjacent exon, such that the total number of skipped nucleotides is again dividable by three. In most cases, the skipping of an adjacent exon is sufficient; however, if this also does not result in a number of skipped nucleotides that is dividable by three, the skipping of yet a further exon, adjacent to the two mentioned, may be necessary. Skipping of four or more exons is possible but often does not yield a lot of the correct protein. Sometimes, it is possible to skip only a part of an exon. This is either the 5′ part of the 3′ part of the exon. This occurs when the exon contains a cryptic 3′ or 5′ splice site that can be activated.
The term “pre-mRNA” refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template in the cell nucleus by transcription. Within the context of the invention, inducing and/or promoting skipping of an exon sequence that codes for a proteolytic cleavage site, as indicated herein, means that at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the mRNA encoding of the targeted protein in a cell will not contain the skipped exon sequence (modified/(modified+unmodified) mRNA). This is preferably assessed by PCR as described in the examples.
An AON hereof that induces skipping of an exon sequence that encodes a proteolytic cleavage site, preferably, comprises a sequence that is complementary to the exon. In some embodiments, the AON induces skipping of an exon in its entirety. In other embodiments, the AON induces skipping of a part of an exon, preferably, the part encodes a proteolytic cleavage site. Preferably, the AON contains a continuous stretch of between 8-50 nucleotides that is complementary to the exon. An AON hereof preferably comprises a stretch of at least 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 nucleotides that is complementary to the exon. In certain embodiments, the AON contains a continuous stretch of between 12-45 nucleotides that is complementary to the exon. More preferably, a stretch of between 15-41 nucleotides. Depending on the chemical modification introduced into the AON the complementary stretch may be at the smaller side of the range or at the larger side. A preferred antisense oligonucleotide, according to the invention, comprises a T-O alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications, such as halogenated derivatives. A most preferred AON, comprises of 2′-O-methyl phosphorothioate ribose. Such AON, typically, do not need to have a very large complementary stretch. Such AON, typically, contain a stretch of between 15-25 complementary nucleotides. As described herein below, another preferred AON hereof comprises a morpholino backbone. AON comprising such backbones typically contain somewhat larger stretches of complementarity. Such AON, typically, contain a stretch of between 25-40 complementary nucleotides. When in this invention reference is made to the range of nucleotides, this range includes the number(s) mentioned. Thus, by way of example, when reference is made to a stretch of between 8-50, this includes 8 and 50.
An AON hereof that is complementary to a target RNA is capable of hybridizing to the target RNA under stringent conditions. Typically, this means that the reverse complement of the AON is at least 90% and, preferably, at least 95% and, more preferably, at least 98% and, most preferably, at least 100% identical to the nucleotide sequence of the target at the targeted sited. An AON hereof, thus preferably, has two or less mismatches with the reverse complement of the target RNA, preferably, it has one or no mismatches with the reverse complement of the target RNA. In another preferred embodiment, the AON may be specifically designed to have one or more mismatches, preferably, one or two mismatches, e.g., in cases where it is necessary to reduce the affinity when the skipping of the 100% complementary AON is more effective than biologically desired in view of the necessary remaining protein activity. A mismatch is defined herein as a nucleotide or nucleotide analogue that does not have the same base pairing capacity in kind, not necessarily in amount, as the nucleotide it replaces. For instance, the base of uracil that replaces a thymine and vice versa, is not a mismatch. A preferred mismatch comprises an inosine. An inosine nucleotide is capable of pairing with any natural base in an RNA, i.e., capable of pairing with an A, C, G or U in the target RNA.
In certain embodiments, the nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six-membered ring, and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane. One study comparing several of these methods, found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium. A modified backbone is typically preferred to increase nuclease resistance of the AON, the target RNA or the AON/target RNA hybrid, or a combination thereof. A modified backbone can also be preferred because of its altered affinity for the target sequence compared to an unmodified backbone. An unmodified backbone can be RNA or DNA, preferably it is an RNA backbone.
It is further preferred that the linkage between the residues in a backbone does not include a phosphorus atom, such as a linkage that is 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.
A preferred nucleotide analogue or equivalent, comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of 7V-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun. 495-497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively, (Egholm et al. (1993) Nature 365:566-568).
A further preferred backbone, comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a six-membered morpholino ring. A most preferred nucleotide analog or equivalent, comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a six-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.
In yet a further embodiment, a nucleotide analogue or equivalent of the invention, comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent, comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3′-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.
A further preferred nucleotide analogue or equivalent of the invention, comprises one or more sugar moieties that are mono- or disubstituted at the 2′,3′ and/or 5′ position, such as a —OH; —F; substituted or unsubstituted, linear or branched lower (Cl-ClO) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; -amino xy; methoxyethoxy; -dimethyl-aminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably, a ribose or a derivative thereof, or a deoxyribose or a derivative thereof. Such preferred derivatized sugar moieties comprise Locked Nucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby, forming a bicyclic sugar moiety. A preferred LNA comprises 2′-0,4′-C-ethylene-bridged nucleic acid (Morita et al. 2001, Nucleic Acid Res., Supplement No. 1:241-242). These substitutions render the nucleotide analogue, or equivalent RNase H and nuclease, resistant and increase the affinity for the target RNA. As is apparent to one of skill in the art, the substitutions provided herein render the double-stranded complex of the antisense oligonucleotide with its target pre-mRNA RNase H resistant. Accordingly, preferred oligonucleotides bind to the pre-mRNA of the protein to form a double-stranded nucleic acid complex and are chemically modified to render the double-stranded nucleic acid complex RNAse H resistant.
It is understood by a skilled person that it is not necessary for all positions in an antisense oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents, may be incorporated in a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide hereof has at least two different types of analogues or equivalents.
As mentioned hereinabove, a preferred AON hereof, comprises a T-O alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications, such as halogenated derivatives. A most preferred AON, comprises of 2′-O-methyl phosphorothioate ribose.
An AON can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids, such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains, such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain, such as a cameloid single domain antigen-binding domain.
Additional flanking sequences may be used to modify the binding of a protein to the AON, or to modify a thermodynamic property of the AON, more preferably, to modify target RNA binding affinity.
AON administration in humans is typically well-tolerated. Clinical manifestations of the administration of AON in human clinical trials have been limited to the local side effects following subcutaneous (SC) injection (on the whole intravenous (i.v.) administration seems to be better tolerated) and generalized side effects, such as fever and chills that similar to the response to interferon administration, respond well to paracetamol. More than 4000 patients with different disorders have been treated so far using systemic delivery of first generation AON (phosphorothioate backbone), and approximately 500 following local administration. The typical dosage used ranged from 0.5 mg/kg every other day for one month in Crohn's disease, to 200 mg twice weekly for three months in rheumatoid arthritis, to higher dosages of 2 mg/kg day in other protocols dealing with malignancies. Fewer patients (approx. 300) have been treated so far using new generation AON (uniform phosphorothioated backbone with flanking 2′ methoxyethoxy wing) delivered systemically at doses comprised between 0.5 and 9 mg/kg per week for up to three weeks.
Delivery of AON to cells of the brain can be achieved by various means. For instance, they can be delivered directly to the brain via intracerebral inoculation (Schneider et al., Journal of Neuroimmunology (2008) 195:21-27), intraparenchymal infusion (Broaddus et al., J. Neurosurg. 1998 April; 88(4):734-42), intrathecal, or intraventricularly. Alternatively, the AON can be coupled to a single domain antibody or the variable domain thereof (VHH) that has the capacity to pass the Blood Brain barrier. Nanotechnology has also been used to deliver oligonucleotides to the brain, e.g., a nanogel consisting of cross-linked PEG and polyethylenimine. Encapsulation of AON in liposomes is also well known to one of skill in the art.
An AON hereof may comprise a sequence that is complementary to part of the pre-mRNA, as defined herein. In a more preferred embodiment, the length of the complementary part of the oligonucleotide is of at least 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, 50 nucleotides. Additional flanking sequences may be used to modify the binding of a protein to the molecule or oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably, to modify target RNA binding affinity. An AON hereof may further comprise additional nucleotides that are not complementary to the target site on the target pre-mRNA. In certain embodiments, an AON contains between 8-50 nucleotides. An AON hereof preferably comprises a stretch of at least 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 nucleotides. In certain embodiments, the AON contains a continuous stretch of between 12-45 nucleotides, more preferably, a stretch of between 15-41 nucleotides. Depending on the chemistry of the backbone, as indicated hereinabove, an AON hereof contains between 15-25 nucleotides. An AON hereof with a morpholino backbone typically contains a stretch of between 25-40 nucleotides. In certain embodiments, the indicated amounts for the number of nucleotides in the AON refers to the length of the complementarity to the target pre-mRNA, preferably to an exon internal sequence, however, the target sequence can also be a 5′ or a 3′ splice site of an exon or an intron sequence, such as preferably a branch point. In another preferred embodiment, the indicated amounts refer to the total number of nucleotides in the AON.
Preferably, the complementary part is at least 50% of the length of the oligonucleotide hereof, more preferably, at least 60%, even more preferably, at least 70%, even more preferably, at least 80%, even more preferably, at least 90% or even more preferably, at least 95%, or even more preferably, 98% and most preferably, up to 100% of the length of the oligonucleotide hereof, with the putative exception of deliberately introduced specific mismatches, e.g., for down-regulating affinity when necessary.
With respect to AON that also contain additional nucleotides, the total number of nucleotides typically does not exceed 50, and the additional nucleotides preferably range in number from between 5-25, preferably from 10-25, more preferably, from 15-25. The additional nucleotides typically are not complementary to the target site on the pre-mRNA but may be complementary to another site on the pre-mRNA or may serve a different purpose and not be complementary to the target pre-mRNA, i.e., less than 95% sequence identity of the additional nucleotides to the reverse complement of the target pre-mRNA.
The proteolytic cleavage site that is to be removed from a protein by a method or AON hereof is preferably a serine endoprotease cleavage site, a metaloendopeptidase cleavage site, a secretase cleavage site and/or a caspase cleavage site. In a particularly preferred embodiment, the cleavage site is a caspase cleavage site or secretase cleavage site. Caspases are a family of intracellular cysteine proteases that play a central role in the initiation and execution of programmed cell death. The term caspases is a short form for Cysteine Aspartate-specific Proteases: their catalytical activity depends on a critical cysteine-residue within a highly conserved active-site pentapeptide QACRG, and the caspases specifically cleave their substrates after Asp residues (also the serine-protease granzyme B has specificity for Asp in the P1 position of substrates). More than ten different members of the caspase family have been identified in mammals. According to a unified nomenclature, the caspases are referred to in the order of their publication: so Caspase-1 is ICE (Interleukin-lbeta-Converting Enzyme), the first aspartate-specific cysteine protease described. The secretase family of proteases is subdivided into three groups, the alpha-, beta- and gamma-secretases. In certain embodiments, the secretase is a gamma-secretase.
The protein from which the proteolytic cleavage site is to be removed can be any protein that contains a proteolytic cleavage site. In certain embodiments, the protein is a mammalian protein, more preferably, a primate protein. In a particularly preferred embodiment, the protein is a human protein. In certain embodiments, the protein is associated with a disease in humans. In a particularly preferred embodiment, the protein is associated with a triplet repeat disease in humans. Preferably, a polyglutamine repeat disease.
In certain embodiments, the protein comprises a caspase cleavage site or secretase cleavage site. Preferably, the protein comprises a caspase-3 or -6 proteolytic cleavage site. Preferably, the protein is a protein that is normally present in the brain of a mammal. In a particularly preferred embodiment, the gene encoding the protein is a mutant gene that encodes a trinucleotide repeat expansion when compared to the gene of a normal individual.
In a particularly preferred embodiment, the protein is a protein encoded by one of the genes listed in Table 1a or 1b. In a particularly preferred embodiment, the gene is a mutant gene that is the causative gene in a polyglutamine disorder, preferably a gene of Table 1a. In a particularly preferred embodiment, the gene is the huntingtin (Htt) gene. Htt is expressed in all mammalian cells. The highest concentrations are found in the brain and testes, with moderate amounts in the liver, heart, and lungs. The function of Htt in humans is as yet not entirely resolved. Htt interacts among others with proteins, which are involved in transcription, cell signaling and intracellular transporting. In humans the gene, and in particular mutants thereof, is associated with Huntington's disease (HD). HD is a progressive neurodegenerative genetic disorder, which affects muscle movement and muscle coordination and leads to cognitive decline and dementia. It typically becomes noticeable in middle age. HD is the most common genetic cause of abnormal involuntary writhing movements called chorea and is much more common in people of Western European descent than in those from Asia or Africa. The disease is caused by an autosomal dominant mutation of the Htt-gene. A child of an affected parent has a 50% risk of inheriting the disease.
For the Htt gene, it is preferred that the caspase-6 proteolytic cleave site encoded by exon Htt exon 12 is removed from the Huntingtin protein. It is preferred that the coding region that codes for the proteolytic cleavage site is removed “in frame,” so as to allow incorporation of the normal downstream amino acid sequence into the mutant protein. In one embodiment, the “in frame” removal is achieved by providing the cell with an AON that enables skipping of exon 12 and an AON that enables skipping of exon 13 of the Htt gene. In another preferred embodiment, the “in frame” removal is achieved by providing the cell with an AON capable of inducing exon skipping directed toward the region delimited by nucleotides 269-297 of exon 12 of the Htt gene. In certain embodiments, the AON is directed toward region delimited by nucleotides 207 until 341 of exon 12. It is preferred that the AON is directed toward the internal region delimited by nucleotides 207 until 341 of exon 12. This includes nucleotides 207 and 341. It has been found in the present invention that AON directed toward the preferred regions induce skipping of the last 135 nucleotides of exon 12, thereby producing an “in frame” complete deletion of two active caspase 3 cleavage sites at amino acid 513 and 552, and removal of the first amino acid of an active caspase 6 site, partially located in exon 12 and partially in exon 13. AON HDEx12_1 (Table 2) activates a cryptic splice site at nucleotide 206 in exon 12, leading to the absence of the remainder of exon 12 from the formed mRNA.
Further provided is an isolated and/or recombinant modified Htt mRNA having a deletion of at least nucleotides 207 until 341 of exon 12. The modified Htt mRNA preferably comprises the exons 1-11, the first 206 nucleotides of exon 12 and exons 13-67. In another preferred embodiment, the modified Htt mRNA comprises the exons 1-11, 14-67.
In another embodiment provided is an isolated and/or recombinant modified Htt protein comprising a deletion of amino acids 538-583. The modified Htt protein preferably comprises the amino acid sequence encoded by exons 1-11, the first 206 nucleotides of exon 12, and exons 13-67. In another preferred embodiment, the modified Htt protein comprises the amino acid sequence encoded by exons 1-11, 14-67.
In yet another embodiment provided is an isolated and/or recombinant cell comprising a modified Htt mRNA and/or a modified Htt protein as indicated herein above. Preferably, the cell comprises an Htt gene comprising a coding region of a polyglutamine repeat, the length of which is associated with HD.
For the ATXN3 gene, it is preferred that the caspase cleavage sites in exon 7 is removed from the protein. It is preferred that the coding region that codes for the proteolytic cleavage site is removed “in frame,” so as to allow incorporation of the normal downstream amino acid into the mutant protein. In one embodiment, the “in frame” removal is achieved by providing the cell with an AON that enables skipping of exon 7 and an AON that enables skipping of exon 8 of the ATXN3 gene.
For the ATN1 gene, it is preferred that the caspase 3 cleavage site near the N-terminus of the protein and the polyglutamine tract (¹⁰⁶DSLD¹⁰⁹) in exon 5 is removed from the protein. It is preferred that the coding region that codes for the proteolytic cleavage site is removed “in frame,” so as to allow incorporation of the normal downstream amino acid into the mutant protein. In one embodiment, the “in frame” removal is achieved by providing the cell with an AON that enables skipping of exon 5 and an AON that enables skipping of exon 6 of the ATN1 gene. In certain embodiments, the AON comprises a sequence as depicted in Table 2 under DPRLA AON.
Further provided is an AON, of preferably between 14-40 nucleotides that induces skipping of an exon that encodes a proteolytic cleavage site in a protein. In certain embodiments, provided is an AON comprising a sequence as depicted in Table 2. The AON is suitable for skipping the indicated exon of the gene. In a particularly preferred embodiment, the AON comprises the sequence of HDEx12_1 of Table 2. In another preferred embodiment, provided is an AON as indicated herein above that is specific for the region identified by a sequence of an AON depicted in Table 2. In certain embodiments, the AON comprises at least 10 consecutive nucleotides of the region identified by an oligonucleotide as depicted in Table 2. In a particularly preferred embodiment, provided is an AON, as indicated hereinabove, that is specific for the region identified by a sequence of HDEx12_1 of Table 2.
Further provided is the use of exon-skipping in a cell for removing a proteolytic cleavage site from a protein. Further provided is the use of an AON that induces skipping of an exon that encodes a proteolytic cleavage site in a protein, for removing the proteolytic cleavage site from the protein in a cell that produces pre-mRNA encoding the protein. Further provided is an oligonucleotide of between 14-40 nucleotides that induces skipping of an exon that encodes a proteolytic cleavage site in a protein for use in the treatment of a disease that is associated with a proteolytic cleavage product of the protein.
In another embodiment, provided is a method for altering the proteolytic processing of a protein that comprises a proteolytic cleavage site comprising providing a cell that produces a pre-mRNA that codes for the protein with an AON that is specific for the pre-mRNA; and that prevents inclusion of the code for the proteolytic cleavage site into mature mRNA produced from the pre-mRNA, the method further comprising allowing translation of the mRNA to produce the protein of which the proteolytic processing is altered.
Further provided is a non-human animal comprising an oligonucleotide hereof. Preferably, the non-human animal comprises a mutant gene that encodes a trinucleotide repeat expansion when compared to the gene of a normal individual.
Further provided is a modified human protein from which a proteolytic cleavage site is removed by means of exon skipping. Further provided is an mRNA encoding a modified human protein from which a proteolytic cleavage site is removed by means of exon skipping.
Further provided is a cell encoding a human protein comprising a proteolytic cleavage site, wherein the cell contains an AON hereof for removing the proteolytic cleavage site from the protein in the cell.
The general nomenclature of cleavage site positions of the substrate were formulated by Schecter and Berger, 1967-68 [Schechter and Berger, 1967], [Schechter and Berger, 1968]. They designate the cleavage site between P1-P1′, incrementing the numbering in the N-terminal direction of the cleaved peptide bond (P2, P3, P4, etc.). On the carboxyl side of the cleavage site, numbering are likewise incremented (P1′, P2′, P3′, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: Exon skipping after transfection with various concentrations HDEx12_1 AON. FIG. 1A) Patient derived HD fibroblasts were treated with 1, 25, 150, and 1000 nM HDEx12_1. β-Actin was taken along as loading control. Increasing the AON concentration from 1 nM to 25 nM resulted in a higher skip percentage from 16% to 92% as was measured by Lab-on-a-Chip. The highest skip percentage of 95% was obtained with 150 nM HDEx12_1. Too high concentration of AON resulted in inefficient skip. In the Mock I control (transfection agent only) no skip is visible as expected. The potency of HDEx12_1 exon 12 skip was also seen in another HD and control fibroblast cell line and human neuroblastoma SH-SY5Y cells. FIG. 1B) Schematic representation of PCR of HD exons 9 to 14. Both schematic representation of normal (top) and shorter, skipped exon 12 (bottom) products are shown.

FIG. 2: Log dose response curve of HDEx12_1 AON in a HD fibroblast cell line. X-axis displays the log concentration (nM) and y-axis the percentage of skip. The half maximum inhibitory value (IC50) of the HDEx12_1 AON was found to be 40 nM. The optimal percentage exon 12 skip was achieved with an AON concentration of 150 nM and higher. Results shown as mean±SEM (n=2-3).

FIGS. 3A and 3B: Sanger sequencing of normal (FIG. 3A) and skipped (SEQ ID NO:228) (FIG. 3B) PCR product (SEQ ID NO:229). HDEx12_1 AON transfection in a HD fibroblast cell line resulted in an in-frame skip of 135 nucleotides, which corresponds with 45 amino acids. The observed skip is caused by the activation of an alternative splice site (AG|GTRAG, see dashed box (positions 6-12 of SEQ ID NO:228)), resulting in an alternative splice site exon isoform. This partial exon 12 skip results in the deletion of an active caspase-3 site ⁵⁴⁹DLND⁵⁵²and partial removal of the first amino acid (Isoleucine) of an active caspase-6 site (⁵⁸³IVLD⁵⁸⁶).

FIG. 4: Partial amino acid sequence of the huntingtin protein (see SEQ ID NO:227). Underlined are the amino acids encoded by

exon

12 and 13. Highlighted is the part of the protein that is currently skipped by the exon 12 AON. In bold is the caspase-3 site ⁵¹⁰DSVD⁵¹³, caspase-3 site ⁵⁴⁹DLND⁵⁵²and caspase-6 site ⁵⁸³IVLD⁵⁸⁶.

FIGS. 5A-5D: Schematic diagram of huntingtin. FIG. 5A) Diagram of complete htt protein. PolyQ indicates the polyglutamine tract. The arrows indicate the caspase cleavage sites and their amino acid positions. FIG. 5B) Amino-terminal part of the htt protein. Htt exon 1 to 17 are depicted. The arrows indicate the caspase cleavage sites and their amino acid positions. FIG. 5C) Schematic representation and amino acid sequence of

htt exon

12 and 13 with the caspase cleavage motifs depicted in bold. Exon boundaries are shown with vertical grey bars (SEQ ID NO:230). FIG. 5D) Partial amino acid and nucleotide sequence of htt exon 12 and 13 (SEQ ID NOS:231 and 233). Caspase cleavage motifs are depicted in bold and exon boundary is shown with vertical grey bar. The light grey highlighted sequence denotes the part which is skipped after HDEx12_1 AON treatment.

DETAILED DESCRIPTION

Examples

AON-Mediated Exon Skipping in Neurodegenerative Diseases to Remove Proteolytic Cleavage Sites. AON-Mediated Exon Skipping in Huntington's Disease to Remove Proteolytic Cleavage Sites from the Huntingtin Protein

Methods

AONs and Primers

All AONs consisted of 2′-O-methyl RNA and full length phosphorothioate backbones.

Cell Cultures and AON Transfection

Patient fibroblast cells and human neuroblastoma cells were transfected with AONs at concentrations ranging between 1-1000 nM, using Polyethylenemine (PEI) ExGen500 according to the manufacturer's instructions, with 3.3 μl PEI per μg of transfected AON. A second transfection was performed 24 hours after the first transfection. RNA was isolated 24 hours after the second transfection and cDNA was synthesized using random hexamer primers.

Cell Lines Used:

FLB73 Human Fibroblast Control
GM04022 Human Fibroblast HD
GM02173 Human Fibroblast HD
SH-SY5Y Neuroblastoma Control
Quantitative Real-Time PCR (qRT-PCR) was carried out using the LIGHTCYCLER® 480 System (Roche) allowing for quantification of gene expression.

Agarose Gel and Sanger Sequencing

All PCR products were run on 2% agarose gel with 100 base pair ladders. Bands were isolated using the QIAGEN® PCR purification kit according to manufacturer's instructions. The samples were then sequenced by Sanger sequencing using the Applied Biosystems BigDyeTerminator v3.1 kit.

Lab-On-a-Chip

Lab-on-a-Chip automated electrophoresis was used to quantify the PCR products using a 2100 Bioanalyzer. Samples were made 1 part β-Actin primed product, as a reference transcript, to 5 parts experimental PCR products. The samples were run on a DNA 1000 chip.

Western Blot

Protein was isolated from cells 72 hours after the first transfection and run on a Western blots, transferred onto a PVDF membrane and immunolabelled with primary antibodies recognizing htt, 1H6 or 4C8 (both 1:1,000 diluted)

Materials

AONs and primers were obtained from Eurogentec, Liege, Belgium.
AON sequences:

HDEx12_1:

(SEQ ID NO: 1)

CGGUGGUGGUCUGGGAGCUGUCGCUGAUG

HDEx12_2:

(SEQ ID NO: 2)

UCACAGCACACACUGCAGG

HDEx13_1:

(SEQ ID NO: 3)

GUUCCUGAAGGCCUCCGAGGCUUCAUCA

HDEx13_2:

(SEQ ID NO: 4)

GGUCCUACUUCUACUCCUUCGGUGU

Patient fibroblast cell lines GM04022 and GM02173 were obtained from Coriell, Institute for Medical Research, Camden, USA and control fibroblast cell line FLB73 from Maaike Vreeswijk, LUMC.

Results

Transfection of AON HDEx12_1 in both patient derived HD fibroblast and human neuroblastoma cells showed an efficient skip (see FIGS. 1A and 1B) of exon 12. The optimal percentage exon 12 skip was achieved with a concentration of 150 nM, but a skip was already visible at 1 nM (see FIG. 2). Sanger sequencing confirmed that the last 135 nucleotides of exon 12 were skipped after transfection of the cells with AON HDEx12_1. This corresponded to deletion of 45 amino acids containing two active caspase 3 sites and the first amino acid of an active caspase 6 site (see FIGS. 3A, 3B, and 4). In silico analysis revealed that the observed skip is likely due to the activation of the alternative splice site AG|GTRAG (positions 6-12 of SEQ ID NO:228) resulting in an alternative splice site exon isoform (see FIGS. 3A and 3B).

CONCLUSIONS

With AON HDEx12_1, we have shown a partial skip of exon 12 of the huntingtin transcript that result in a truncated but in frame protein product. Using different cell lines we have confirmed this partial exon 12 skip by Sanger sequencing and in silico analysis revealed an alternative splice site in exon 12 that is likely the cause of this partial skip. This skipped protein product misses two complete caspase-3 cleavage sites located in exon 12, and the first amino acid of the caspase-6 cleavage site that is located on the border of exon 12 and 13. Recent mouse model data showed that the preferred site of in vivo htt cleavage to be at amino acid 552, which is used in vitro by either caspase-3 or caspase-2¹and that mutation of the last amino acid of the caspase 6 cleavage site at amino acid position 586 reduces toxicity in an HD model.²
Functional analysis will be performed to determine whether AON HDEx12_1 can reduce the toxicity of mutant huntingtin and to determine the level of prevention of formation of toxic N-terminal huntingtin fragments. Also other AONs will be tested to completely skip exons 12 and 13 of the huntingtin transcript.

REFERENCES CITED

1. Wellington, C. L. et al. Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells. J. Biol. Chem. 275:19831-19838 (2000).
2. Graham, R. K. et al. Cleavage at the Caspase-6 Site Is Required for Neuronal Dysfunction and Degeneration Due to Mutant Huntingtin. Cell 125:1179-1191 (2006).

TABLE 1a

Polyglutamine (PolyQ) Diseases

		Normal	Pathogenic
		PolyQ	PolyQ
Type	Gene	repeats	repeats

DRPLA (Dentatorubropallidoluysian	ATN1 or DRPLA	6-35	49-88
atrophy)
HD (Huntington's disease)	Htt (Huntingtin)	10-35	35+
SBMA (Spinobulbar muscular atrophy	Androgen receptor on	9-36	38-62
or Kennedy disease)	the X chromosome.
SCA1 (Spinocerebellar ataxia Type 1)	ATXN1	6-35	49-88
SCA2 (Spinocerebellar ataxia Type 2)	ATXN2	14-32	33-77
SCA3 (Spinocerebellar ataxia Type 3 or	ATXN3	12-40	55-86
Machado-Joseph disease)
SCA6 (Spinocerebellar ataxia Type 6)	CACNA1A	4-18	21-30
SCA7 (Spinocerebellar ataxia Type 7)	ATXN7	7-17	38-120
SCA17 (Spinocerebellar ataxia Type 17)	TBP	25-42	47-63

TABLE 1b

Non-Polyglutamine Diseases
Unstable repeat disorders caused by loss-of-function, RNA-mediated, or unknown mechanism

	MIM	Repeat	Gene	Normal	Expanded	Main clinical features
Disease	Number	unit	product	repeat	repeat	length

Loss of function mechanism

FRAXA	309550	(CGC)_n	FMRP	6-60	>200 (full	Mental retardation,
					mutation)	macroorchidsm, connective
						tissue defects, behavioral
						abnormalities
FRAXE	309548	(CCG)_n	FMR2	4-39	200-900	Mental retardation
FRDA	229300	(GAA)_n	Frataxin	6-32	200-1700	Sensory ataxia,
						cardiomyopathy, diabetes

RNA-mediated pathogenesis

DM1	160900	(CTG)_n	DMPK	5-37	50-10,000	Myotonia, weakness,
						cardiac conduction defects,
						insulin resistance, cataracts,
						testicular atrophy, and
						mental retardation in
						congenital form
FXTAS	309550	(CGG)_n	FMR1 RNA	6-60	60-200	Ataxia, tremor,
					(premutation)	Parkinsonism, and dementia

Unknown pathogenic mechanism

SCA8	608768	(CTG)_n	SCA8 RNA	16-34	>74	Ataxia, slurred speech,
						nystagmus
SCA12	604326	(CAG)_n	PPP2R2B	7-45	55-78	Ataxia and seizures
HDL2	606438	(CTG)_n	Junctophilin	7-28	66-78	Similar to HD

Annual Review of Neuroscience, Vol. 30: 575-621 (Volume publication date July 2007)
Trinucleotide Repeat Disorders, Harry T. Orr and Huda Y. Zoghbi

TABLE 2

List of AON

HDEx12_1: CGGUGGUGGUCUGGGAGCUGUCGCUGAUG (SEQ ID

NO: 1)

HDEx12_2: UCACAGCACACACUGCAGG (SEQ ID NO: 2)

HDEx13_1: GUUCCUGAAGGCCUCCGAGGCUUCAUCA (SEQ ID

NO: 3)

HDEx13_2: GGUCCUACUUCUACUCCUUCGGUGU (SEQ ID NO:4)

HDEx12_2 is a comparative example of an oligo-

nucleotide having the nucleotide sequence of Htt

in the sense strand.

DRPLA AONs:

1	DRPLAEx5_18	GUC GCU GCU GCC AUC AUC AU
		(SEQ ID NO: 5)

2	DRPLAEx5_128	AAG AGG AAG CAG GAG GCA GA
		(SEQ ID NO: 6)

3	DRPLAEx5_81	GGA GGA GCC UGG AAC AUU CG
		(SEQ ID NO: 7)

1	DRPLAEx6_80	AAG CUC GCG CUC CUU CUC GC
		(SEQ ID NO: 8)

2	DRPLAEx6_1	CGA GUU GAA GCC GCG AUC CA
		(SEQ ID NO: 9)

3	DRPLAEx6_84	GUU CAA GCU CGC GCU CCU UC
		(SEQ ID NO: 10)

HDEx AON are oligonucleotides for skipping

exons

12 or 13 of the Htt gene.

DRPLA AON are oligonucleotides for skipping exons

5 or 6 of the DRPLA/ATN1 gene.

Table 3 provides further oligonucleotides for exon skipping.
APP: amyloid precursor protein in Alzheimer's disease (AD); ATN1: Atrophin 1 in DRPLA; ATNX3: Ataxin 3 for SCA3; ATXN7: Ataxin 7 in SCAT; TBP: TATA binding protein for SCA17; and HTT in Huntington's disease (HD)

TABLE 3

AON sequences targeting proteins involved in neurodegenerative diseases

			SEQ ID		SEQ ID
Disease	AON Name	Target Sequence	NO:	AON Sequence	NO:

AD	hAPPEx15_1	GTTCTGGGTTGACAAATATCAAG	11	CUUGAUAUUUGUCAACCCAGAAC	12

AD	hAPPEx15_2	CGGAGGAGATCTCTGAAGTGAAG	13	CUUCACUUCAGAGAUCUCCUCCG	14

AD	hAPPEx15_3	GATGCAGAATTCCGACATGAC	15	GUCAUGUCGGAAUUCUGCAUC	16

AD	hAPPEx15_4	CTCAGGATATGAAGTTCATCATC	17	GAUGAUGAACUUCAUAUCCUGAG	18

AD	hAPPEx16_1	GCAATCATTGGACTCATGGT	19	ACCAUGAGUCCAAUGAUUGC	20

AD	hAPPEx16_2	GATCGTCATCACCTTGGTGA	21	UCACCAAGGUGAUGACGAUC	22

AD	hAPPEx16_3	GTACACATCCATTCATCATGGTG	23	CACCAUGAUGAAUGGAUGUGUAC	24

AD	hAPPEx16_4	GCAGAAGATGTGGGTTCAAAC	25	GUUUGAACCCACAUCUUCUGC	26

AD	hAPPEx16_5	GGTGATGCTGAAGAAGAAACAG	27	CUGUUUCUUCUUCAGCAUCACC	28

AD	hAPPEx16_6	TCATCATGGTGTGGTGGAGGTAG	29	CUACCUCCACCACACCAUGAUGA	30

DRPLA	hATN1Ex5_1	CTCCCTCGGCCACAGTCTCCCT	31	AGGGAGACUGUGGCCGAGGGAG	32

DRPLA	hATN1Ex5_2	GCGGAGCCTTAATGATGATGGC	33	GCCAUCAUCAUUAAGGCUCCGC	34

DRPLA	hATN1Ex5_3	AGCAGCGACCCTAGGGATATCG	35	CGAUAUCCCUAGGGUCGCUGCU	36

DRPLA	hATN1Ex5_4	AGGACAACCGAAGCACGTCCC	37	GGGACGUGCUUCGGUUGUCCU	38

DRPLA	hATN1Ex5_5	TGGAAGTGTGGAGAATGACTCTG	39	CAGAGUCAUUCUCCACACUUCCA	40

DRPLA	hATN1Ex5_6	ATCTTCTGGCCTGTCCCAGGGC	41	GCCCUGGGACAGGCCAGAAGAU	42

DRPLA	hATN1Ex5_7	CGACAGCCAGAGGCTAGCTTTGA	43	UCAAAGCUAGCCUCUGGCUGUCG	44

DRPLA	hATN1Ex5_8	CTCGAATGTTCCAGGCTCCTCC	45	GGAGGAGCCUGGAACAUUCGAG	46

DRPLA	hATN1Ex5_9	TCTATCCTGGGGGCACTGGTGG	47	CCACCAGUGCCCCCAGGAUAGA	48

DRPLA	hATN1Ex5_10	TGGACCCCCAATGGGTCCCAAG	49	CUUGGGACCCAUUGGGGGUCCA	50

DRPLA	hATN1Ex5_11	AGGGGCTGCCTCATCAGTGG	51	CCACUGAUGAGGCAGCCCCU	52

DRPLA	hATN1Ex5_12	AAGCTCTGGGGCTAGTGGTGCTC	53	GAGCACCACUAGCCCCAGAGCUU	54

DRPLA	hATN1Ex5_13	ACAAAGCCGCCTACCACTCCAG	55	CUGGAGUGGUAGGCGGCUUUGU	56

DRPLA	hATN1Ex5_14	CTCCACCACCAGCCAACTTCC	57	GGAAGUUGGCUGGUGGUGGAG	58

DRPLA	hATN1Ex5_15	CCAACCACTACCTGGTCATCTG	59	CAGAUGACCAGGUAGUGGUUGG	60

DRPLA	hATN1Ex5_16	TGGCCCAGAGAAGGGCCCAAC	61	GUUGGGCCCUUCUCUGGGCCA	62

DRPLA	hATN1Ex5_17	TTCCTCTTCTGCTCCAGCGCC	63	GGCGCUGGAGCAGAAGAGGAA	64

DRPLA	hATN1Ex5_18	GTTTCCTTATTCATCCTCTAG	65	CUAGAGGAUGAAUAAGGAAAC	66

DRPLA	hATN1Ex5_19	GCCTCTCTGTCTCCAATCAGC	67	GCUGAUUGGAGACAGAGAGGC	68

DRPLA	hATN1Ex5_20	CCATCCCAGGCTGTGTGGAG	69	CUCCACACAGCCUGGGAUGG	70

DRPLA	hATN1Ex5_21	TCTACTGGGGCCCAGTCCACCG	71	CGGUGGACUGGGCCCCAGUAGA	72

DRPLA	hATN1Ex5_22	GCATCACGGAAACTCTGGGCC	73	GGCCCAGAGUUUCCGUGAUGC	74

DRPLA	hATN1Ex5_23	CCACTGGAGGGCGGTAGCTCC	75	GGAGCUACCGCCCUCCAGUGG	76

DRPLA	hATN1Ex5_24	CTCCCTGGGGTCTCTGAGGCC	77	GGCCUCAGAGACCCCAGGGAG	78

DRPLA	hATN1Ex5_25	CACCAGGGCCAGCACACCTGC	79	GCAGGUGUGCUGGCCCUGGUG	80

DRPLA	hATN1Ex5_26	GTGTCCTACAGCCAAGCAGGCC	81	GGCCUGCUUGGCUGUAGGACAC	82

DRPLA	hATN1Ex5_27	CAAGGGTCCTACCCATGTTCAC	83	GUGAACAUGGGUAGGACCCUUG	84

DRPLA	hATN1Ex5_28	CACCGGTGCCTACGGTCACCAC	85	GUGGUGACCGUAGGCACCGGUG	86

DRPLA	hATN1Ex5_29	CTCTTCGGCTACCCTTTCCAC	87	GUGGAAAGGGUAGCCGAAGAG	88

DRPLA	hATN1Ex5_30	GGTCATTGCCACCGTGGCTTC	89	GAAGCCACGGUGGCAAUGACC	90

DRPLA	hATN1Ex5_31	CCACCGTACGGAAAGAGAGCC	91	GGCUCUCUUUCCGUACGGUGG	92

DRPLA	hATN1Ex5_32	CCACCGGGCTATCGAGGAACCTC	93	GAGGUUCCUCGAUAGCCCGGUGG	94

DRPLA	hATN1Ex5_33	CAGGCCCAGGGACCTTCAAGCC	95	GGCUUGAAGGUCCCUGGGCCUG	96

DRPLA	hATN1Ex5_34	CCACCGTGGGACCTGGGCCCCTG	97	CAGGGGCCCAGGUCCCACGGUGG	98

DRPLA	hATN1Ex5_35	GCCACCTGCGGGGCCCTCAGGC	99	GCCUGAGGGCCCCGCAGGUGGC	100

DRPLA	hATN1Ex5_36	CCATCGCTGCCACCACCACCT	101	AGGUGGUGGUGGCAGCGAUGG	102

DRPLA	hATN1Ex5_37	CCTGCCTCAGGGCCGCCCCTG	103	CAGGGGCGGCCCUGAGGCAGG	104

DRPLA	hATN1Ex5_38	GCCGGCTGAGGAGTATGAGACC	105	GGUCUCAUACUCCUCAGCCGGC	106

DRPLA	hATN1Ex5_39	CCAAGGTGGTAGATGTACCCA	107	UGGGUACAUCUACCACCUUGG	108

DRPLA	hATN1Ex5_40	GCCATGCCAGTCAGTCTGCCAG	109	CUGGCAGACUGACUGGCAUGGC	110

DRPLA	hATN1Ex6_1	CCTGGATCGCGGCTTCAACTC	111	GAGUUGAAGCCGCGAUCCAGG	112

DRPLA	hATN1Ex6_2	CCTGTACTTCGTGCCACTGGAGG	113	CCUCCAGUGGCACGAAGUACAGG	114

DRPLA	hATN1Ex6_3	GACCTGGTGGAGAAGGTGCGGCG	115	CGCCGCACCUUCUCCACCAGGUC	116

DRPLA	hATN1Ex6_4	CGCGAAGAAAAGGAGCGCGAGCG	117	CGCUCGCGCUCCUUUUCUUCGCG	118

DRPLA	hATN1Ex6_5	GCGAGCGGGAACGCGAGAAAG	119	CUUUCUCGCGUUCCCGCUCGC	120

DRPLA	hATN1Ex6_6	GCGAGAAGGAGCGCGAGCTTG	121	CAAGCUCGCGCUCCUUCUCGC	122

S CA3	hATXN3Ex7_1	TTGTCGTTAAGGGTGATCTGC	123	GCAGAUCACCCUUAACGACAA	124

S CA3	hATXN3Ex7_2	CTGCCAGATTGCGAAGCTGA	125	UCAGCUUCGCAAUCUGGCAG	126

S CA3	hATXN3Ex7_3	GACCAACTCCTGCAGATGATT	127	AAUCAUCUGCAGGAGUUGGUC	128

S CA3	hATXN3Ex7_4	GGTCCAACAGATGCATCGAC	129	GUCGAUGCAUCUGUUGGACC	130

S CA3	hATXN3Ex7_5	GCACAACTAAAAGAGCAAAG	131	CUUUGCUCUUUUAGUUGUGC	132

S CA3	hATXN3Ex8_1	GTTAGAAGCAAATGATGGCTC	133	GAGCCAUCAUUUGCUUCUAAC	134

S CA3	hATXN3Ex8_2	CTCAGGAATGTTAGACGAAG	135	CUUCGUCUAACAUUCCUGAG	136

S CA3	hATXN3Ex8_3	GAGGAGGATTTGCAGAGGGC	137	GCCCUCUGCAAAUCCUCCUC	138

S CA3	hATXN3Ex8_4	GAGGAAGCAGATCTCCGCAG	139	CUGCGGAGAUCUGCUUCCUC	140

S CA3	hATXN3Ex8_5	GGCTATTCAGCTAAGTATGCAAG	141	CUUGCAUACUUAGCUGAAUAGCC	142

S CA3	hATXN3Ex9_1	GGTAGTTCCAGAAACATATCTC	143	GAGAUAUGUUUCUGGAACUACC	144

S CA3	hATXN3Ex9_2	GCTTCGGAAGAGACGAGAAGC	145	GCUUCUCGUCUCUUCCGAAGC	146

S CA3	hATXN3Ex10_1	CAGCAGCAAAAGCAGCAACAGC	147	GCUGUUGCUGCUUUUGCUGCUG	148

S CA3	hATXN3Ex10_2	GACCTATCAGGACAGAGTTC	149	GAACUCUGUCCUGAUAGGUC	150

S CA7	hATXN7Ex3_1	GAGCGGAAAGAATGTCGGAGC	151	GCUCCGACAUUCUUUCCGCUC	152

S CA7	hATXN7Ex3_2	AGCGGGCCGCGGATGACGTCA	153	UGACGUCAUCCGCGGCCCGCU	154

S CA7	hATXN7Ex3_3	AGCAGCCGCCGCCTCCGCAG	155	CUGCGGAGGCGGCGGCUGCU	156

S CA7	hATXN7Ex3_4	ACACGGCCGGAGGACGGCG	157	CGCCGUCCUCCGGCCGUGU	158

S CA7	hATXN7Ex3_5	GCGCCGCCTCCACCTCGGCCG	159	CGGCCGAGGUGGAGGCGGCGC	160

S CA7	hATXN7Ex3_6	ACCTCGGCCGCCGCAATGGCGA	161	UCGCCAUUGCGGCGGCCGAGGU	162

S CA7	hATXN7Ex3_7	GGCCTCTGCCCAGTCCTGAAGT	163	ACUUCAGGACUGGGCAGAGGCC	164

S CA7	hATXN7Ex3_8	TGATGCTGGGACAGTCGTGGAAT	165	AUUCCACGACUGUCCCAGCAUCA	166

S CA7	hATXN7Ex3_9	AGGCTTCCAAACTTCCTGGGAAG	167	CUUCCCAGGAAGUUUGGAAGCCU	168

HD	hHTTEx12_1	CATCAGCGACAGCTCCCAGACCACCACCG	169	CGGUGGUGGUCUGGGAGCUGUCGCUGAUG	170

HD	hHTTEx12_2	TCACAGCACACACTGCAGGC	171	GCCUGCAGUGUGUGCUGUGA	172

HD	hHTTEx12_3	GGTCAGCAGGTCATGACATCAT	173	AUGAUGUCAUGACCUGCUGACC	174

HD	hHTTEx12_4	AGAGCTGGCTGCTTCTTCAG	175	CUGAAGAAGCAGCCAGCUCU	176

HD	hHTTEx12_5	GATGAGGAGGATATCTTGAG	177	CUCAAGAUAUCCUCCUCAUC	178

HD	hHTTEx12_6	TCAGTGAAGGATGAGATCAGTGG	179	CCACUGAUCUCAUCCUUCACUGA	180

HD	hHTTEx12_7	ATGGACCTGAATGATGGGAC	181	GUCCCAUCAUUCAGGUCCAU	182

HD	hHTTEx12_8	TGACAAGCTCTGCCACTGAT	183	AUCAGUGGCAGAGCUUGUCA	184

HD	hHTTEx12_9	TCCAGCCAGGTCAGCGCCGT	185	ACGGCGCUGACCUGGCUGGA	186

HD	hHTTEx12_10	ACTCAGTGGATCTGGCCAGCT	187	AGCUGGCCAGAUCCACUGAGU	188

HD	hHTTEx13_1	CCTGCAGATTGGACAGCC	189	GGCUGUCCAAUCUGCAGG	190

HD	hHTTEx13_2	GGTACCGACAACCAGTATTT	191	AAAUACUGGUUGUCGGUACC	192

HD	hHTTEx14_1	AACATGAGTCACTGCAGGCAG	193	CUGCCUGCAGUGACUCAUGUU	194

HD	hHTTEx14_2	GCCTTCTGACAGCAGTGTTGAT	195	AUCAACACUGCUGUCAGAAGGC	196

HD	hHTTEx14_3	GTTGAGAGATGAAGCTACTG	197	CAGUAGCUUCAUCUCUCAAC	198

SCA17	hTBPEx3_1:	GCCATGACTCCCGGAATCCCTA	199	UAGGGAUUCCGGGAGUCAUGGC	200

SCA17	hTBPEx3_2:	CCTATCTTTAGTCCAATGATGC	201	GCAUCAUUGGACUAAAGAUAGG	202

SCA17	hTBPEx3_3:	TATGGCACTGGACTGACCCCAC	203	GUGGGGUCAGUCCAGUGCCAUA	204

SCA17	hTBPEx3_4:	GCAGCTGCAGCCGTTCAGCAG	205	CUGCUGAACGGCUGCAGCUGC	206

SCA17	hTBPEx3_5:	GTTCAGCAGTCAACGTCCCAGC	207	GCUGGGACGUUGACUGCUGAAC	208

SCA17	hTBPEx3_6:	AACCTCAGGCCAGGCACCACAG	209	CUGUGGUGCCUGGCCUGAGGUU	210

SCA17	hTBPEx3_7:	GCACCACAGCTCTTCCACTCA	211	UGAGUGGAAGAGCUGUGGUGC	212

SCA17	hTBPEx3_8:	CTCACAGACTCTCACAACTGC	213	GCAGUUGUGAGAGUCUGUGAG	214

SCA17	hTBPEx3_9:	GGCACCACTCCACTGTATCCCT	215	AGGGAUACAGUGGAGUGGUGCC	216

SCA17	hTBPEx3_10:	CATCACTCCTGCCACGCCAGCT	217	AGCUGGCGUGGCAGGAGUGAUG	218

SCA17	hTBPEx3_11:	AGAGTTCTGGGATTGTACCGCA	219	UGCGGUACAAUCCCAGAACUCU	220

SCA17	hTBPEx4_1:	TGTATCCACAGTGAATCTTGGT	221	ACCAAGAUUCACUGUGGAUACA	222

SCA17	hTBPEx4_2:	GGTTGTAAACTTGACCTAAAG	223	CUUUAGGUCAAGUUUACAACC	224

SCA17	hTBPEx4_3:	CATTGCACTTCGTGCCCGAAACG	225	CGUUUCGGGCACGAAGUGCAAUG	226

Claims

1.-20. (canceled)

21. An oligonucleotide of between fourteen (14) and forty (40) nucleotides that induces skipping of an exon or a part thereof that encodes a proteolytic cleavage site in a protein involved in a disease that is associated with a proteolytic cleavage product of the protein, wherein the oligonucleotide binds to pre-mRNA of the protein to form a double-stranded nucleic acid complex, wherein the oligonucleotide is chemically modified to render the double-stranded nucleic acid complex RNase H resistant, and wherein the disease is a polyglutamine disorder.

22. The oligonucleotide of claim 21, wherein the polyglutamine disorder is Huntington's disease (“HD”) or Alzheimer's disease (“AD”).

23. The oligonucleotide of claim 21, wherein the polyglutamine disorder is Huntington's disease (“HD”), and wherein part of exon 12 of human huntingtin pre-mRNA is skipped.

24. The oligonucleotide of claim 21, wherein the polyglutamine disorder is Huntington's disease (“HD”), and wherein nucleotides 207 to 341 of exon 12 of human huntingtin pre-mRNA is skipped.

25. The oligonucleotide of claim 21, wherein the polyglutamine disorder is Huntington's disease (“HD”), and wherein the oligonucleotide comprises a polynucleotide selected from the group consisting of SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, and SEQ ID NO: 188.

26. The oligonucleotide of claim 21, wherein the polyglutamine disorder is Huntington's disease (“HD”), and wherein the oligonucleotide consists of a polynucleotide selected from the group consisting of SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, and SEQ ID NO: 188.

27. The oligonucleotide of claim 21, wherein the proteolytic cleavage site is a caspase-3 cleavage site or a caspase-6 cleavage site.

28. The oligonucleotide of claim 21, wherein at least one nucleotide of the oligonucleotide is chemically modified by a 2′-O-methyl substitution.

29. The oligonucleotide of claim 27, wherein each nucleotide of the oligonucleotide is chemically modified by a 2′-O-methyl substitution.

30. The oligonucleotide of claim 21, wherein at least one nucleotide of the oligonucleotide is chemically modified by a 2′-O-methoxyethyl substitution.

31. The oligonucleotide of claim 29, wherein each nucleotide of the oligonucleotide is chemically modified by a 2′-O-methoxyethyl substitution.

32. The oligonucleotide of claim 21, wherein all internucleoside linkages of the oligonucleotide are phosphorothioated.

33. The oligonucleotide of claim 21, wherein the oligonucleotide is a uniformly 2′-O-methoxyethylribose modified phosphorothioate oligonucleotide.