ANTISENSE OLIGONUCLEOTIDE DIRECTED REMOVAL OF PROTEOLYTIC CLEAVAGE SITES, THE HCHWA-D MUTATION, AND TRINUCLEOTIDE REPEAT EXPANSIONS

Abstract

Described are methods for removing a proteolytic cleavage site, the HCHWA-D mutation or the amino acids encoded by a trinucleotide repeat expansion 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 comprises the proteolytic cleavage site, HCHWA-D mutation or trinucleotide repeat expansion, respectively, the method further comprising allowing translation of mRNA produced from the pre-mRNA.

Claims

1. A method for treating an individual suffering from a disease that is associated with a mutant gene comprising a trinucleotide repeat expansion when compared to the gene of a normal individual, the method comprising: administering to the individual a therapeutically effective amount of one or more anti-sense oligonucleotides that induce skipping of an exonic sequence that comprises the trinucleotide repeat expansion.

2. The method according to claim 1, wherein the mutant gene is the causative gene of a polyglutamine disorder.

3. The method according to claim 2, wherein the gene is the ATXN3 gene and exonic sequences from exons 9 and 10 thereof are skipped.

4. A method for removing amino acids encoded by a trinucleotide repeat expansion from a mutant protein, the method comprising: providing a cell that expresses pre-mRNA encoding the mutant protein with an anti-sense oligonucleotide that induces skipping of an exonic sequence that comprises the trinucleotide repeat expansion, and allowing translation of mRNA produced from the pre-mRNA.

5. The method according to claim 4, wherein the trinucleotide repeat expansion is a polyglutamine expansion.

6. At least one oligonucleotide of between 14-40 nucleotides that induces skipping of an exonic sequence that comprises a trinucleotide repeat expansion in a pre-mRNA.

7. The at least one oligonucleotide of claim 6, wherein the at least one oligonucleotide binds to the pre-mRNA of protein to form a double-stranded nucleic acid complex and wherein the at least one oligonucleotide is chemically modified to render the double-stranded nucleic acid complex RNAse H resistant.

8. The at least one oligonucleotide of claim 6, comprising: a first oligonucleotide that induces skipping of an exonic sequence from exon 9 of ATXN3, and a second oligonucleotide that induces skipping of an exonic sequence from exon 10 of ATXN3 comprising a trinucleotide repeat expansion.

9. A method for treating an individual afflicted with HCHWA-D mutation (hereditary cerebral hemorrhage with amyloidosis, Dutch type), the method comprising: administering to the individual: a therapeutically effective amount of one or more anti-sense oligonucleotides that induce skipping of the exonic sequence that comprises the HCHWA-D mutation, and/or a therapeutically effective amount of one or more cells comprising the oligonucleotides.

10. The method according to claim 9, wherein the oligonucleotide induces skipping of an exonic sequence corresponding to exon 16 of APP751.

11. A method for removing the HCHWA-D mutation from mutant APP protein, the method comprising: providing a cell that expresses pre-mRNA encoding the mutant APP protein with an anti-sense oligonucleotide that induces skipping of the exonic sequence that comprises the HCHWA-D mutation, and allowing translation of mRNA produced from the pre-mRNA in the cell.

12. The method according to claim 11, wherein the oligonucleotide induces skipping of an exonic sequence corresponding to exon 16 of APP751.

13. At least one oligonucleotide of between 14-40 nucleotides that induces skipping of an exonic sequence that comprises the HCHWA-D mutation of mutant APP protein.

14. The at least one oligonucleotide of claim 13, wherein the oligonucleotide binds to the pre-mRNA of the mutant APP protein to form a double-stranded nucleic acid complex, and wherein the oligonucleotide is chemically modified to render the double-stranded nucleic acid complex RNAse H resistant.

15. The at least one oligonucleotide of claim 13, wherein the at least one oligonucleotide comprises an oligonucleotide that induces skipping of an exonic sequence corresponding to exon 16 of APP751.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0090] 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. -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-SYSY 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.

[0091] 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).

[0092] 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 .sup.549DLND.sup.552 and partial removal of the first amino acid (Isoleucine) of an active caspase-6 site (.sup.583IVLD.sup.586).

[0093] 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 in red is the part of the protein that is currently skipped by the exon 12 AON. In bold is the caspase-3 site .sup.510DSVD.sup.513, caspase-3 site .sup.549DLND.sup.552 and caspase-6 site .sup.583IVLD.sup.586.

[0094] 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.

[0095] FIG. 6: Schematic representation of caspase motif hotspot in the htt protein and the skipping of exon 12 and 13. (Panel a) The active caspase-6 site at amino acid position 586 (IVLD) is encoded by the last 3 nucleotides of exon 12 and the first 9 nucleotides of exon 13. Exon 12 also encodes two active caspase-3 sites at amino acids 513 (DSVD) and 552 (DLND). (Panel b) To remove all three proteolytic cleavage sites from the htt protein, both exon 12 and 13 have to be skipped by using 3 AONs. (Panel c) AON12.1 targets a region in the 3 part of htt exon 12. This results in the activation of a 5 cryptic splice site and an in-frame exclusion of the 3 part of exon 12. The resulting protein lacks the caspase-3 site at amino acid 552 (DLND), and the isoleucine (I) of the active caspase-6 site at amino acid 586 (IVLD) is replaced by a glutamine (Q).

[0096] FIG. 7: Optimal AON concentrations. Human fibroblasts were transfected with concentrations ranging from 10 to 200 nM per htt AON. RNA was isolated after 24 hours and Lab-on-a-Chip analysis was performed to calculate exon skip levels for AON12.1 and AON12.2 combined (Panel a), and AON13 (Panel b). (*P<0.05, **P<0.01, ***P<0.001, n=4).

[0097] FIG. 8: Transfection to induce double and single exon skipping. Control and HD patient fibroblasts were transfected with htt AONs, control AON, and non-transfected cells (Mock) and RNA was isolated after 24 hours. (Panel a) Agarose gel analysis of the htt transcript with primers flanking exon 12 and 13. Transfection with 100 nM per AON resulted in a product lacking both exon 12 and 13 (AON12.1+12.2+13) or a shorter than expected PCR product after transfection with 100 nM AON12.1, lacking the 3 part of exon 12. (Panel b) Lab-on-a-Chip analysis was performed to calculate partial exon 12 skip levels in control cells transfected with AON12.1 concentrations ranging from 10 to 200 nM. (Panel c) Skip efficiencies determined by Lab-on-a-Chip after 50 nM AON treatment in HD cells. (Panel d) Skip efficiencies determined by Lab-on-a-Chip after 50 nM AON treatment in control cells. Both partial skip (AON12.1) and full skip percentages (AON12.1, AON12.2, and AON13) are shown in Panels c and d (**P<0.01, ***P<0.001, n=4). Partial skip (AON12.1) (Panel e) (SEQ ID NO:249) and full skip (AON12.1, AON12.2, and AON13) (Panel f) (SEQ ID NO:250) confirmed by Sanger sequencing.

[0098] FIG. 9: Formation of modified htt protein after partial exon 12 skipping that is resistant to caspase-6 cleavage. Human control fibroblasts were transfected with 50 nM AON12.1 and control AONs. (Panel a) Transfection with AON12.1 resulted in the appearance of a htt protein that is shorter than the full length protein and runs around 343 kDa. (Panel b) Levels of normal (black bars) and shorter (white bar) htt protein after transfection with AON12.1 determined by Odyssey software quantification (** P<0.01, n =6). (Panel c) In vitro caspase-6 cleavage assay shows a 586 amino acids N-terminal htt fragment appearance of 95 kDa that increases with increasing concentration of caspase-6. In samples from cells treated with AON12.1 this N-terminal htt fragment of 95 kDa is reduced, while an unrelated caspase-6 fragment at 35 kDa remains unchanged. (Panel d) Quantification of Panel c, determined by Odyssey software, using the 35 kDa caspase-6 fragment as reference (*P<0.05, n=4).

[0099] FIG. 10: Skipping murine htt exon 12 and 13 in vitro. Mouse C2C12 cells were transfected with murine htt AONs, control AON, scrambled AON, and not transfected (Mock). (Panel a) Agarose gel analysis of the htt transcript with primers flanking exon 12 and 13. Skipping of htt exon 12 and 13 is seen after transfection with mAON12.1, mAON12.2, and mAON13. (Panel b) Lab-on-a-Chip analysis of double-exon skipping after AON treatment (***P<0.001, n=4).

[0100] FIG. 11: A single bilateral injection of Alexa488 AON into the striatum resulted in widespread distribution in the mouse midbrain. AON-Alexa488 is shown in green. NeuN (Panel a) and GFAP (Panel b) are shown in red. Nuclei were counterstained with DAPI as shown in blue. Scale bar=10 m.

[0101] FIG. 12: Reduction of mouse htt exon 12 and 13 after a single local injection into the mouse striatum. A single injection consisting of mAON12.1, mAON12.2 and mAON13 (10 each) or 30 g scrambled AON was injected bilaterally into the mouse striatum. After seven days, the mice were sacrificed and the presence of exon 12, 13 and 27 in the htt transcript was examined by qRT-PCR. (**P<0.01, n=5).

[0102] FIG. 13: Single exon skipping of ataxin-3 pre-mRNA in vitro. Control fibroblasts were transfected with ataxin-3 AONs, control AON, and non-transfected (mock) and RNA was isolated after 24 hours. (Panel a) Agarose gel analysis of the ataxin-3 transcript with primers flanking exon 9 and 10 (full-length, grey arrowhead). Transfection with 50 nM AON against exon 9 resulted in a product lacking the entire exon 9 (AON9.1, white arrowhead) or lacking the 3 part of exon 9 (AON9.2, two white arrowheads). Transfection with 50 nM AON10 resulted in a product lacking exon 10 (three white arrowheads). Fibroblasts were transfected with concentrations ranging from 10 to 200 nM per ataxin-3 AON and Lab-on-a-Chip analysis was performed to calculate exon skip levels for (Panel b) AON9.1, (Panel c) AON9.2, and (Panel d) AON10. Mean SD, data were evaluated using paired student t-test, *P<0.05, **P<0.01, ***P<0.001, relative to mock transfection, n=4.

[0103] FIG. 14: Double exon skipping of ataxin-3 pre-mRNA in vitro. (Panel a) Schematic representation of two approaches to induce in-frame skipping of the CAG repeat-containing exon. (Panel b) Skip of exon 9 and 10 (AON9.1 +AON10) (SEQ ID NO:251) confirmed by Sanger sequencing. (Panel c) Partial skip of exon 9 and complete skip of exon 10 (AON9.2 +AON10) (SEQ ID NO:252) confirmed by Sanger sequencing. (Panel d) Agarose gel analysis of the ataxin-3 transcript with primers flanking exon 9 and 10. Transfection of control fibroblasts resulted in a product lacking both exon 9 and 10 (AON9.1 +AON10, black arrowhead) or lacking the 3 part of exon 9 and exon 10 (AON9.2 +AON10, white arrowhead). (Panel e) Lab-on-a-Chip analysis was performed to calculate exon skip levels in control cells. Mean+SD, data were evaluated using paired student t-test, **P<0.01, ***P<0.001, relative to mock, n=4.

[0104] FIG. 15: Modified ataxin-3 protein after exon 9 and 10 skipping. Human control and SCA3 fibroblasts were transfected with 50 nM of each AON. (Panel a) Transfection with AON9.1 and AON10, or AON9.2 and AON10 resulted in modified ataxin-3 proteins of 35 kDa (ataxin-3 72aa) and 37 kDa (ataxin-3 59aa), respectively. The modified protein products were shown using an ataxin-3 specific antibody. The reduction in polyQ-containing mutant ataxin-3 was shown with the polyQ antibody 1C2. Densitometric analysis was used after transfection with AONs. Ataxin-3 72aa (white bars) and ataxin-3 59aa (black bars) in (Panel b) control and (Panel c) SCA3 cells. Mean+SD, data were evaluated using paired student t-test, *P<0.05, **P<0.01, ***P<0.001, relative to mock, n=5.

[0105] FIG. 16: Full-length and modified ataxin-3 protein displays identical ubiquitin binding. (Panel a) Schematic representation of the known functional domains of the ataxin-3 protein involved in deubiquitination. The ataxin-3 protein consists of an N-terminal (Josephin) domain with ubiquitin protease activity and a C-terminal tail with the polyQ repeat and three ubiquitin interacting motifs (UIMs). After exon skipping (ataxin-3 59aa), the polyQ repeat is removed, leaving the Josephin domain and UIMs intact. (Panel b) Overview of a leucine (L) to alanine (Panel a) substitution in UIM 1 (L229A), UIM 2 (L249A) or both (L229A/L249A) in full-length ataxin-3 and ataxin-3 59aa. (Panel c) Ubiquitin binding assay. HIS-tagged full-length ataxin-3 and ataxin-3 59aa-bound ubiquitylated proteins were analyzed by Western blot. HIS control and beads only were taken along as negative control. The modified ataxin-3 59aa lacking the polyQ repeat showed identical ubiquitylated protein binding as unmodified ataxin-3. (n=3).

[0106] FIG. 17: Double exon skipping of murine ataxin-3 pre-mRNA in vitro. Mouse C2C12 cells were transfected with murine ataxin-3 AONs, control AON, scrambled AON, and not transfected (Mock). (Panel a) Agarose gel analysis of the ataxin-3 transcript with primers flanking exon 9 and 10. Skipping of ataxin-3 exon 9 and 10 was seen after transfection with mAON9.1 and mAON10. (Panel b) Sanger sequencing confirmed the precise skipping of exon 9 and 10 (SEQ ID NO:253). (Panel c) Transfection with mouse AON9.1 and AON10 resulted in the appearance of a modified ataxin-3 protein of 34 kDa.

[0107] FIG. 18: Reduction of mouse ataxin-3 exon 9 in vivo. Seven days after a single injection consisting of mAON9 and mAON10 (20 g each) into the mouse cerebral ventricle. qRT-PCR analysis of cerebellar tissue showed reduced exon 9 and 10 transcript levels, whereas exon 4 and 11 levels were not affected. Mean+SD, data were evaluated using paired student t-test, *P<0.05, n=3.

[0108] FIG. 19: Schematic of APP pre-mRNA and HCHWA-D mutation. The exon-intron structure of APP751 is depicted in this pre-mRNA schematic representation (SEQ ID NO:254). The shape of the exon-boxes depict the reading frame. The HCHWA-D mutation is located in exon 16 of APP751.

[0109] FIG. 20: APP isoforms in human and mouse. Schematic representation of the exon-intron structure of two different human APP isoforms (APP751 and APP770), and mouse APP (SEQ ID NO:255; NO:254 and NO:256, respectively). The exon containing the HCHWA-D mutation is indicated by the arrow head. Below the exon-intron scheme is the amino acid sequence encoded for by the exons surrounding the HCHWA-D mutation-containing exon, where the HCHWA-D mutation is indicated in italics, and the gamma-secretase cleavage site is indicated by underline.

DETAILED DESCRIPTION

Examples

Example 1: 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

[0110] All AONs consisted of 2-O-methyl RNA and full length phosphorothioate backbones.

Cell Cultures and AON Transfection

[0111] Patient fibroblast cells and human neuroblastoma cells were transfected with AONs at concentrations ranging between 1 and 1000 nM using Polyethylenimine (PEI) ExGen500 according to the manufacturer's instructions, with 3.3 l PEI per g 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:

[0112] FLB73 Human Fibroblast Control

[0113] GM04022 Human Fibroblast HD

[0114] GM02173 Human Fibroblast HD

[0115] SH-SY5Y Neuroblastoma Control

[0116] 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

[0117] All PCR products were run on 2% agarose gel with 100 base pair ladders. Bands were isolated using the QTAgen 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

[0118] Lab-on-a-Chip automated electrophoresis was used to quantify the PCR products using a 2100 Bioanalyzer. Samples were made 1 part -Actin primed product, as a reference transcript, to 5 parts experimental PCR products. The samples were run on a DNA 1000 chip.

Western blot

[0119] Protein was isolated from cells 72 hours after the first transfection and run on a Western blots, transferred onto a PVDF membrane and immunolabeled with primary antibodies recognizing htt, 1H6 or 4C8 (both 1:1,000 diluted)

Materials

[0120] AONs and primers were obtained from Eurogentec, Liege, Belgium.

TABLE-US-00001 AONsequences: HDEx12_1: (SEQIDNO:1) CGGUGGUGGUCUGGGAGCUGUCGCUGAUG HDEx12_2: (SEQIDNO:2) UCACAGCACACACUGCAGG HDEx13_1: (SEQIDNO:3) GUUCCUGAAGGCCUCCGAGGCUUCAUCA HDEx13_2: (SEQIDNO:4) GGUCCUACUUCUACUCCUUCGGUGU

[0121] 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

[0122] 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, 3B).

Conclusions

[0123] With AON HDEx12_1, a partial skip of exon 12 of the huntingtin transcript was shown that results in a truncated but in-frame protein product. Using different cell lines, this partial exon 12 skip was confirmed 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-2 .sup.1 and that mutation of the last amino acid of the caspase 6 cleavage site at amino acid position 586 reduces toxicity in an HD model .sup.2.

[0124] 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.

Example 2: AON Mediated Skipping of htt Exon 12 or 13 in Human Fibroblasts

[0125] The caspase-6 site at amino acid position 586 previously shown to be important in disease pathology is encoded partly in exon 12 and partly in exon 13. Exon 12 also encodes two active caspase-3 sites at amino acids 513 and 552 (10, 33). Skipping of both exon 12 and 13 would maintain the open reading frame and therefore is anticipated to generate a shorter htt protein lacking these 3 caspase sites (see FIG. 6). The AONs used in this study are shown below.

TABLE-US-00002 AONNameSequence(5-3): hHTTEx12_7 (SEQIDNO:182) GUCCCAUCAUUCAGGUCCAU hHTTEx12_5 (SEQIDNO:178) CUCAAGAUAUCCUCCUCAUC hHTTEx13_1 (SEQIDNO:190) GGCUGUCCAAUCUGCAGG ControlAON (SEQIDNO:238) UCCUUUCAUCUCUGGGCUC mAON12.1 (SEQIDNO:239) GGCUCAAGAUGUCCUCCUCAUCC mAON12.2 (SEQIDNO:240) UUUCAGAACUGUCCGAAGGAGUC mAON13 (SEQIDNO:241) GGCUGUCCUAUCUGCAUG ScrambledAON (SEQIDNO:242) CUGAACUGGUCUACAGCUC Alexa488AON (SEQIDNO:243) GGUACACCUAGCGGAACAAU

[0126] AONs were transfected in human fibroblasts, total RNA was isolated after 24 hours and cDNA was amplified using htt primers flanking the skipped exons to examine skipping efficiencies. When transfected individually, none of the AONs induced exon 12 skipping. A complete exon 12 skip of 341 base pairs could be achieved by combining two AONs (hHTTEx12_7 and hHTTEx12_5). The most efficient complete exon 12 skip of 30.9% (0.3%) was achieved by transfecting 100 nM of each hHTTEx12_7 and hHTTEx12_5 (FIG. 7, Panel a).

[0127] Skipping efficiency of htt exon 13 by hHTTEx13_1 was (45.2% 3.4%) at a concentration of 50 nM (FIG. 7, Panel b). Skipped products were confirmed by Sanger sequencing. With a full skip of both exon 12 and 13 the mRNA reading frame is maintained. With a combination of three AONs, an efficient skip of htt exon 12 and exon 13 was achieved. This resulted in a skip of 465 base pairs (FIG. 8, Panel a) that was confirmed by Sanger sequencing (FIG. 8, Panel f). The efficiency of this double skip was 62.4% (3.2%) and 58.0% (19.1%) in HD and control cells, respectively (FIG. 8, Panels c and d). Partial exon 12 skipping results in a shorter htt protein resistant to caspase 6 cleavage. Interestingly, hHTTEx12_7, targeting the 3 part of exon 12 resulted in a partial skip of exon 12 of 135 base pairs (FIG. 8, Panel a) that was confirmed by Sanger sequencing (FIG. 8, Panel e). The highest skipping percentage of hHTTEx12_7 in control cells was 59.9% (0.7%) at a concentration of 50 nM (FIG. 8, Panel b). The efficiency of this partial skip in HD cells was 62.2% (3.6%) (FIG. 8C). This partial exclusion of the 3 part of htt exon 12 can be explained by activation of a cryptic 5 splice site present within exon 12 (AG|GTCAG) (positions 6-12 of SEQ ID NO:228).

[0128] Western blot analysis using the 4C8 antibody indeed revealed an additional band of approximately 343 kDa after transfection with hHTTEx12_7 (FIG. 9, Panel a). The 5 kDa shorter htt protein is in concordance with the calculated 45 amino acid skip after AON12.1 transfection. This shorter htt protein represents 27.9% (15.1%) of total htt protein levels (FIG. 9, Panel b).

[0129] To test if the modified htt protein was resistant to caspase-6 cleavage at amino acid position 586, an in vitro caspase-6 assay was performed. Protein was isolated from human fibroblasts three days after treatment with 50 nM of hHTTEx12_7. Htt protein fragments were detected by Western blotting using the 4C8 antibody. After samples were incubated with recombinant active caspase-6, N-terminal htt fragments of 98 kDa were detected (FIG. 9, Panel c). Previous studies showed that these fragments are involved in neuronal dysfunction and neurodegeneration in HD. Samples treated with hHTTEx12_7 resulted in a 31.9% (21.5%) reduction of these 98 kDa htt fragments, while the shorter htt fragment of 35 kDa, which is not cleaved at amino acid 586, remained unchanged (FIG. 9, Panel d). This shows that mutating the first amino acid of the 586 amino acid caspase-6 motif is sufficient to prevent proteolytic cleavage.

Example 3: Removal of the 586 Caspase-6 Cleavage Site from Mouse htt Protein In Vitro and In Vivo

[0130] To investigate the potential of htt exon skipping in vivo and to test if removal of the amino acid sequence surrounding the 586 caspase-6 cleavage site could be harmful in vivo, AONs homologues to the mouse sequence was designed. Since mice do not exhibit the cryptic splice site that is responsible for the partial skip in human cells, the full skip of exon 12 and 13 as was described for the human cells was investigated. Transfection of 200 nM of each mouse specific htt AON targeting exon 12 and 13 in mouse C2C12 cells showed a skip of both exons with an efficiency of 86.8% (5.6) (FIG. 10, Panels a and b).

[0131] To investigate distribution of the AON in the mouse brain, 10 g of Alexa Fluor 488 labeled control AON was injected bilaterally into the striatum of a control mouse. The mouse was sacrificed after one week, the brain was perfused, and sections were immunolabeled using the neuronal marker NeuN and astrocyte marker glial fibrillary acidic protein (GFAP). Examination under the fluorescence microscope showed AON distribution throughout the midbrain in both astrocytes and neuronal cells (see FIG. 11, Panels a and b).

[0132] Next, a single dose of 30 g scrambled AON or 30 g AON mix (10 g per AON) was injected bilaterally into the mouse striatum. After seven days, the mice were sacrificed and expression levels of exon 12 and 13 in the mouse htt transcript were assessed by qRT-PCR (FIG. 12). Exon 12 was significantly reduced by 21.5% (08.5%) and exon 13 was significantly reduced by 23.1% (8.3%). Exon 27, downstream of the area targeted for skipping, was not reduced showing that a single intrastriatal administration of AONs already resulted in a skip of htt exon 12 and 13.

Material and methods

In Vivo Injection into Mice

[0133] Mouse htt specific AONs (mAON12.1, mAON12.2, and mAON13) and scrambled control AONs were injected in anesthetized C57bl/6j male mice between the ages of 12 and 14 weeks (Janvier SAS, France). Animals were singly housed in individually ventilated cages (IVC) at a 12 hour light cycle with lights on at 7 a.m. Food and water were available ad libitum. Animals were anesthetized with a cocktail of Hypnorm-Dormicum-demineralized water in a volume ratio of 1.33:1:3. The depth of anesthesia was confirmed by examining the paw and tail reflexes. When mice were deeply anesthetized they were mounted on a Kopf stereotact (David Kopf instruments, Tujunga, USA). A total of 30 g AON mix diluted in 2.5 l sterile saline was bilaterally injected at the exact locations 0.50 mm frontal from bregma, 2.0 mm medio-lateral, and 3.5 mm dorso-ventral. For injections, customized borosilicate glass micro-capillary tips of approximately 100 m in diameter, connected to a Hamilton needle (5 l, 30 gauge) were used. The Hamilton syringe was connected to an injection pump (Harvard apparatus, Holliston, Mass., USA) which controlled the injection rate set at 0.5 l/minute. After surgery the animals were returned to the home cage and remained undisturbed until sacrifice, with the exception of daily weighing in order to monitor their recovery from surgery. After seven days, the mice were sacrificed by intraperitoneal injection of overdose Euthasol (ASTfarma, Oudewater, the Netherlands) and brain tissue isolated and snap frozen till further analysis. To determine AON distribution, two mice were injected with 10 g of Alexa Fluor 488 labeled control AON. After seven days, the mice were sacrificed, perfused and brain isolated and frozen till further analysis.

Immunohistochemistry on Mouse Brain Sections

[0134] To assess AON distribution, brains were removed and post fixated overnight in 4% paraformaldehyde (PFA) (Sigma, St. Louis, USA) in PBS at 4 C. Subsequently they were cryoprotected in 15% and 30% sucrose in PBS, snap frozen on dry ice and stored at 80 C. Brains were cut into 30 m sections on a Leica cryostat and sections stored in 0.1% sodium azide in PBS. Sections were stained free floating and after three washes in PBS containing 0.2% TRITON X-100 (PBS-Triton) were incubated overnight at 4 C. with mouse anti-NeuN (Millipore) or rabbit anti-GFAP (Sigma), both diluted 1:5000 in PBS-Triton with 1% normal goat serum and 0.4% Thimerosal (Sigma). Next, sections were washed, incubated for three hours with rabbit anti-Alexa594 (Invitrogen Life Technologies). After three more washes, sections were mounted on glass slides with a DAPI/DABCO solution and examined on a Leica confocal microscope.

Calculations and Statistical Analysis

[0135] RNA and protein skipping percentages were calculated using the following formula: Skipping % =(Molarity skipped product/(Total molarity full length product+skipped product))*100%. The 95 kDa N-terminal htt fragment levels were calculated using the 35 kDa caspase-6 fragment as reference. The skipping percentages were analyzed using a paired two-sided Student t test. Differences were considered significant when P<0.05.

Example 4: AON Mediated Skipping of Ataxin-3 Exon 9 and 10 In Vitro

[0136] The CAG repeat in the ATXN3 gene is located in exon 10, which is 119 nucleotides in length. Thus skipping will disrupt the reading frame. To preserve the reading frame exon 9 (97 nucleotides) and 10 need to be skipped simultaneously (FIG. 14, Panel a). Various AONs were designed targeting exon internal sequences of ataxin-3 exon 9 and 10 and transfected in human fibroblasts (Table 1). PCR analysis revealed a 97 nucleotide skip after transfection with 100 nM of AON9.1 (efficiency=59.2% 1.0%) (FIG. 13, Panels a and b). Sanger sequencing confirmed that this was a skip of exon 9. Transfection with 100 nM AON9.2 resulted in a skip of 55 nucleotides (efficiency=62.3% 3.7%) instead of the anticipated 97 nucleotides (FIG. 13, Panels a and c). Sanger sequencing revealed that this fragment was a partial skip product that still contained the 5 part of exon 9. In silico analysis showed the existence of a cryptic 5 splice site AG|GTCCA in exon 9 that could explain the occurrence of this shorter fragment .sup.24. Successful skipping of exon 10 was achieved with 50 nM AON10 (efficiency=96.3% 0.3%) (FIG. 13, Panels a and d), as confirmed by Sanger sequencing.

[0137] Co-transfection of AON9.1 and AON10 and AON9.2 and AON10 resulted in a skip of respectively 216 and 174 nucleotides (FIG. 14, Panels b and c). The efficiency of the AON9.1 and AON10 induced double skip was 77.0% (0.9%) in control fibroblasts (FIG. 14, Panels d and e). The efficiency of AON9.2 and AON10 co-transfection was 97.8% (0.8%) in control fibroblasts (FIG. 14, Panels d and e). The unexpected in-frame partial skip of exon 9 with AON9.2 resulted in an alternative approach to remove the CAG repeat containing exon from the ataxin-3 protein (FIG. 14, Panels a-e).

Modified Ataxin-3 Protein Maintains its Ubiquitin Binding Capacity

[0138] To investigate if AON transfection resulted in a modified ataxin-3 protein, control and SCA3 fibroblasts were transfected with AONs targeting exon 9 and 10 and protein was isolated three days after transfection. No negative effect was seen on cell viability after AON treatment in either control or SCA3 fibroblasts. Western blot analysis using an ataxin-3-specific antibody revealed a modified band of approximately 35 kDa after the complete skip of exon 9 and 10 (ataxin-3 72aa) (FIG. 15, Panel a); 11.4% (5.1%) and 6.2% (1.9%) of total ataxin-3 protein levels consisted of this modified ataxin-3 72aa protein in, respectively, control and SCA3 fibroblasts (FIG. 15, Panels b and c).

[0139] The partial exon skip resulted in a novel 37 kDa protein (ataxin-3 59aa) (FIG. 15, Panel a). 27.1% (9.0%) and 15.9% (3.2%) of total ataxin-3 protein levels consisted of this 59 amino acids shorter ataxin-3 protein in, respectively, control and SCA3 cells (FIG. 15, Panels b and c). The ataxin-3 72aa protein was also formed, suggesting that AON9.2 and AON10 transfection also resulted in some ataxin-3 72aa protein. The consistent lower percentage of exon skipping in SCA3 cells were caused by the lower AON transfection efficiencies in the diseased cells as compared to control cells.

[0140] A significant reduction in expanded polyQ containing ataxin-3 was shown using the 1C2 antibody that recognizes long glutamine stretches (FIG. 15, Panel a) in the samples with the full and partial exon skip approaches. This indicates a reduction of expanded polyQ-containing ataxin-3 in SCA3 patient derived fibroblasts after AON transfection.

[0141] The polyQ repeat in the ataxin-3 protein is located between the second and third UIM (FIG. 16, Panel a). Both full and partial exon skip approaches resulted in removal of the polyQ repeat, preserving the Josephin domain, nuclear export signal (NES), and UIMs. To investigate whether the ubiquitin binding capacities of the UIMs in ataxin-3 are still intact after protein modification, poly-ubiquitin chains were incubated with purified cell free produced full-length ataxin-3 and ataxin-3 459aa protein. As negative controls, three different ataxin-3 protein products were produced containing 1 amino acid substitutions from leucine (L) to alanine (A) in UIM 1 (L229A), UIM 2 (L249A), or both (L229A/L249A) (FIG. 4). Single amino acid changes in UIM 1 (L229A) already showed reduced binding of ataxin-3 to poly-ubiquitin chains, whereas double UIM mutated ataxin-3 (L229A/L249A) resulted in a nearly complete elimination poly-ubiquitin binding (FIG. 16, Panel c). The negative HIS control protein did not bind ubiquitylated proteins as expected. Ataxin-3 59aa bound poly-ubiquitin chains comparable to full-length ataxin-3, indicating that its ubiquitin binding capacity after protein modification is still intact (FIG. 16, Panel c).

AON Mediated Skipping of Ataxin-3 Exon 9 and 10 in Mouse

[0142] To examine ataxin-3 exon skipping in the mouse brain and to determine if the modified protein is not harmful, AONs specific to the mouse sequence were designed. Since mice do not exhibit the cryptic splice site that is responsible for the partial exon 9 skip in the human transcript, only the full skip of exon 9 and 10 was investigated. Transfection of 200 nM of each murine AON9 (mAON9) and AON10 (mAON10) in mouse C2C12 cells showed a skip of both exons with an efficiency of 31.7% (2.4%) (FIG. 17, Panel a). Sanger sequencing confirmed this in-frame double exon skip (FIG. 17, Panel b). Transfection with mAON9 and mAON10 resulted in formation of a modified protein of 34 kDa (FIG. 17, Panel c).

[0143] Next, a single intra-cerebral ventricular (ICV) injection was administered of 40 ataxin-3 AON mix (20 g per AON) or 40 g scrambled AON. After 7 days, the mice were sacrificed and skipping efficiency in the cerebellum was assessed by qRT-PCR (FIG. 18). Exon 9 was found significantly reduced by 44.5% (7.6%) and exon 10 was reduced by 35.9% (14.1%) after a single ICV injection of AONs as compared to scrambled AON. Exon 4, upstream, and exon 11, downstream of the area targeted for skipping were not reduced, demonstrating a specific skip of ataxin-3 exon 9 and 10 in vivo.

[0144] In the current study, a novel approach to reduce toxicity of the mutant ataxin-3 protein is shown through skipping of the CAG repeat containing exon in the ataxin-3 transcript. The resulting modified ataxin-3 protein lacks the polyQ repeat that is toxic when expanded, but maintains its ubiquitin binding properties. ICV administration of these AONs in mice resulted in skipping of the CAG repeat-containing exon in the cerebellum of control mice, proving distribution and efficiency of ataxin-3 exon skipping after ICV injection in vivo.

[0145] There was no negative effect on cell viability after AON treatment in both control and SCA3 fibroblasts and also no overt toxicity in vivo.

Additional Materials and Methods

Cell Culture and Transfection

[0146] Patient derived fibroblasts from SCA3 patients (GM06151, purchased from Coriell Cell Repositories, Camden, USA) and controls (FLB73, a kind gift from Dr. M. P. G. Vreeswijk, LUMC) were cultured at 37 C. and 5% CO.sub.2 in Minimal Essential Medium (MEM) (Gibco Invitrogen, Carlsbad, USA) with 15% heat inactivated Fetal Bovine Serum (FBS)

[0147] (Clontech, Palo Alto, Calif., USA), 1% Glutamax (Gibco) and 100 U/ml penicillin/streptomycin (P/S) (Gibco). Mouse myoblasts C2C12 (ATCC, Teddington, UK) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) with 10% FBS, 1% glucose, 2% Glutamax and 100 U/ml P/S.

[0148] AON transfection was performed in a six-well plate with 3l of Lipofectamine 2000 (Life Technologies, Paisley, UK) per well. AON and Lipofectamine 2000 were diluted in MEM to a total volume of 500 l and mixtures were prepared according to the manufacturer's instruction. Four different transfection conditions were used: 1) transfection with 1-200 nM AONs, 2) transfection with non-relevant h40AON2 directed against exon 40 of the DMD gene (Control AON) .sup.36, 3) transfection with scrambled AON (Scrambled), and 4) transfection without AON (Mock). For AON sequences, see Table 1. Mixtures were added to a total volume of 1 ml of MEM. Four hours after transfection, medium was replaced with fresh medium containing 5% FBS. All AONs consisted of 2-O-methyl RNA and contained a full-length phosphorothioate modified backbone (Eurogentec, Liege, Belgium).

TABLE-US-00003 TABLE1 AONsequencesusedfortransfectionand injection AONName Sequence(5-3) AON9.1 GAGAUAUGUUUCUGGAACUACC hATXN3Ex9_1 (SEQIDNO:144) AON9.2 GCUUCUCGUCUCUUCCGAAGC hATXN3Ex9_2 (SEQIDNO:146) AON10 GCUGUUGCUGCUUUUGCUGCUG hATXN3Ex10_1 (SEQIDNO:148) ControlAON UCCUUUCAUCUCUGGGCUC (SEQIDNO:238) mAON9.1 GCUUCUCGUCUCCUCCGCAGC (SEQIDNO:247) mAON10 GAACUUGUGGUCGGUCUUUCAC (SEQIDNO:248) ScrambledAON CUGAACUGGUCUACAGCUC (SEQIDNO:242)

TABLE-US-00004 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-US-00005 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).sub.n FMRP 6-60 >200 (full Mental retardation, mutation) macroorchidsm, connective tissue defects, behavioral abnormalities FRAXE 309548 (CCG).sub.n FMR2 4-39 200-900 Mental retardation FRDA 229300 (GAA).sub.n Frataxin 6-32 200-1700 Sensory ataxia, cardiomyopathy, diabetes RNA-mediated pathogenesis DM1 160900 (CTG).sub.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).sub.n FMR1 RNA 6-60 60-200 Ataxia, tremor, (premutation) Parkinsonism, and dementia Unknown pathogenic mechanism SCA8 608768 (CTG).sub.n SCA8 RNA 16-34 >74 Ataxia, slurred speech, nystagmus SCA12 604326 (CAG).sub.n PPP2R2B 7-45 55-78 Ataxia and seizures HDL2 606438 (CTG).sub.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-US-00006 TABLE2 ListofAON HDEx12_1: (SEQIDNO:1) CGGUGGUGGUCUGGGAGCUGUCGCUGAUG HDEx12_2: (SEQIDNO:2) UCACAGCACACACUGCAGG HDEx13_1: (SEQIDNO:3) GUUCCUGAAGGCCUCCGAGGCUUCAUCA HDEx13_2: (SEQIDNO:4) GGUCCUACUUCUACUCCUUCGGUGU

[0149] HDEx12_2 is a comparative example of an oligonucleotide having the nucleotide sequence of Htt in the sense strand.

TABLE-US-00007 DRPLAAONs: 1DRPLAEx5_18 (SEQIDNO:5) GUCGCUGCUGCCAUCAUCAU 2DRPLAEx5_128 (SEQIDNO:6) AAGAGGAAGCAGGAGGCAGA 3DRPLAEx5_81 (SEQIDNO:7) GGAGGAGCCUGGAACAUUCG 1DRPLAEx6_80 (SEQIDNO:8) AAGCUCGCGCUCCUUCUCGC 2DRPLAEx6_1 (SEQIDNO:9) CGAGUUGAAGCCGCGAUCCA 3DRPLAEx6_84 (SEQIDNO:10) GUUCAAGCUCGCGCUCCUUC

[0150] HDEx AON are oligonucleotides for skipping exons 12 or 13 of the Htt gene.

[0151] DRPLA AON are oligonucleotides for skipping exons 5 or 6 of the DRPLA/ATN1 gene.

[0152] Table 3 provides further oligonucleotides for exon skipping.

[0153] 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-US-00008 TABLE3 AONsequencestargetingproteinsinvolvedinneurodegenerativediseases Disease AONName TargetSequence AONSequence AD hAPPEx15_1 GTTCTGGGTTGACAAATATCAAG CUUGAUAUUUGUCAACCCAGAAC (SEQIDNO:11) (SEQIDNO:12) AD hAPPEx15_2 CGGAGGAGATCTCTGAAGTGAAG CUUCACUUCAGAGAUCUCCUCCG (SEQIDNO:13) (SEQIDNO:14) AD hAPPEx15_3 GATGCAGAATTCCGACATGAC GUCAUGUCGGAAUUCUGCAUC (SEQIDNO:15) (SEQIDNO:16) AD hAPPEx15_4 CTCAGGATATGAAGTTCATCATC GAUGAUGAACUUCAUAUCCUGAG (SEQIDNO:17) (SEQIDNO:18) AD hAPPEx16_1 GCAATCATTGGACTCATGGT ACCAUGAGUCCAAUGAUUGC (SEQIDNO:19) (SEQIDNO:20) AD hAPPEx16_2 GATCGTCATCACCTTGGTGA UCACCAAGGUGAUGACGAUC (SEQIDNO:21) (SEQIDNO:22) AD hAPPEx16_3 GTACACATCCATTCATCATGGTG CACCAUGAUGAAUGGAUGUGUAC (SEQIDNO:23) (SEQIDNO:24) AD hAPPEx16_4 GCAGAAGATGTGGGTTCAAAC GUUUGAACCCACAUCUUCUGC (SEQIDNO:25) (SEQIDNO:26) AD hAPPEx16_5 GGTGATGCTGAAGAAGAAACAG CUGUUUCUUCUUCAGCAUCACC (SEQIDNO:27) (SEQIDNO:28) AD hAPPEx16_6 TCATCATGGTGTGGTGGAGGTAG CUACCUCCACCACACCAUGAUGA (SEQIDNO:29) (SEQIDNO:30) DRPLA hATN1Ex5_1 CTCCCTCGGCCACAGTCTCCCT AGGGAGACUGUGGCCGAGGGAG (SEQIDNO:31) (SEQIDNO:32) DRPLA hATN1Ex5_2 GCGGAGCCTTAATGATGATGGC GCCAUCAUCAUUAAGGCUCCGC (SEQIDNO:33) (SEQIDNO:34) DRPLA hATN1Ex5_3 AGCAGCGACCCTAGGGATATCG CGAUAUCCCUAGGGUCGCUGCU (SEQIDNO:35) (SEQIDNO:36) DRPLA hATN1Ex5_4 AGGACAACCGAAGCACGTCCC GGGACGUGCUUCGGUUGUCCU (SEQIDNO:37) (SEQIDNO:38) DRPLA hATN1Ex5_5 TGGAAGTGTGGAGAATGACTCTG CAGAGUCAUUCUCCACACUUCCA (SEQIDNO:39) (SEQIDNO:40) DRPLA hATN1Ex5_6 ATCTTCTGGCCTGTCCCAGGGC GCCCUGGGACAGGCCAGAAGAU (SEQIDNO:41) (SEQIDNO:42) DRPLA hATN1Ex5_7 CGACAGCCAGAGGCTAGCTTTGA UCAAAGCUAGCCUCUGGCUGUCG (SEQIDNO:43) (SEQIDNO:44) DRPLA hATN1Ex5_8 CTCGAATGTTCCAGGCTCCTCC GGAGGAGCCUGGAACAUUCGAG (SEQIDNO:45) (SEQIDNO:46) DRPLA hATN1Ex5_9 TCTATCCTGGGGGCACTGGTGG CCACCAGUGCCCCCAGGAUAGA (SEQIDNO:47) (SEQIDNO:48) DRPLA hATN1Ex5_10 TGGACCCCCAATGGGTCCCAAG CUUGGGACCCAUUGGGGGUCCA (SEQIDNO:49) (SEQIDNO:50) DRPLA hATN1Ex5_11 AGGGGCTGCCTCATCAGTGG CCACUGAUGAGGCAGCCCCU (SEQIDNO:51) (SEQIDNO:52) DRPLA hATN1Ex5_12 AAGCTCTGGGGCTAGTGGTGCTC GAGCACCACUAGCCCCAGAGCUU (SEQIDNO:53) (SEQIDNO:54) DRPLA hATN1Ex5_13 ACAAAGCCGCCTACCACTCCAG CUGGAGUGGUAGGCGGCUUUGU (SEQIDNO:55) (SEQIDNO:56) DRPLA hATN1Ex5_14 CTCCACCACCAGCCAACTTCC GGAAGUUGGCUGGUGGUGGAG (SEQIDNO:57) (SEQIDNO:58) DRPLA hATN1Ex5_15 CCAACCACTACCTGGTCATCTG CAGAUGACCAGGUAGUGGUUGG (SEQIDNO:59) (SEQIDNO:60) DRPLA hATN1Ex5_16 TGGCCCAGAGAAGGGCCCAAC GUUGGGCCCUUCUCUGGGCCA (SEQIDNO:61) (SEQIDNO:62) DRPLA hATN1Ex5_17 TTCCTCTTCTGCTCCAGCGCC GGCGCUGGAGCAGAAGAGGAA (SEQIDNO:63) (SEQIDNO:64) DRPLA hATN1Ex5_18 GTTTCCTTATTCATCCTCTAG CUAGAGGAUGAAUAAGGAAAC (SEQIDNO:65) (SEQIDNO:66) DRPLA hATN1Ex5_19 GCCTCTCTGTCTCCAATCAGC GCUGAUUGGAGACAGAGAGGC (SEQIDNO:67) (SEQIDNO:68) DRPLA hATN1Ex5_20 CCATCCCAGGCTGTGTGGAG CUCCACACAGCCUGGGAUGG (SEQIDNO:69) (SEQIDNO:70) DRPLA hATN1Ex5_21 TCTACTGGGGCCCAGTCCACCG CGGUGGACUGGGCCCCAGUAGA (SEQIDNO:71) (SEQIDNO:72) DRPLA hATN1Ex5_22 GCATCACGGAAACTCTGGGCC GGCCCAGAGUUUCCGUGAUGC (SEQIDNO:73) (SEQIDNO:74) DRPLA hATN1Ex5_23 CCACTGGAGGGCGGTAGCTCC GGAGCUACCGCCCUCCAGUGG (SEQIDNO:75) (SEQIDNO:76) DRPLA hATN1Ex5_24 CTCCCTGGGGTCTCTGAGGCC GGCCUCAGAGACCCCAGGGAG (SEQIDNO:77) (SEQIDNO:78) DRPLA hATN1Ex5_25 CACCAGGGCCAGCACACCTGC GCAGGUGUGCUGGCCCUGGUG (SEQIDNO:79) (SEQIDNO:80) DRPLA hATN1Ex5_26 GTGTCCTACAGCCAAGCAGGCC GGCCUGCUUGGCUGUAGGACAC (SEQIDNO:81) (SEQIDNO:82) DRPLA hATN1Ex5_27 CAAGGGTCCTACCCATGTTCAC GUGAACAUGGGUAGGACCCUUG (SEQIDNO:83) (SEQIDNO:84) DRPLA hATN1Ex5_28 CACCGGTGCCTACGGTCACCAC GUGGUGACCGUAGGCACCGGUG (SEQIDNO:85) (SEQIDNO:86) DRPLA hATN1Ex5_29 CTCTTCGGCTACCCTTTCCAC GUGGAAAGGGUAGCCGAAGAG (SEQIDNO:87) (SEQIDNO:88) DRPLA hATN1Ex5_30 GGTCATTGCCACCGTGGCTTC GAAGCCACGGUGGCAAUGACC (SEQIDNO:89) (SEQIDNO:90) DRPLA hATN1Ex5_31 CCACCGTACGGAAAGAGAGCC GGCUCUCUUUCCGUACGGUGG (SEQIDNO:91) (SEQIDNO:92) DRPLA hATN1Ex5_32 CCACCGGGCTATCGAGGAACCTC GAGGUUCCUCGAUAGCCCGGUGG (SEQIDNO:93) (SEQIDNO:94) DRPLA hATN1Ex5_33 CAGGCCCAGGGACCTTCAAGCC GGCUUGAAGGUCCCUGGGCCUG (SEQIDNO:95) (SEQIDNO:96) DRPLA hATN1Ex5_34 CCACCGTGGGACCTGGGCCCCTG CAGGGGCCCAGGUCCCACGGUGG (SEQIDNO:97) (SEQIDNO:98) DRPLA hATN1Ex5_35 GCCACCTGCGGGGCCCTCAGGC GCCUGAGGGCCCCGCAGGUGGC (SEQIDNO:99) (SEQIDNO:100) DRPLA hATN1Ex5_36 CCATCGCTGCCACCACCACCT AGGUGGUGGUGGCAGCGAUGG (SEQIDNO:101) (SEQIDNO:102) DRPLA hATN1Ex5_37 CCTGCCTCAGGGCCGCCCCTG CAGGGGCGGCCCUGAGGCAGG (SEQIDNO:103) (SEQIDNO:104) DRPLA hATN1Ex5_38 GCCGGCTGAGGAGTATGAGACC GGUCUCAUACUCCUCAGCCGGC (SEQIDNO:105) (SEQIDNO:106) DRPLA hATN1Ex5_39 CCAAGGTGGTAGATGTACCCA UGGGUACAUCUACCACCUUGG (SEQIDNO:107) (SEQIDNO:108) DRPLA hATN1Ex5_40 GCCATGCCAGTCAGTCTGCCAG CUGGCAGACUGACUGGCAUGGC (SEQIDNO:109) (SEQIDNO:110) DRPLA hATN1Ex6_1 CCTGGATCGCGGCTTCAACTC GAGUUGAAGCCGCGAUCCAGG (SEQIDNO:111) (SEQIDNO:112) DRPLA hATN1Ex6_2 CCTGTACTTCGTGCCACTGGAGG CCUCCAGUGGCACGAAGUACAGG (SEQIDNO:113) (SEQIDNO:114) DRPLA hATN1Ex6_3 GACCTGGTGGAGAAGGTGCGGCG CGCCGCACCUUCUCCACCAGGUC (SEQIDNO:115) (SEQIDNO:116) DRPLA hATN1Ex6_4 CGCGAAGAAAAGGAGCGCGAGCG CGCUCGCGCUCCUUUUCUUCGCG (SEQIDNO:117) (SEQIDNO:118) DRPLA hATN1Ex6_5 GCGAGCGGGAACGCGAGAAAG CUUUCUCGCGUUCCCGCUCGC (SEQIDNO:119) (SEQIDNO:120) DRPLA hATN1Ex6_6 GCGAGAAGGAGCGCGAGCTTG CAAGCUCGCGCUCCUUCUCGC (SEQIDNO:121) (SEQIDNO:122) SCA3 hATXN3Ex7_1 TTGTCGTTAAGGGTGATCTGC GCAGAUCACCCUUAACGACAA (SEQIDNO:123) (SEQIDNO:124) SCA3 hATXN3Ex7_2 CTGCCAGATTGCGAAGCTGA UCAGCUUCGCAAUCUGGCAG (SEQIDNO:125) (SEQIDNO:126) SCA3 hATXN3Ex7_3 GACCAACTCCTGCAGATGATT AAUCAUCUGCAGGAGUUGGUC (SEQIDNO:127) (SEQIDNO:128) SCA3 hATXN3Ex7_4 GGTCCAACAGATGCATCGAC GUCGAUGCAUCUGUUGGACC (SEQIDNO:129) (SEQIDNO:130) SCA3 hATXN3Ex7_5 GCACAACTAAAAGAGCAAAG CUUUGCUCUUUUAGUUGUGC (SEQIDNO:131) (SEQIDNO:132) SCA3 hATXN3Ex8_1 GTTAGAAGCAAATGATGGCTC GAGCCAUCAUUUGCUUCUAAC (SEQIDNO:133) (SEQIDNO:134) SCA3 hATXN3Ex8_2 CTCAGGAATGTTAGACGAAG CUUCGUCUAACAUUCCUGAG (SEQIDNO:135) (SEQIDNO:136) SCA3 hATXN3Ex8_3 GAGGAGGATTTGCAGAGGGC GCCCUCUGCAAAUCCUCCUC (SEQIDNO:137) (SEQIDNO:138) SCA3 hATXN3Ex8_4 GAGGAAGCAGATCTCCGCAG CUGCGGAGAUCUGCUUCCUC (SEQIDNO:139) (SEQIDNO:140) SCA3 hATXN3Ex8_5 GGCTATTCAGCTAAGTATGCAAG CUUGCAUACUUAGCUGAAUAGCC (SEQIDNO:141) (SEQIDNO:142) SCA3 hATXN3Ex9_1 GGTAGTTCCAGAAACATATCTC GAGAUAUGUUUCUGGAACUACC (SEQIDNO:143) (SEQIDNO:144) SCA3 hATXN3Ex9_2 GCTTCGGAAGAGACGAGAAGC GCUUCUCGUCUCUUCCGAAGC (SEQIDNO:145) (SEQIDNO:146) SCA3 hATXN3Ex10_1 CAGCAGCAAAAGCAGCAACAGC GCUGUUGCUGCUUUUGCUGCUG (SEQIDNO:147) (SEQIDNO:148) SCA3 hATXN3Ex10_2 GACCTATCAGGACAGAGTTC GAACUCUGUCCUGAUAGGUC (SEQIDNO:149) (SEQIDNO:150) SCA7 hATXN7Ex3_1 GAGCGGAAAGAATGTCGGAGC GCUCCGACAUUCUUUCCGCUC (SEQIDNO:151) (SEQIDNO:152) SCA7 hATXN7Ex3_2 AGCGGGCCGCGGATGACGTCA UGACGUCAUCCGCGGCCCGCU (SEQIDNO:153) (SEQIDNO:154) SCA7 hATXN7Ex3_3 AGCAGCCGCCGCCTCCGCAG CUGCGGAGGCGGCGGCUGCU (SEQIDNO:155) (SEQIDNO:156) SCA7 hATXN7Ex3_4 ACACGGCCGGAGGACGGCG CGCCGUCCUCCGGCCGUGU (SEQIDNO:157) (SEQIDNO:158) SCA7 hATXN7Ex3_5 GCGCCGCCTCCACCTCGGCCG CGGCCGAGGUGGAGGCGGCGC (SEQIDNO:159) (SEQIDNO:160) SCA7 hATXN7Ex3_6 ACCTCGGCCGCCGCAATGGCGA UCGCCAUUGCGGCGGCCGAGGU (SEQIDNO:161) (SEQIDNO:162) SCA7 hATXN7Ex3_7 GGCCTCTGCCCAGTCCTGAAGT ACUUCAGGACUGGGCAGAGGCC (SEQIDNO:163) (SEQIDNO:164) SCA7 hATXN7Ex3_8 TGATGCTGGGACAGTCGTGGAAT AUUCCACGACUGUCCCAGCAUCA (SEQIDNO:165) (SEQIDNO:166) SCA7 hATXN7Ex3_9 AGGCTTCCAAACTTCCTGGGAAG CUUCCCAGGAAGUUUGGAAGCCU (SEQIDNO:167) (SEQIDNO:168) HD hHTTEx12_1 CATCAGCGACAGCTCCCAGACCACCACCG CGGUGGUGGUCUGGGAGCUGUCGCUGAUG (SEQIDNO:169) (SEQIDNO:170) HD hHTTEx12_2 TCACAGCACACACTGCAGGC GCCUGCAGUGUGUGCUGUGA (SEQIDNO:171) (SEQIDNO:172) HD hHTTEx12_3 GGTCAGCAGGTCATGACATCAT AUGAUGUCAUGACCUGCUGACC (SEQIDNO:173) (SEQIDNO:174) HD hHTTEx12_4 AGAGCTGGCTGCTTCTTCAG CUGAAGAAGCAGCCAGCUCU (SEQIDNO:175) (SEQIDNO:176) HD hHTTEx12_5 GATGAGGAGGATATCTTGAG CUCAAGAUAUCCUCCUCAUC (SEQIDNO:177) (SEQIDNO:178) HD hHTTEx12_6 TCAGTGAAGGATGAGATCAGTGG CCACUGAUCUCAUCCUUCACUGA (SEQIDNO:179) (SEQIDNO:180) HD hHTTEx12_7 ATGGACCTGAATGATGGGAC GUCCCAUCAUUCAGGUCCAU (SEQIDNO:181) (SEQIDNO:182) HD hHTTEx12_8 TGACAAGCTCTGCCACTGAT AUCAGUGGCAGAGCUUGUCA (SEQIDNO:183) (SEQIDNO:184) HD hHTTEx12_9 TCCAGCCAGGTCAGCGCCGT ACGGCGCUGACCUGGCUGGA (SEQIDNO:185) (SEQIDNO:186) HD hHTTEx12_10 ACTCAGTGGATCTGGCCAGCT AGCUGGCCAGAUCCACUGAGU (SEQIDNO:187) (SEQIDNO:188) HD hHTTEx13_1 CCTGCAGATTGGACAGCC GGCUGUCCAAUCUGCAGG (SEQIDNO:189) (SEQIDNO:190) HD hHTTEx13_2 GGTACCGACAACCAGTATTT AAAUACUGGUUGUCGGUACC (SEQIDNO:191) (SEQIDNO:192) HD hHTTEx14_1 AACATGAGTCACTGCAGGCAG CUGCCUGCAGUGACUCAUGUU (SEQIDNO:193) (SEQIDNO:194) HD hHTTEx14_2 GCCTTCTGACAGCAGTGTTGAT AUCAACACUGCUGUCAGAAGGC (SEQIDNO:195) (SEQIDNO:196) HD hHTTEx14_3 GTTGAGAGATGAAGCTACTG CAGUAGCUUCAUCUCUCAAC (SEQIDNO:197) (SEQIDNO:198) SCA17 hTBPEx3_1: GCCATGACTCCCGGAATCCCTA UAGGGAUUCCGGGAGUCAUGGC (SEQIDNO:199) (SEQIDNO:200) SCA17 hTBPEx3_2: CCTATCTTTAGTCCAATGATGC GCAUCAUUGGACUAAAGAUAGG (SEQIDNO:201) (SEQIDNO:202) SCA17 hTBPEx3_3: TATGGCACTGGACTGACCCCAC GUGGGGUCAGUCCAGUGCCAUA (SEQIDNO:203) (SEQIDNO:204) SCA17 hTBPEx3_4: GCAGCTGCAGCCGTTCAGCAG CUGCUGAACGGCUGCAGCUGC (SEQIDNO:205) (SEQIDNO:206) SCA17 hTBPEx3_5: GTTCAGCAGTCAACGTCCCAGC GCUGGGACGUUGACUGCUGAAC (SEQIDNO:207) (SEQIDNO:208) SCA17 hTBPEx3_6: AACCTCAGGCCAGGCACCACAG CUGUGGUGCCUGGCCUGAGGUU (SEQIDNO:209) (SEQIDNO:210) SCA17 hTBPEx3_7: GCACCACAGCTCTTCCACTCA UGAGUGGAAGAGCUGUGGUGC (SEQIDNO:211) (SEQIDNO:212) SCA17 hTBPEx3_8: CTCACAGACTCTCACAACTGC GCAGUUGUGAGAGUCUGUGAG (SEQIDNO:213) (SEQIDNO:214) SCA17 hTBPEx3_9: GGCACCACTCCACTGTATCCCT AGGGAUACAGUGGAGUGGUGCC (SEQIDNO:215) (SEQIDNO:216) SCA17 hTBPEx3_10: CATCACTCCTGCCACGCCAGCT AGCUGGCGUGGCAGGAGUGAUG (SEQIDNO:217) (SEQIDNO:218) SCA17 hTBPEx3_11: AGAGTTCTGGGATTGTACCGCA UGCGGUACAAUCCCAGAACUCU (SEQIDNO:219) (SEQIDNO:220) SCA17 hTBPEx4_1: TGTATCCACAGTGAATCTTGGT ACCAAGAUUCACUGUGGAUACA (SEQIDNO:221) (SEQIDNO:222) SCA17 hTBPEx4_2: GGTTGTAAACTTGACCTAAAG CUUUAGGUCAAGUUUACAACC (SEQIDNO:223) (SEQIDNO:224) SCA17 hTBPEx4_3: CATTGCACTTCGTGCCCGAAACG CGUUUCGGGCACGAAGUGCAAUG (SEQIDNO:225) (SEQIDNO:226)

REFERENCES CITED

[0154] 1. Wellington, C. L. et al. Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and non-neuronal cells. J. Biol. Chem. 275, 19831-19838 (2000).

[0155] 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).