ANTISENSE OLIGONUCLEOTIDE DIRECTED REMOVAL OF PROTEOLYTIC CLEAVAGE SITES FROM PROTEINS

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.

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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] 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-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.

[0057] 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 meanSEM (n=2-3).

[0058] 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).

[0059] 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 .sup.510DSVD.sup.513, caspase-3 site .sup.549DLND.sup.552 and caspase-6 site .sup.583IVLD.sup.586.

[0060] 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

[0061] 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

[0062] All AONs consisted of 2-O-methyl RNA and full length phosphorothioate backbones.

Cell Cultures and AON Transfection

[0063] 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 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:

[0064] FLB73 Human Fibroblast Control

[0065] GM04022 Human Fibroblast HD

[0066] GM02173 Human Fibroblast HD

[0067] SH-SY5Y Neuroblastoma Control

[0068] 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

[0069] 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

[0070] 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

[0071] 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

[0072] AONs and primers were obtained from Eurogentec, Liege, Belgium.

[0073] AON sequences:

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

[0074] 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

[0075] 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

[0076] 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-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

[0077] 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

[0078] 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). [0079] 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-US-00002 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-00003 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-00004 TABLE2 ListofAON HDEx12_1:CGGUGGUGGUCUGGGAGCUGUCGCUGAUG(SEQID NO:1) HDEx12_2:UCACAGCACACACUGCAGG(SEQIDNO:2) HDEx13_1:GUUCCUGAAGGCCUCCGAGGCUUCAUCA(SEQID NO:3) HDEx13_2:GGUCCUACUUCUACUCCUUCGGUGU(SEQIDNO:4) HDEx12_2isacomparativeexampleofanoligo- nucleotidehavingthenucleotidesequenceofHtt inthesensestrand. DRPLAAONs: 1 DRPLAEx5_18 GUCGCUGCUGCCAUCAUCAU (SEQIDNO:5) 2 DRPLAEx5_128 AAGAGGAAGCAGGAGGCAGA (SEQIDNO:6) 3 DRPLAEx5_81 GGAGGAGCCUGGAACAUUCG (SEQIDNO:7) 1 DRPLAEx6_80 AAGCUCGCGCUCCUUCUCGC (SEQIDNO:8) 2 DRPLAEx6_1 CGAGUUGAAGCCGCGAUCCA (SEQIDNO:9) 3 DRPLAEx6_84 GUUCAAGCUCGCGCUCCUUC (SEQIDNO:10) HDExAONareoligonucleotidesforskippingexons 12or13oftheHttgene. DRPLAAONareoligonucleotidesforskippingexons 5or6oftheDRPLA/ATN1gene.

[0080] Table 3 provides further oligonucleotides for exon skipping.

[0081] 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-00005 TABLE3 AONsequencestargetingproteinsinvolvedinneurodegenerativediseases SEQID SEQID Disease AONName TargetSequence NO: AONSequence 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 SCA3 hATXN3Ex7_1 TTGTCGTTAAGGGTGATCTGC 123 GCAGAUCACCCUUAACGACAA 124 SCA3 hATXN3Ex7_2 CTGCCAGATTGCGAAGCTGA 125 UCAGCUUCGCAAUCUGGCAG 126 SCA3 hATXN3Ex7_3 GACCAACTCCTGCAGATGATT 127 AAUCAUCUGCAGGAGUUGGUC 128 SCA3 hATXN3Ex7_4 GGTCCAACAGATGCATCGAC 129 GUCGAUGCAUCUGUUGGACC 130 SCA3 hATXN3Ex7_5 GCACAACTAAAAGAGCAAAG 131 CUUUGCUCUUUUAGUUGUGC 132 SCA3 hATXN3Ex8_1 GTTAGAAGCAAATGATGGCTC 133 GAGCCAUCAUUUGCUUCUAAC 134 SCA3 hATXN3Ex8_2 CTCAGGAATGTTAGACGAAG 135 CUUCGUCUAACAUUCCUGAG 136 SCA3 hATXN3Ex8_3 GAGGAGGATTTGCAGAGGGC 137 GCCCUCUGCAAAUCCUCCUC 138 SCA3 hATXN3Ex8_4 GAGGAAGCAGATCTCCGCAG 139 CUGCGGAGAUCUGCUUCCUC 140 SCA3 hATXN3Ex8_5 GGCTATTCAGCTAAGTATGCAAG 141 CUUGCAUACUUAGCUGAAUAGCC 142 SCA3 hATXN3Ex9_1 GGTAGTTCCAGAAACATATCTC 143 GAGAUAUGUUUCUGGAACUACC 144 SCA3 hATXN3Ex9_2 GCTTCGGAAGAGACGAGAAGC 145 GCUUCUCGUCUCUUCCGAAGC 146 SCA3 hATXN3Ex10_1 CAGCAGCAAAAGCAGCAACAGC 147 GCUGUUGCUGCUUUUGCUGCUG 148 SCA3 hATXN3Ex10_2 GACCTATCAGGACAGAGTTC 149 GAACUCUGUCCUGAUAGGUC 150 SCA7 hATXN7Ex3_1 GAGCGGAAAGAATGTCGGAGC 151 GCUCCGACAUUCUUUCCGCUC 152 SCA7 hATXN7Ex3_2 AGCGGGCCGCGGATGACGTCA 153 UGACGUCAUCCGCGGCCCGCU 154 SCA7 hATXN7Ex3_3 AGCAGCCGCCGCCTCCGCAG 155 CUGCGGAGGCGGCGGCUGCU 156 SCA7 hATXN7Ex3_4 ACACGGCCGGAGGACGGCG 157 CGCCGUCCUCCGGCCGUGU 158 SCA7 hATXN7Ex3_5 GCGCCGCCTCCACCTCGGCCG 159 CGGCCGAGGUGGAGGCGGCGC 160 SCA7 hATXN7Ex3_6 ACCTCGGCCGCCGCAATGGCGA 161 UCGCCAUUGCGGCGGCCGAGGU 162 SCA7 hATXN7Ex3_7 GGCCTCTGCCCAGTCCTGAAGT 163 ACUUCAGGACUGGGCAGAGGCC 164 SCA7 hATXN7Ex3_8 TGATGCTGGGACAGTCGTGGAAT 165 AUUCCACGACUGUCCCAGCAUCA 166 SCA7 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