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RNA MODULATING OLIGONUCLEOTIDES WITH IMPROVED CHARACTERISTICS FOR THE TREATMENT OF NEUROMUSCULAR DISORDERS

20200149043 ยท 2020-05-14

    Inventors

    Cpc classification

    International classification

    Abstract

    The current invention provides an improved oligonucleotide and its use for treating, ameliorating, preventing, delaying and/or treating a human cis-element repeat instability associated genetic neuromuscular or neurodegenerative disorder.

    Claims

    1-15. (canceled)

    16. A method for treating, delaying, ameliorating and/or preventing a spinocerebellar ataxia (SCA), spinal and bulbar muscular atrophy (SBMA), or dentatorubral-pallidoluysian atrophy (DRPLA) in a subject, comprising providing to the subject an antisense oligonucleotide comprising a 2-O-methyl RNA nucleotide residue, having a backbone wherein at least one phosphate moiety is replaced by a phosphorothioate moiety, and comprising one or more 5-methylpyrimidine and/or one or more 2,6-diaminopurine bases, wherein said oligonucleotide is able to hybridize to a (CAG)n repetitive nucleotide unit.

    17. The method of claim 16, wherein said oligonucleotide comprises or consists of a repetitive nucleotide unit (XYG)m, wherein m is an integer from 4 to 12 and each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil, wherein at least one X is 5-methylcytosine and/or at least one Y is 5-methyluracil.

    18. The method of claim 17, wherein said oligonucleotide is such that each X is 5-methylcytosine and/or each Y is 5-methyluracil.

    19. The method of claim 17, wherein said oligonucleotide is such that m is 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12.

    20. The method of claim 19, wherein said oligonucleotide comprises or consists of a repetitive nucleotide unit (XYG).sub.7, wherein each X is 5-methylcytosine and each Y is a uracil (SEQ ID NO: 2), or each X is a cytosine and each Y is 5-methyluracil (SEQ ID NO:3).

    21. The method of claim 19, wherein said oligonucleotide is such that m is 7.

    22. The method of claim 20, wherein the sequence of said oligonucleotide comprises or consists of any of SEQ ID NO: 90-118.

    23. The method of claim 22, wherein the base sequence of said oligonucleotide comprises or consists of SEQ ID NO:90.

    24. The method of claim 16, wherein said oligonucleotide is a single-stranded oligonucleotide.

    25. The method of claim 16 wherein said oligonucleotide has a length of from 12 to 36 nucleotides.

    26. The method of claim 16, wherein said oligonucleotide remain in association to its target when there are up to 20% of mismatched nucleotides.

    27. The method of claim 16, wherein said oligonucleotide is at least 90% reverse complementary with said repetitive nucleotide unit.

    28. The method of claim 16, wherein said oligonucleotide has an improved parameter by comparison to a corresponding oligonucleotide comprising a 2-O-methyl RNA nucleotide residue and having a backbone wherein at least one phosphate moiety is replaced by a phosphorothioate moiety, without a 5-methylcytosine, and/or a 5-methyluracil and/or a 2,6-diaminopurine.

    29. The method of claim 16, wherein said oligonucleotide reduces a detectable amount of mutant transcript and/or reduces the translation of said mutant transcript and thus the amount of mutant protein.

    30. The method of claim 29, wherein said reduction of mutant protein reduces protein aggregates in nucleus and/or cytoplasm.

    31. The method of claim 16, wherein said oligonucleotide is present in a composition.

    32. The method of claim 31, wherein said composition comprises at least one excipient that may further aid in enhancing the targeting and/or delivery of said composition and/or said oligonucleotide to a tissue and/or cell and/or into a tissue and/or cell.

    33. The method of claim 16, wherein said oligonucleotide preferentially hybridizes to a disease-associated or disease-causing transcript and leaves a function of a normal transcript relatively unaffected.

    34. The method of claim 16, wherein said subject has DRPLA, SBMA, or SCA type 1, 2, 3, 6, 7, 12 or 17.

    35. The method of claim 34, wherein said SCA type 1 is caused by CAG repeat expansions in the transcripts of ATXN1 (SEQ ID NO:81), said SCA type 2 is caused by CAG repeat expansions in the transcripts of ATXN2 (SEQ ID NO: 82), said SCA type 3 is caused by CAG repeat expansions in the transcripts of ATXN3 (SEQ ID NO: 83), said SCA type 6 is caused by CAG repeat expansions in the transcripts of CACNA1A (SEQ ID NO:84), said SCA type 7 is caused by CAG repeat expansions in the transcripts of ATXN7 (SEQ ID NO: 85), said SCA type 12 is caused by CAG repeat expansions in the transcripts of PPP2R2B (SEQ ID NO: 86), and said SCA type 17 is caused by CAG repeat expansions in the transcripts of TBP (SEQ ID NO: 87) genes.

    36. The method of claim 16, wherein said SBMA is caused by CAG repeat expansions in the transcripts of AR (SEQ ID NO: 88), and said DRPLA is caused by CAG repeat expansions in the transcripts of ATN1 (SEQ ID NO: 89) genes.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0335] FIGS. 1A-1B. In vitro activity assay for (XYG).sub.7 in which X=5-methylcytosine and Y=U (PS659 SEQ ID NO:90; derived from SEQ ID NO:2) and (XYG).sub.7 in which X=C and Y is 5-methyluracil (PS661 SEQ ID NO: 97; derived from SEQ ID NO:3). PS659 (FIG. 1A) and PS661 (FIG. 1B) were transfected into HD fibroblasts (GM04022) at increasing concentrations (0.5-200 nM). Efficacy and selectivity was determined with RT-PCR and lab-on-a-chip analysis. Silencing of the expanded ((CAG).sub.44) and healthy ((CAG).sub.18) HTT transcripts were compared to the relative HTT transcript levels in mock samples. For all AONs n=2 except for mock (n=3).

    [0336] FIGS. 2A-2D. In vivo efficacy of PS659 ((XYG).sub.7 in which X=5-methylcytosine and Y=U; SEQ ID NO:2) in a transgenic HD rat model. Transgenic HD rats ((CAG).sub.51 repeat) received 15 times an intraventricular injection with PS659 (SEQ ID NO:90 derived from SEQ ID NO: 2), during 18 weeks at a final dose of 200 g per injection, control HD rats received vehicle only. Rats were sacrificed one week after the final injection. From all rats tissue was isolated and HTT levels were determined with Q-RT-PCR analysis. Reduced levels of HTT transcript were found in (FIG. 2A) cortex, (FIG. 2B) hippocampus, (FIG. 2C) olfactory bulb and (FIG. 2D) thalamus after PS659 treatment compared to control.

    TABLE-US-00001 TABLE 1 General structures of AONs. X = C or 5-methylcytosine Y = U or 5-methyl- uracil, Z = A or 2,6-diaminopurine, I = inosine, and Q = abasic monomer. Target Repeat AON Sequence (5 .fwdarw. 3) SEQ ID NO (CAG)n (XYG)7 (PS57) 1 X = C, Y = U (XYG)7 (PS659) 2 X = 5-methylcytosine, Y = U (XYG)7 (PS661) 3 X = C, Y = 5-methyluracil (XYG)4 4 (XYG)5 5 (XYG)6 6 (XYG)7 7 (XYG)8 8 (XYG)9 9 (XYG)10 10 (XYG)11 11 (XYG)12 12 (GCG)n (XGX)4 13 (XGX)5 14 (XGX)6 15 (XGX)7 16 (XGX)8 17 (XGX)9 18 (XGX)10 19 (XGX)11 20 (XGX)12 21 (CGG)n (XXG)4 22 (XXG)5 23 (XXG)6 24 (XXG)7 25 (XXG)8 26 (XXG)9 27 (XXG)10 28 (XXG)11 29 (XXG)12 30 (GAA)n (YYX)4 31 (YYX)5 32 (YYX)6 33 (YYX)7 34 (YYX)8 35 (YYX)9 36 (YYX)10 37 (YYX)11 38 (YYX)12 39 (GCC)n (GGX)4 40 (GGX)5 41 (GGX)6 42 (GGX)7 43 (GGX)8 44 (GGX)9 45 (GGX)10 46 (GGX)11 47 (GGX)12 48 (CCG)n (XGG)4 49 (XGG)5 50 (XGG)6 51 (XGG)7 52 (XGG)8 53 (XGG)9 54 (XGG)10 55 (XGG)11 56 (XGG)12 57 (AUUCU)n (ZGZZY)3 58 (ZGZZY)4 59 (ZGZZY)5 60 (ZGZZY)6 61 (ZGZZY)7 62 (CCUG)n (XZGG)3 63 (XZGG)4 64 (XZGG)5 65 (XZGG)6 66 (XZGG)7 67 (XZGG)8 68 (XZGG)9 69 (GGGGCC)n (GGXUXX)3 216 (GGXUXX)4 217 (GGXIXX)4 218 (GGXQXX)4 219 Note: All AONs with SEQ ID NO: 4-69, or 216-219 comprise at least one base modification selected from 5-methylcytosine, 5-methyluracil, and 2,6-diaminopurine.

    TABLE-US-00002 TABLE2 GeneralstructuresofAONs.AllAONsare2-O-methylphosphorothioate AONswhereinCis5-methylcytosine,Uis5-methyluracil,Ais2,6- diaminopurine,IisinosineandQisanabasicmonomer. Target AON SEQ Repeat ID AONSequence(5.fwdarw.3) IDNO (CAG)n PS659 CUGCUGCUGCUGCUGCUGCUG 90 CUGCUGCUGCUGCUGCUGCUG 91 CUGCUGCUGCUGCUGCUGCUG 92 CUGCUGCUGCUGCUGCUGCUG 93 CUGCUGCUGCUGCUGCUGCUG 94 CUGCUGCUGCUGCUGCUGCUG 95 CUGCUGCUGCUGCUGCUGCUG 96 PS661 CUGCUGCUGCUGCUGCUGCUG 97 CUGCUGCUGCUGCUGCUGCUG 98 CUGCUGCUGCUGCUGCUGCUG 99 CUGCUGCUGCUGCUGCUGCUG 100 CUGCUGCUGCUGCUGCUGCUG 101 CUGCUGCUGCUGCUGCUGCUG 102 CUGCUGCUGCUGCUGCUGCUG 103 PS660 CUGCUGCUGCUGCUGCUGCUG 104 CUGCUGCUGCUGCUGCUGCUG 105 CUGCUGCUGCUGCUGCUGCUG 106 PS684 CUGCUGCUGCUGCUGCUGCUG 107 CUGCUGCUGCUGCUGCUGCUGQQQQ 220 CUGCUGCUGCUGCUGCUGCUG 108 CUGCUGCUGCUGCUGCUGCUGQQQQ 221 CUGCUGCUGCUGCUGCUGCUGCUG 109 CUGCUGCUGCUGCUGCUGCUGCUG 110 CUGCUGCUGCUGCUGCUGCUGCUGCUG 111 CUGCUGCUGCUGCUGCUGCUGCUGCUG 112 CUGCUGCUGCUGCUGCUGCUGCUGCUGCUG 113 CUGCUGCUGCUGCUGCUGCUGCUGCUGCUG 114 CUGCUGCUGCUGCUGCUGCUGCUGCUGCUG 115 CUG CUGCUGCUGCUGCUGCUGCUGCUGCUGCUG 116 CUG CUGCUGCUGCUGCUGCUGCUGCUGCUGCUG 117 CUGCUG CUGCUGCUGCUGCUGCUGCUGCUGCUGCUG 118 CUGCUG (GCG)n CGCCGCCGCCGC 119 CGCCGCCGCCGC 120 CGCCGCCGCCGCCGC 121 CGCCGCCGCCGCCGC 122 CGCCGCCGCCGCCGCCGC 123 CGCCGCCGCCGCCGCCGC 124 CGCCGCCGCCGCCGCCGCCGC 125 CGCCGCCGCCGCCGCCGCCGC 126 CGCCGCCGCCGCCGCCGCCGCCGC 127 CGCCGCCGCCGCCGCCGCCGCCGC 128 CGCCGCCGCCGCCGCCGCCGCCGCCGC 129 CGCCGCCGCCGCCGCCGCCGCCGCCGC 130 CGCCGCCGCCGCCGCCGCCGCCGCCGCCGC 131 CGCCGCCGCCGCCGCCGCCGCCGCCGCCGC 132 (CGG)n CCGCCGCCGCCG 133 CCGCCGCCGCCG 134 CCGCCGCCGCCGCCG 135 CCGCCGCCGCCGCCG 136 CCGCCGCCGCCGCCGCCG 137 CCGCCGCCGCCGCCGCCG 138 CCGCCGCCGCCGCCGCCGCCG 139 CCGCCGCCGCCGCCGCCGCCG 140 CCGCCGCCGCCGCCGCCGCCGCCG 141 CCGCCGCCGCCGCCGCCGCCGCCG 142 CCGCCGCCGCCGCCGCCGCCGCCGCCG 143 CCGCCGCCGCCGCCGCCGCCGCCGCCG 144 CCGCCGCCGCCGCCGCCGCCGCCGCCGCCG 145 CCGCCGCCGCCGCCGCCGCCGCCGCCGCCG 146 (GAA)n UUCUUCUUCUUC 147 UUCUUCUUCUUC 148 UUCUUCUUCUUCUUC 149 UUCUUCUUCUUCUUC 150 UUCUUCUUCUUCUUCUUC 151 UUCUUCUUCUUCUUCUUC 152 UUCUUCUUCUUCUUCUUCUUC 153 UUCUUCUUCUUCUUCUUCUUC 154 UUCUUCUUCUUCUUCUUCUUC 155 UUCUUCUUCUUCUUCUUCUUC 156 UUCUUCUUCUUCUUCUUCUUC 157 UUCUUCUUCUUCUUCUUCUUCUUC 158 UUCUUCUUCUUCUUCUUCUUCUUC 159 UUCUUCUUCUUCUUCUUCUUCUUCUUC 160 UUCUUCUUCUUCUUCUUCUUCUUCUUC 161 UUCUUCUUCUUCUUCUUCUUCUUCUUCUUC 162 UUCUUCUUCUUCUUCUUCUUCUUCUUCUUC 163 UUCUUCUUCUUCUUCUUCUUCUUCUUCUUC 164 UUC UUCUUCUUCUUCUUCUUCUUCUUCUUCUUC 165 UUC UUCUUCUUCUUCUUCUUCUUCUUCUUCUUC 166 UUCUUC UUCUUCUUCUUCUUCUUCUUCUUCUUCUUC 167 UUCUUC (GCC)n GGCGGCGGCGGC 168 GGCGGCGGCGGCGGC 169 GGCGGCGGCGGCGGCGGC 170 GGCGGCGGCGGCGGCGGCGGC 171 GGCGGCGGCGGCGGCGGCGGC 172 GGCGGCGGCGGCGGCGGCGGC 173 GGCGGCGGCGGCGGCGGCGGC 174 GGCGGCGGCGGCGGCGGCGGCGGC 175 GGCGGCGGCGGCGGCGGCGGCGGCGGC 176 GGCGGCGGCGGCGGCGGCGGCGGCGGCGGC 177 (CCG)n CGGCGGCGGCGG 178 CGGCGGCGGCGGCGG 179 CGGCGGCGGCGGCGGCGG 180 CGGCGGCGGCGGCGGCGGCGG 181 CGGCGGCGGCGGCGGCGGCGGCGG 182 CGGCGGCGGCGGCGGCGGCGGCGGCGG 183 CGGCGGCGGCGGCGGCGGCGGCGGCGGCGG 184 (AUUCU)n AGAAUAGAAUAGAAU 185 AGAAUAGAAUAGAAUAGAAU 186 AGAAUAGAAUAGAAUAGAAU 187 AGAAUAGAAUAGAAUAGAAU 188 AGAAUAGAAUAGAAUAGAAU 189 AGAAUAGAAUAGAAUAGAAUAGAAU 190 AGAAUAGAAUAGAAUAGAAUAGAAUAGAAU 191 AGAAUAGAAUAGAAUAGAAUAGAAUAGAAU 192 AGAAU (CCUG)n CAGGCAGGCAGG 193 CAGGCAGGCAGG 194 CAGGCAGGCAGGCAGG 195 CAGGCAGGCAGGCAGG 196 CAGGCAGGCAGGCAGGCAGG 197 CAGGCAGGCAGGCAGGCAGG 198 CAGGCAGGCAGGCAGGCAGG 199 CAGGCAGGCAGGCAGGCAGG 200 CAGGCAGGCAGGCAGGCAGGCAGG 201 CAGGCAGGCAGGCAGGCAGGCAGG 202 CAGGCAGGCAGGCAGGCAGGCAGGCAGG 203 CAGGCAGGCAGGCAGGCAGGCAGGCAGG 204 CAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGG 205 CAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGG 206 CAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGG 207 CAGG CAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGG 208 CAGG (GGGGCC)n PS1252 GGCUCCGGCUCCGGCUC 209 GGCQCCGGCQCCGGCQCC 210 GGCUCCGGCUCCGGCUCC 211 GGCUCCGGCUCCGGCUCCGGCUCC 212 GGCQCCGGCQCCGGCQCCGGCQCC 213 GGCICCGGCICCGGCICCGGCICC 214 GGCCUCGGCCUCGGCCUCGGCCUC 215

    EXAMPLES

    Example 1

    Introduction

    [0337] The particular characteristics of a chosen antisense oligonucleotide (AON) chemistry may at least in part enhance binding affinity and stability, enhance activity, improve safety, and/or reduce cost of goods by reducing length or improving synthesis and/or purification procedures. This example describes the comparative analysis of the activity of AONs designed to target the expanded (CAG).sub.n repeat in HTT transcripts in HD fibroblasts in vitro, and includes AONs with either 5-methylcytosines (XYG).sub.7, wherein X is 5-methylcytosine and Y=U being also identified as SEQ ID NO:90 (and derived from SEQ ID NO:2), or 5-methyluracils (XYG).sub.7, wherein X=C and Y=5-methyluracil being also identified as SEQ ID NO: 97 (and derived from SEQ ID NO:3).

    [0338] Materials and Methods

    [0339] Cell Culture.

    [0340] Patient derived HD fibroblasts (GM04022) (purchased from Coriell Cell Repositories, Camden, USA) 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) (Clontech, Palo Alto USA), 1% Glutamax (Gibco) and 100 U/ml penicillin/streptomycin (P/S) (Gibco).

    [0341] Oligonucleotides.

    [0342] The AONs were fully 2-O-methyl phosphorothioate modified: PS659; (XYG).sub.7, wherein X is 5-methylcytosine and Y=U being also identified as SEQ ID NO: 90 (and derived from SEQ ID NO:2), and PS661; (XYG).sub.7, wherein X=C and Y=5-methyluracil being also identified as SEQ ID NO:97 (and derived from SEQ ID NO:3).

    [0343] Transfection.

    [0344] Cells were transfected with AONs complexed with PEI (2 L per g AON, in 0.15 M NaCl). AON-PEI complex was added in MEM medium with 5% FBS to cells to a final AON concentration varying from 0.5-200 nM. Fresh medium was supplemented after four hours and after 24 hours RNA was isolated.

    [0345] RNA Isolation.

    [0346] RNA from cultured cells was isolated using the Aurum Total RNA Mini Kit (Bio-Rad, Hercules, Calif.) according to the manufacturer's protocol.

    [0347] RT-PCR and Lab-On-a-Chip Analysis.

    [0348] Approximately 200 ng RNA was subjected to cDNA synthesis with random hexamers using the SuperScript first-strand synthesis system (Invitrogen) in a total volume of 20 L. PCR was performed with primers for HTT (across the CAG repeat) and -actin. The PCR program started with a 4 min initial denaturation at 95 C., followed by 35 cycles of 30 sec denaturation at 94 C., 30 sec annealing at 60 C., 45 sec elongation at 72 C., after which a final elongation step was performed at 72 C. for 7 min. Lab-on-a-Chip was performed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany), using the Agilent DNA 1000 Kit. Expression levels were normalized for -actin levels and relative to transcript levels without transfection. The following primers were used:

    TABLE-US-00003 HTTforward; (SEQIDNO:70) 5-ATGGCGACCCTGGAAAAGCTGAT-3 HTTreverse: (SEQIDNO:71) 5-TGAGGCAGCAGCGGCTG-3 -actinforward; (SEQIDNO:72) 5-GGACTTCGAGCAAGAGATGG-3 -actinreverse; (SEQIDNO:73) 5-AGCACTGTGTTGGCGTACAG-3

    [0349] Results

    [0350] Both PS659 (SEQ ID NO: 90 derived from SEQ ID NO:2) and PS661 (SEQ ID NO: 97 derived from SEQ ID NO:3) were highly effective and reduced the HTT transcripts in HD fibroblasts in a dose-dependent manner (FIGS. 1A-1B). Both AONs also showed a preference for the allele with the expanded CAG repeats. PS659 (SEQ ID NO: 90 derived from SEQ ID NO:2) was more effective and more allele-specific at lower concentrations (strongest effect at 5 nM) (FIG. 1A) than PS661 (SEQ ID NO: 97 derived from SEQ ID NO:3) (strongest effect at 20 nM) (FIG. 1B).

    Example 2

    Introduction

    [0351] PS659 (XYG).sub.7, wherein X is 5-methylcytosine and Y=U also identified as SEQ ID NO: 90 (derived from SEQ ID NO:2), was selected from in vitro studies as most efficient and safe candidate. This example describes its activity in a transgenic HD rat model after a series of direct intraventricular injections.

    [0352] Materials and Methods

    [0353] Animals.

    [0354] Transgenic HD rats carry a truncated Huntington cDNA fragment with 51 CAG repeats under the control of the native rat Huntington promoter. The expressed gene product is about 75 kDa, corresponding to 22% of the full-length Huntington (cDNA position 324-2321, amino acid position 1-709/825, corresponding to exon 1-16), under the control of 886 bp of the rat Huntington promoter (von Hrsten S. et al.). All animal experiments were approved by the Institutional Animal Care and Use Committees of the Maastricht University, Maastricht.

    [0355] Oligonucleotides.

    [0356] PS659 (XYG).sub.7, wherein X is 5-methylcytosine and Y=U also identified as SEQ ID NO: 90 (derived from SEQ ID NO:2), is a fully 2-O-methyl phosphorothioate modified AON.

    [0357] In Vivo Treatment.

    [0358] Transgenic HD rats received 15 times an intraventricular injection at a final dose of 200 g PS659 also identified as SEQ ID NO: 90 (derived from SEQ ID NO:2) during 18 weeks. Control HD rats received vehicle only. Rats were sacrificed one week after the final injection.

    [0359] RNA Isolation.

    [0360] RNA from brain tissue was isolated using RNA-Bee reagent (Tel Test, Inc). In brief, tissue samples were homogenized in MagNA Lyser green bead tubes (Roche) by adding RNA-Bee (50 mg tissue/mL RNA-Bee) and homogenizing using a MagNA Lyser instrument (Roche). Lysate was transferred to a new tube, chloroform (SIGMA) was added (0.2 mL per mL RNA-Bee), mixed, incubated on ice for 5 minutes and centrifuged at 13,000 rpm for 15 minutes at 4 C. The upper aqueous phase was collected and an equal volume isopropanol (SIGMA) was added, followed by a 1 hour incubation period at 4 C. and centrifugation (13,000 rpm, 15 min, 4 C.). The RNA precipitate was washed with 70% (v/v) ethanol (BioSolve), air dried and dissolved in MilliQ.

    [0361] Quantitative RT-PCR Analysis.

    [0362] Approximately 200 ng was subjected to cDNA synthesis with random hexamers using the SuperScript first-strand synthesis system (Invitrogen) in a total volume of 20 L. 3 L of 1/40 cDNA dilution preparation was subsequently used in a quantitative PCR analysis according to standard procedures in presence of iQ SYBR Green Supermix (Bio-Rad). Quantitative PCR primers were designed based on NCBI database sequence information. Product identity was confirmed by DNA sequencing. The signal for Rab2 and YWHAZ was used for normalization. The following primers were used:

    TABLE-US-00004 RatHtt-F; (SEQIDNO:74) 5-CGCCGCCTCCTCAGCTTC-3 RatHtt-R; (SEQIDNO:75) 5-GAGAGTTCCTTCTTTGGTCGGTGC-3 Rab2-F; (SEQIDNO:76) 5-TGGGAAACAGATAAAACTCCAGA-3 Rab2-R; (SEQIDNO:77) 5-AATATGACCTTGTGATAGAACGAAAG-3 YWHAZ-F; (SEQIDNO:78) 5-AAATGAGCTGGTGCAGAAGG-3 YWHAZ-R; (SEQIDNO:79) 5-GGCTGCCATGTCATCGTAT-3

    [0363] Results

    [0364] PS659 (also identified as SEQ ID NO: 90 or derived from SEQ ID NO: 2) reduced transgenic Htt transcript levels in cortex (FIG. 2A), hippocampus (FIG. 2B), olfactory bulb (FIG. 2C) as well as in thalamus (FIG. 2D) when compared to saline treated rats. These results demonstrate that PS659 (also identified as SEQ ID NO: 90 or derived from SEQ ID NO; 2) is effective in vivo after direct intraventricular injection.

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