RNA modulating oligonucleotides with improved characteristics for the treatment of neuromuscular disorders

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. A 2′-O-methyl phosphorothioate oligoribonucleotide consisting of the base sequence (XYG).sub.7, wherein each X is 5-methyl cytosine and each Y is uracil (SEQ ID NO: 2) or wherein each X is cytosine and each Y is 5-methyluracil (SEQ ID No:3).

2. An oligoribonucleotide according to claim 1, wherein said oligoribonucleotide is single stranded.

3. A composition comprising an oligoribonucleotide according to claim 1.

4. A composition according to claim 3, said composition comprising at least one excipient for enhancing the targeting and/or delivery of said composition to a tissue and/or cell and/or into a tissue and/or cell.

5. A method for treating a subject having a human cis-element repeat instability associated genetic disorder by administering an oligoribonucleotide according to claim 2 to said subject.

6. The method of claim 5, wherein said subject has the human cis-element repeat instability associated genetic disorder-Huntington Disease.

Description

LEGENDS TO THE FIGURE

(1) 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 (1A) and PS661 (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).

(2) 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 μg 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 (2A) cortex, (2B) hippocampus, (2C) olfactory bulb and (2D) thalamus after PS659 treatment compared to control.

(3) TABLE-US-00001 TABLE 1 General structures of AONs. X = C or 5-methylcytosine Y = U or 5- methyluracil, 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.

(4) TABLE-US-00002 TABLE 2 General structures of AONs. All AONs are 2′-O-methyl phosphorothioate AONs wherein C is 5-methylcytosine, U is 5-methyluracil, A is 2,6- diaminopurine, I is inosine and Q is an abasic monomer. Target AON SEQ Repeat ID AON Sequence (5′.fwdarw.3′) ID NO (CAG)n PS659 CUG CUG CUG CUG CUG CUG CUG 90 CUG CUG CUG CUG CUG CUG CUG 91 CUG CUG CUG CUG CUG CUG CUG 92 CUG CUG CUG CUG CUG CUG CUG 93 CUG CUG CUG CUG CUG CUG CUG 94 CUG CUG CUG CUG CUG CUG CUG 95 CUG CUG CUG CUG CUG CUG CUG 96 PS661 CUG CUG CUG CUG CUG CUG CUG 97 CUG CUG CUG CUG CUG CUG CUG 98 CUG CUG CUG CUG CUG CUG CUG 99 CUG CUG CUG CUG CUG CUG CUG 100 CUG CUG CUG CUG CUG CUG CUG 101 CUG CUG CUG CUG CUG CUG CUG 102 CUG CUG CUG CUG CUG CUG CUG 103 PS660 CUG CUG CUG CUG CUG CUG CUG 104 CUG CUG CUG CUG CUG CUG CUG 105 CUG CUG CUG CUG CUG CUG CUG 106 PS684 CUG CUG CUG CUG CUG CUG CUG 107 CUG CUG CUG CUG CUG CUG CUG QQQQ 220 CUG CUG CUG CUG CUG CUG CUG 108 CUG CUG CUG CUG CUG CUG CUG QQQQ 221 CUG CUG CUG CUG CUG CUG CUG CUG 109 CUG CUG CUG CUG CUG CUG CUG CUG 110 CUG CUG CUG CUG CUG CUG CUG CUG CUG 111 CUG CUG CUG CUG CUG CUG CUG CUG CUG 112 CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG 113 CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG 114 CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG  115 CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG  116 CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG  117 CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG  118 CUG (GCG)n CGC CGC CGC CGC 119 CGC CGC CGC CGC 120 CGC CGC CGC CGC CGC 121 CGC CGC CGC CGC CGC 122 CGC CGC CGC CGC CGC CGC 123 CGC CGC CGC CGC CGC CGC 124 CGC CGC CGC CGC CGC CGC CGC 125 CGC CGC CGC CGC CGC CGC CGC 126 CGC CGC CGC CGC CGC CGC CGC CGC 127 CGC CGC CGC CGC CGC CGC CGC CGC 128 CGC CGC CGC CGC CGC CGC CGC CGC CGC 129 CGC CGC CGC CGC CGC CGC CGC CGC CGC 130 CGC CGC CGC CGC CGC CGC CGC CGC CGC CGC 131 CGC CGC CGC CGC CGC CGC CGC CGC CGC CGC 132 (CGG)n CCG CCG CCG CCG 133 CCG CCG CCG CCG 134 CCG CCG CCG CCG CCG 135 CCG CCG CCG CCG CCG 136 CCG CCG CCG CCG CCG CCG 137 CCG CCG CCG CCG CCG CCG 138 CCG CCG CCG CCG CCG CCG CCG 139 CCG CCG CCG CCG CCG CCG CCG 140 CCG CCG CCG CCG CCG CCG CCG CCG 141 CCG CCG CCG CCG CCG CCG CCG CCG 142 CCG CCG CCG CCG CCG CCG CCG CCG CCG 143 CCG CCG CCG CCG CCG CCG CCG CCG CCG 144 CCG CCG CCG CCG CCG CCG CCG CCG CCG CCG 145 CCG CCG CCG CCG CCG CCG CCG CCG CCG CCG 146 (GAA)n UUC UUC UUC UUC 147 UUC UUC UUC UUC 148 UUC UUC UUC UUC UUC 149 UUC UUC UUC UUC UUC 150 UUC UUC UUC UUC UUC UUC 151 UUC UUC UUC UUC UUC UUC 152 UUC UUC UUC UUC UUC UUC UUC 153 UUC UUC UUC UUC UUC UUC UUC 154 UUC UUC UUC UUC UUC UUC UUC 155 UUC UUC UUC UUC UUC UUC UUC 156 UUC UUC UUC UUC UUC UUC UUC 157 UUC UUC UUC UUC UUC UUC UUC UUC 158 UUC UUC UUC UUC UUC UUC UUC UUC 159 UUC UUC UUC UUC UUC UUC UUC UUC UUC 160 UUC UUC UUC UUC UUC UUC UUC UUC UUC 161 UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC  162 UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 163 UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 164 UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 165 UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 166 UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC 167 UUC (GCC)n GGC GGC GGC GGC 168 GGC GGC GGC GGC GGC 169 GGC GGC GGC GGC GGC GGC 170 GGC GGC GGC GGC GGC GGC GGC 171 GGC GGC GGC GGC GGC GGC GGC 172 GGC GGC GGC GGC GGC GGC GGC 173 GGC GGC GGC GGC GGC GGC GGC 174 GGC GGC GGC GGC GGC GGC GGC GGC 175 GGC GGC GGC GGC GGC GGC GGC GGC GGC 176 GGC GGC GGC GGC GGC GGC GGC GGC GGC GGC 177 (CCG)n CGG CGG CGG CGG 178 CGG CGG CGG CGG CGG 179 CGG CGG CGG CGG CGG CGG 180 CGG CGG CGG CGG CGG CGG CGG 181 CGG CGG CGG CGG CGG CGG CGG CGG 182 CGG CGG CGG CGG CGG CGG CGG CGG CGG 183 CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG 184 (AUUCU)n AGAAU AGAAU AGAAU 185 AGAAU AGAAU AGAAU AGAAU 186 AGAAU AGAAU AGAAU AGAAU 187 AGAAU AGAAU AGAAU AGAAU 188 AGAAU AGAAU AGAAU AGAAU 189 AGAAU AGAAU AGAAU AGAAU AGAAU 190 AGAAU AGAAU AGAAU AGAAU AGAAU AGAAU 191 AGAAU AGAAU AGAAU AGAAU AGAAU AGAAU 192 AGAAU (CCUG)n CAGG CAGG CAGG 193 CAGG CAGG CAGG 194 CAGG CAGG CAGG CAGG 195 CAGG CAGG CAGG CAGG 196 CAGG CAGG CAGG CAGG CAGG 197 CAGG CAGG CAGG CAGG CAGG 198 CAGG CAGG CAGG CAGG CAGG 199 CAGG CAGG CAGG CAGG CAGG 200 CAGG CAGG CAGG CAGG CAGG CAGG 201 CAGG CAGG CAGG CAGG CAGG CAGG 202 CAGG CAGG CAGG CAGG CAGG CAGG CAGG 203 CAGG CAGG CAGG CAGG CAGG CAGG CAGG 204 CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG 205 CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG 206 CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG 207 CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG 208 CAGG (GGGGCC)n PS1252 GGCUCC GGCUCC GGCUC 209 GGCQCC GGCQCC GGCQCC 210 GGCUCC GGCUCC GGCUCC 211 GGCUCC GGCUCC GGCUCC GGCUCC 212 GGCQCC GGCQCC GGCQCC GGCQCC 213 GGCICC GGCICC GGCICC GGCICC 214 GGCCUC GGCCUC GGCCUC GGCCUC 215

EXAMPLES

Example 1

Introduction

(5) 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).

(6) Materials and Methods

(7) Cell Culture.

(8) 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 Um′ penicillin/streptomycin (P/S) (Gibco).

(9) Oligonucleotides.

(10) 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).

(11) Transfection.

(12) Cells were transfected with AONs complexed with PEI (2 μL per μg 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.

(13) RNA Isolation.

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

(15) RT-PCR and Lab-on-a-Chip Analysis.

(16) 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 μL. PCR was performed with primers for HTT (across the CAG repeat) and β-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 β-actin levels and relative to transcript levels without transfection. The following primers were used:

(17) TABLE-US-00003 HTT forward; (SEQ ID NO: 70) 5′-ATGGCGACCCTGGAAAAGCTGAT-3′ HTT reverse: (SEQ ID NO: 71) 5′-TGAGGCAGCAGCGGCTG-3′ β-actin forward; (SEQ ID NO: 72) 5′-GGACTTCGAGCAAGAGATGG-3′ β-actin reverse; (SEQ ID NO: 73) 5′-AGCACTGTGTTGGCGTACAG-3′
Results

(18) 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 (FIG. 1a, b). 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) (1a) than PS661 (SEQ ID NO: 97 derived from SEQ ID NO:3) (strongest effect at 20 nM) (1b).

Example 2

Introduction

(19) 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.

(20) Materials and Methods

(21) Animals.

(22) 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 Horsten S. et al.). All animal experiments were approved by the Institutional Animal Care and Use Committees of the Maastricht University, Maastricht.

(23) Oligonucleotides.

(24) 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.

(25) In Vivo Treatment.

(26) Transgenic HD rats received 15 times an intraventricular injection at a final dose of 200 ng 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.

(27) RNA Isolation.

(28) 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.

(29) Quantitative RT-PCR Analysis.

(30) 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 μL 3 μL 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:

(31) TABLE-US-00004 Rat Htt-F; (SEQ ID NO: 74) 5′-CGCCGCCTCCTCAGCTTC-3′ Rat Htt-R; (SEQ ID NO: 75) 5′-GAGAGTTCCTTCTTTGGTCGGTGC-3′ Rab2-F; (SEQ ID NO: 76) 5′-TGGGAAACAGATAAAACTCCAGA-3′ Rab2-R; (SEQ ID NO: 77) 5′-AATATGACCTTGTGATAGAACGAAAG-3′ YWHAZ-F; (SEQ ID NO: 78) 5′-AAATGAGCTGGTGCAGAAGG-3′ YWHAZ-R; (SEQ ID NO: 79) 5′-GGCTGCCATGTCATCGTAT-3′
Results

(32) 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. 3d) 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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