Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat

Abstract

Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.

Using non-coding small RNAs to develop therapies for Huntington's disease

“…The mechanism of selective silencing of chemically modified ASOs and miRNA-like siRNA might be different from traditional siRNA processing. Both ASOs and siRNA with central-site mismatch inhibited HD protein without any significant effect on HD mRNA, [24][25][26] which indicates translational repression rather than transcriptional degradation (Figure 3). For translational inhibition, it is best to have a target near the start codon of an mRNA.…”

“…22 Pursuing this reasoning, single-strand antisense oligoribonucleotides (ASOs), or two strands of siRNA (with or without unmatched sites), were designed to silence HD alleles with unusually long CAG repeats. 18,[22][23][24][25][26] ASOs contain a single nucleic acid strand that were heavily modified to enhance stability, target binding and biodistribution. 18,23,24 Locked nucleic acids-modified ASOs with chemical conjugation with peptide (peptide nucleic acid) were initially reported to have up to six-fold allele selectivity to potently silence the mutant, but not the wild-type HD allele in fibroblasts from HD patients.…”

“…18,[22][23][24][25][26] ASOs contain a single nucleic acid strand that were heavily modified to enhance stability, target binding and biodistribution. 18,23,24 Locked nucleic acids-modified ASOs with chemical conjugation with peptide (peptide nucleic acid) were initially reported to have up to six-fold allele selectivity to potently silence the mutant, but not the wild-type HD allele in fibroblasts from HD patients. 18 However, after comparing with 10 chemical modification methods, even the most efficient chemically modified ASO had limited potency and were unable to reach greater than eight-fold allele selectivity.…”

“…18 However, after comparing with 10 chemical modification methods, even the most efficient chemically modified ASO had limited potency and were unable to reach greater than eight-fold allele selectivity. 24 Recently, it was found that using mismatch-containing duplex siRNAs, robust selectivity (410-to 20-fold) and potencies for inhibiting mutant huntingtin expression (o10 nM) in two HD skin fibroblast cell lines were achieved. 25,26 Adapting endogenous miRNA-like targeting with mismatches design in siRNA guide strand produced a better selectivity.…”

“…Allele-specific silencing of the mutant species has been achieved in these cells with both endogenous wild-type and mutant HD (Figure 1). [15][16][17][18][19][20][21][22][23][24][25][26] APPLICATION OF RNAi IN HD TRANSGENIC ANIMAL MODELS To date, all published studies on non-coding RNAs therapy in HD transgenic mice have used animals expressing short fragments of human huntingtin protein (instead of the full-length huntingtin) with an expanded CAG repeat. These mice exhibit a rapidly progressing syndrome reminiscent of HD in humans.…”