Allele-selective inhibition of ataxin-3 (ATX3) expression by antisense oligomers and duplex RNAs

Abstract

Spinocerebellar ataxia-3 (also known as Machado-Joseph disease) is an incurable neurodegenerative disorder caused by expression of a mutant variant of ataxin-3 (ATX3) protein. Inhibiting expression of ATX3 would provide a therapeutic strategy, but indiscriminant inhibition of both wild-type and mutant ATX3 might lead to undesirable side effects. An ideal silencing agent would block expression of mutant ATX3 while leaving expression of wild-type ATX3 intact. We have previously observed that peptide nucleic acid (PNA) conjugates targeting the expanded CAG repeat within ATX3 mRNA block expression of both alleles. We have now identified additional PNAs capable of inhibiting ATX3 expression that vary in length and in the nature of the conjugated cation chain. We can also achieve potent and selective inhibition using duplex RNAs containing one or more mismatches relative to the CAG repeat. Anti-CAG antisense bridged nucleic acid oligonucleotides that lack a cationic domain are potent inhibitors but are not allele-selective. Allele-selective inhibitors of ATX3 expression provide insights into the mechanism of selectivity and promising lead compounds for further development and in vivo investigation.

Figure 1

Chemical structures of (a) PNA, carba-LNA and cEt; (b) peptide and peptoid.

Figure 2. Effects of PNA length on ATX3 expression

All PNAs are conjugated to peptide d-K₈ at their C-terminus. (a–f) PNAs with varied length were tested in patient fibroblast cell line GM06151 (CAG 74 mutant repeats/24 wild-type repeats) at increasing concentrations. Representative western blot images are presented. Quantification and a nonlinear fitting curve of ATX3 expression is plotted from multiple experiments. Error bars are standard error of the mean (SEM).

Figure 3. Effects of peptide orientation and chemistry on ATX3 expression

19-base PNAs (a–c) or 7-base PNAs (d–f) with 8 lysines or arginines at their N or C-terminus were tested in patient fibroblast cell line GM06151 (CAG 74 mutant repeats/24 wild-type repeats) at increasing concentrations. Representative western blot images are presented. Quantification and a nonlinear fitting curve of ATX3 expression is plotted from multiple experiments. Error bars are SEM.

Figure 4. Effects of PNAs with different conjugate designs on ATX3 expression

13-base PNA conjugates (a–c) or 19-base PNA conjugates (d,e) were tested in patient fibroblasts GM06151 (CAG 74 mutant repeats/24 wild-type repeats) at increasing concentrations. Representative western blot images are presented. Quantification and a nonlinear fitting curve of ATX3 expression is plotted from multiple experiments. Error bars are SEM.

Figure 5. Effect of single stranded BNA oligonucleotides

Effects on ATX3 protein levels after adding increasing amount of (a) carba-LNA, or (b) cET in fibroblasts GM06151 (CAG 74 mutant repeats/24 wild-type repeats). The BNAs were introduced into cells by PepMute transfection reagent. Representative western blot images are presented. Quantification and a nonlinear fitting curve of ATX3 expression is plotted from multiple experiments. Error bars are SEM.

Figure 6. Western analysis of ATX3 expression in fibroblasts GM06151 (CAG 74 mutant repeats/24 wild-type repeats) after treating with mismatch-containing siRNAs

(a) Effects of 25 nM siRNAs on ATX3 expression. (b) Comparison of 25 nM siRNAs with different numbers of centrally mismatched bases. (c) siRNAs P9, P11 P910, PM3, and PM4 selectively inhibit mutant ATX3 expression. Representative gel images are presented. Quantification and a nonlinear fitting curve of ATX3 expression is plotted from multiple experiments. Error bars are SEM.

Figure 7. Selective siRNAs have little effects on ATX3 mRNA levels

qPCR analysis of ATX3 mRNA levels after treatment with siRNAs at 12 nM.