Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs

Abstract

Expanded trinucleotide repeats cause many neurological diseases. These include Machado-Joseph disease (MJD) and Huntington's disease (HD), which are caused by expanded CAG repeats within an allele of the ataxin-3 (ATXN3) and huntingtin (HTT) genes, respectively. Silencing expression of these genes is a promising therapeutic strategy, but indiscriminate inhibition of both the mutant and wild-type alleles may lead to toxicity, and allele-specific approaches have required polymorphisms that differ among individuals. We report that peptide nucleic acid and locked nucleic acid antisense oligomers that target CAG repeats can preferentially inhibit mutant ataxin-3 and HTT protein expression in cultured cells. Duplex RNAs were less selective than single-stranded oligomers. The activity of the peptide nucleic acids does not involve inhibition of transcription, and differences in mRNA secondary structure or the number of oligomer binding sites may be important. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length may offer new options for developing treatments for MJD, HD and related hereditary diseases.

Figure 1. PNAs, LNAs, and inhibition of HTT expression by PNA-peptide conjugates

Unless otherwise noted, experiments use GM04281 fibroblast cells that are heterozygous for mutant HTT expression and graphs are quantifications of triplicate independent experiments. (a) Chemical structures of PNA and LNA. (b) Schematic of target sites for PNA and LNA oligomers within HTT mRNA. (c) Western analysis showing that wild-type and mutant HTT protein can be separated by gel electrophoresis. Lane 1 shows HTT from GM04795 cells, a fibroblast cell line that is homozygous for wild-type HTT. Lane 2 shows HTT from GM04281 cells, a fibroblast cell line that is heterozygous for mutant HTT. (d) Effect on HTT expression of adding 5 µM PNA-peptide conjugates. (e) Effect on HTT expression of adding (e) REP19 or (f) 3J/HTT. For (e) and (f), examples of western gels used for quantitation are shown in Supplementary Fig. 6 online. (g) Timecourse of inhibition of HTT expression by REP19 (1 µM). (h) Effect of adding REP19 on expression of other proteins with mRNAs that contain CAG repeats. (i) Effect of adding REP19 on cell toxicity measured by monitoring levels of caspase 3. *p < 0.05, ***p < 0.001 relative to negative control PNA/-CTL1. n = 3. (j) Glutamate-induced apoptosis of WT and YAC128 MSN. The fraction of TUNEL-positive is shown for WT (open bars) and YAC128 (filled bars) MSN. Experiments were repeated five times and data from a blinded cell count is shown. Data were evaluated using One-Way ANOVA. Statistical difference was considered to be significant if p ≤0.05. n.s. – not statistically significant.

Figure 2. Effect of PNA modification, LNAs, and siRNAs on selectivity

All quantitation of western analysis of protein levels in GM04281 fibroblast cells is derived from at least three independent experiments. Examples of western gels used for quantitation are shown in Supplementary Fig. 6 online. Effect on HTT expression of adding increasing concentrations of (a) REP16, (b) REP13, (c) REP19Arg, (d) REP19N, (e) LNA/REP, (f) LNA/3J, (g) siRNA/REP and, (h) siRNA/5J.

Figure 3. Selectivity is affected by the number of repeats

All quantitation of western analysis of protein levels in fibroblast cells is derived from at least three independent experiments. Examples of Western gels used for quantitation are shown in Supplementary Fig. 9 online. (a) Separation by SDS-PAGE of wild-type and mutant HTT protein variants in four different patient-derived cell lines. For (b)–(g) effect on HTT protein expression of adding (b) REP19 to GM04869 cells, (c) REP19N to GM04869 cells, (d) REP19 to GM04719 cells, (e) REP19N to GM04719 cells, (f) REP19 to GM04717 cells, and (g) REP19N to GM04717 cells.

Figure 4. Potent and selective inhibition of mutant ataxin-3

All data show analysis of ataxin-3 expression in GM06151 fibroblast cells. Examples of Western gels used for quantitation are shown in Supplementary Figure 10 online. (a) Schematic of target sites for PNAs within ataxin-3 mRNA, Inhibition of ataxin-3 expression by (b) PNA conjugate REP19, (c) PNA conjugate 3J/ATX, (d) PNA conjugate 5J/ATX, and (e) siRNA/REP.