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.

Figure 1. Strategy for targeting the expanded CAG repeat region of mutant HTT mRNA to elicit allele-selective inhibition

(A) Schematic of the HTT mRNA and the CAG repeat region that is targeted with complementary CTG-repeat ASOs. UTR, untranslated region. (B) Chemically modified base, backbone and nucleosides incorporated into ASOs tested for allele-selective inhibition of HTT expression.

Figure 2. Selective inhibition of mutant HTT expression is observed with LNA-modified ASOs of varying sequence, length or modification configuration

(A) Properties of LNA-modified ASOs that are complementary to the HTT mRNA CAG repeat sequence. ASO, antisense oligonucleotide alone; ASO:RNA, antisense oligonucleotide duplexed with complementary RNA. DNA22 is an unmodified DNA oligo and (−)CTL is a scrambled sequence containing LNA at every third base. Bases enclosed by boxes indicate LNA modification position. T_m values were experimentally determined by UV melt analysis for ASOs and differential scanning calorimetry (DSC) for ASO:RNA duplexes. Error is standard deviation. IC₅₀ values are calculated from Western blot quantification. Values are reported in mean +/− standard error of the mean (SEM). N/A, not applicable; n.d., not determined due to insufficient cooperativity. (B–G) HTT protein expression in patient-derived fibroblast cells containing 69 CAG repeats in the mutant allele (GM04281) in response to increasing doses of control and inhibitory ASOs. Quantification and a non-linear fit of HTT expression levels from multiple dose responses are plotted. Sequence and modification configuration are indicated above the graph. Error bars are SEM.

Figure 3. The BNA modifications LNA, cEt, and carba-LNA support allele-selective HTT inhibition

(A) Properties of ASOs containing various modifications. ASO, antisense oligonucleotide alone; ASO:RNA, antisense oligonucleotide duplexed with complementary RNA. Position of nucleotide modifications within the ASO sequence are indicated as follows: boxed, LNA; circled, cEt; preceded by a “+”, carba-LNA; bracketed, ENA; italicized, MOE; lowercase, ANA; bold, 2'F-RNA; underlined, 2'F-ANA. T_m values were experimentally determined by UV melt analysis for ASOs and differential scanning calorimetry (DSC) for ASO:RNA duplexes and the MOE and MOE-cEt ASOs. Error is standard deviation. IC₅₀ values are calculated from Western blot quantification. Values are reported in mean +/− standard error of the mean (SEM). n.d., not determined due to insufficient cooperativity. (B–D) HTT protein expression in patient-derived fibroblast cells containing 69 CAG repeats in the mutant allele (GM04281) in response to increasing doses of inhibitory modified ASO. Quantification and a non-linear fit of HTT expression levels from multiple dose responses are plotted. Sequence and modification configuration are indicated above the graph. Error bars are SEM.

Figure 4. Phosphorothioate backbone modification is compatible with allele-selective inhibition

(A) Properties of CTG-repeat ASOs containing select modifications and a uniform phosphorothioate (PS) backbone. ASO, antisense oligonucleotide alone; ASO:RNA, antisense oligonucleotide duplexed with complementary RNA. Modification positions within the sequence are indicated as follows: boxed, LNA; circled, cET; italicized, MOE; bold, 2'F-RNA; preceded by a “+”, carba-LNA; mC, 5-methyl cytosine. T_m values were experimentally determined by UV melt analysis for ASOs and differential scanning calorimetry (DSC) for ASO:RNA duplexes and the MOE-PS and MOE-cEt-PS ASOs. Error is standard deviation. IC₅₀ values are calculated from Western blot quantification. Values are reported in mean +/− standard error of the mean (SEM). (B–E) HTT protein expression in patient-derived fibroblast cells containing 69 CAG repeats in the mutant allele (GM04281) in response to increasing doses of inhibitory phosphorothioate-modified ASOs. Quantification and a non-linear fit of HTT expression levels from multiple dose responses are plotted. Sequence and modification configuration are indicated above the graph. Error bars are SEM.

Figure 5. Allele-selective ASOs discriminate between mutant and wild-type alleles in patient-derived fibroblasts with 44 or 41 CAG repeats in the mutant HTT mRNA

(A) Allele-selectivity and potency of three selective ASOs when targeting mutant HTT mRNAs with only 44 or 41 CAG repeats. IC₅₀ values are calculated from Western blot quantification. Values are reported in mean +/− standard error of the mean (SEM). (B–G) HTT protein expression in patient-derived fibroblast cells containing 44 (GM04719) or 41 (GM04717) CAG repeats in the mutant allele in response to increasing doses of inhibitory ASOs. Quantification and a non-linear fit of HTT expression levels from multiple dose responses are plotted. Error bars are SEM.

Figure 6. Allele-selective ASOs leave HTT mRNA intact and do not support RNase H activity in vitro

(A) HTT mRNA levels are not substantially affected by treatment with allele-selective ASOs as determined by quantitative RT-PCR. Amplification of HTT cDNA was normalized to GAPDH amplification. NT, non-transfected. siHdh1 (transfected at 25 nM) is a duplex siRNA targeted to sequence downstream of the HTT CAG repeat (63). Error bars are standard deviation of triplicate experiments. (B) Allele-selective ASOs do not support RNase H cleavage in vitro. Allele-selective ASOs or control oligonucleotides were incubated with an in vitro transcribed and 5′-radiolabeled 69 CAG repeat-containing HTT mRNA 5′ end transcripts followed by treatment with RNase H enzyme. Reaction products were resolved on a denaturing polyacrylamide gel then visualized by phosphorimager.

Figure 7. The allele-selective ASO LNA(T) forms a stable self-structure and RNA:ASO duplex

(A) UV melt analysis of LNA(T), an allele-selective inhibitor of HTT expression, in the absence of an RNA complement. (B) UV melts of LNA(T) at concentrations spanning almost two orders of magnitude show no concentration dependence for T_m, suggesting an intramolecular hairpin folded structure. T_m A and T_m B correspond to the indicated transitions in panel A. (C) Differential scanning calorimetry (DSC) of LNA(T) base-paired to a fully complementary RNA. The ASO:RNA duplex is exceptionally stable, with a T_m of ~ 97°C. Values were fit to a 2-state unfolding model to calculate ΔH at the T_m.

Figure 8. Multiple ASOs bind CAG repeat-containing HTT 5′-end transcripts in vitro

(A–B) Electrophoretic mobility shift assays (EMSA) demonstrate cooperative and near stoichiometric binding of multiple ASOs to CAG repeat sequence in HTT mRNA 5′ end transcripts, as well as a case where no binding is observed. In vitro transcribed and 5′-radiolabeled wild-type and mutant 5′-end HTT mRNA transcripts were incubated with increasing concentrations of ASO. RNA:ASO complexes were resolved on native polyacrylamide gels and visualized by phosphorimager. ASO concentrations are indicated above the gels and shifted bands identified to the right. (C) Quantification of LNA(T) binding REP17 and REP69 HTT mRNA transcripts indicates cooperative ASO binding. ASO-bound RNA in gel shifts from panels A and B were quantified and plotted as a function of concentration. Fitting to the Hill equation revealed a sigmoidal binding curve and Hill coefficients (n) near 2, suggesting cooperative ASO binding. (D) Putative steric translational blocking mechanism. Translation is repressed on a mutant expanded CAG repeat HTT mRNA due to a proposed steric inhibition of translation by cumulative ASO binding.