Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide

Abstract

Expansions of a G₄C₂ repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Using C9-ALS/FTD patient-derived cells and C9ORF72 BAC transgenic mice, we generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G₄C₂ repeat-containing transcripts and effectively suppress tissue levels of poly(GP) dipeptides. ASOs with reduced phosphorothioate content showed improved tolerability without sacrificing efficacy. In a single patient harboring mutant C9ORF72 with the G₄C₂ repeat expansion, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of cerebrospinal fluid poly(GP). This report provides insight into the effect of nucleic acid chemistry on toxicity and, to our knowledge, for the first time demonstrates the feasibility of clinical suppression of the C9ORF72 gene. Additional clinical trials will be required to demonstrate safety and efficacy of this therapy in patients with C9ORF72 gene mutations.

Figure 1.. G₄C₂-targeting LNA- and MOE-modified ASOs reduce the C9ORF72 repeat-containing transcripts in patient derived fibroblasts, C9BAC mouse derived neurons, and C9BAC mice.

(a) Schematic of C9ORF72 transcript variants and of the two protein isoforms as named in PubMed. The repeat expansion (red triangles) located in the first intron is expressed in variants 1 and 3 (V1-V3). Variant 2 (V2), the most abundant, starts form a different transcription start site (black arrow) and does not include the repeat expansion. V2 and V3 encode the main C9ORF72 protein isoform. Grey boxes represent untranslated regions (UTR); light blue boxes, exons; black lines, introns. ASOs used in this study (green bars) target the intronic region flanking the repeat expansion. (b) LNA and 2’-O-MOE ASOs dramatically reduce the level of repeat-containing transcripts at a dose of 100nM 72 hrs after lipid-mediated delivery in patient derived fibroblasts as measured by qRT-PCR. n=6, each data point is derived from a separate well. ****p<0.0001, comparing any treatment group to either control group, based on one-way ANOVA with Dunnett’s multiple comparisons test. (c) LNA and 2’-O-MOE ASOs significantly reduce the level of repeat-containing transcripts after two weeks treatment with 1 μM ASO without lipid assistance (gymnotic delivery) in primary cortical neurons derived from C9BAC mice. n=3, each data point is derived from a separate well. ns, not significant (p=0.23), comparing NTC group to PBS group, based on one-way ANOVA with Dunnett’s multiple comparisons test. (d-g) Expression of V1-V3 repeat containing transcripts (d, f) and all transcripts (e, g) in cortex and spinal cord quantified by qRT-PCR in mice infused with PBS (dark grey), NTC ASO (light grey), ASO3 (green) or ASO5 (blue) at the indicated dose. For each dose level, n= 5–7, except NTC group (n=3). (h) Relative expression of polyGP in the cortex of mice treated with ASO3 (green) and ASO5 (blue) assayed by sandwich immunoassay. Data are represented as mean ± SEM. (i) % of body weight loss at end point relative to before treatment. In all panels, *p<0.05, **p<0.01, ***p<0.001, comparing treatment groups to PBS group, based on one-way ANOVA with Dunnett’s multiple comparisons test. All replicates shown as individual data points. In panels d-i, each data point is derived from a separate animal.

Figure 2.. Analogues of ASO5 with reduced phosphorothioate content maintain robust and durable biological activity after CNS administration in heterozygous C9BAC mice.

(a-f) A single-dose infusion shows robust silencing of V1-V3 repeat containing transcripts (a, d) with minimal change in overall C9ORF72 RNA levels (b, e) in cortex and spinal cord quantified by qRT-PCR, along with a robust drop in levels of polyGP (c,f) in mice treated with PBS (dark grey), ASO5 (light blue), ASO5-1 (medium blue) and ASO5-2 (dark blue) eight weeks after administration of 30nmol of each ASO. For each ASO group, n=5–7. (g-h) Dose-dependent silencing of C9ORF72 transcripts (g) and polyGP (h) after a single injection of 1, 5, 15, 30, or 60 nmol of ASO5-2 relative to PBS control. ASO 5–2 was administered into the lateral ventricle of 5–6-month-old heterozygous C9BAC mice and expression of RNA and dipeptide levels was evaluated after three weeks. For each ASO group, n=5–7. (i-j) A time course experiment was performed in heterozygous C9BAC mice treated with 30nmol of ASO5-2: tissues were collected and analyzed at 24 h or 3, 8, 12 and 20 weeks after treatment; an 80 nmol dose was also harvested at 20 weeks only. Expression of V1,V3 repeat-containing transcripts (i) and polyGP (j) was analyzed in cortex 24 h or 3, 8, 12 or 20 weeks after a single dose injection of ASO5-2. In all panels, *p<0.05, **p<0.01, ***p<0.001, comparing treatment groups to PBS group, based on one-way ANOVA with Dunnett’s multiple comparisons test. In panels a-f, h-j, each data point is derived from a separate animal.

Figure 3.. Clinical summary and afinersen (ASO5-2) dosing.

A single patient received multiple doses of ASO5-2 as indicated with arrows below the graph. The patient’s ALSFRS-R score before and during treatment is shown as a blue line and points. The patient’s poly(GP) DPR level, relative to the baseline (100%) is shown as a red line and points.