Stereopure Antisense Oligonucleotides Preferentially Knockdown G4C2 Repeat-Containing C9ORF72 Transcripts: A Potential Therapeutic Approach for the Treatment of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) (N2.002)

Objective: To specifically target transcripts that contain the G 4 C 2 repeat expansion using stereopure antisense oligonucleotides (ASOs), thereby preventing production of toxic RNA foci and dipeptide repeat (DPR) proteins with minimal alteration of C9orf72 protein levels. Background: A large G 4 C 2 repeat expansion in the first intronic region of the C9ORF72 gene is the most common genetic cause of ALS and FTD. The G 4 C 2 repeat expansion reduces the normal expression of the C9ORF72 gene, causes the production of repeat-containing transcripts forming nuclear RNA foci and being translated into DPRs. Design/Methods: Using a luciferase reporter assay and patient-derived induced pluripotent stem cell (iPSC) neurons, we identified ASO sequences that preferentially reduced repeat-containing transcripts compared to the total transcripts. In vitro activity of lead sequences was then confirmed under transfection conditions in patient-derived fibroblasts and under gymnotic conditions in primary neurons. In vivo efficacy and distribution studies were performed in transgenic mice carrying a human bacterial artificial chromosome (BAC) construct containing the full human C9ORF72 gene with a repeat expansion. Results: Optimization of ASO chemistry and the chirality of the phosphorothioate backbone resulted in potent stereopure ASOs, with sub-nanomolar activity in the reporter assay, and nanomolar activity in C9-ALS patient-derived fibroblasts and neurons, as well as in primary neurons from C9BAC mice. Stereopure ASOs demonstrated improved in vitro metabolic stability compared to stereorandom ASOs in mouse brain homogenates. Intracerebroventricular injection of these ASOs into C9orf72 BAC transgenic mice resulted in substantial and sustained reduction of repeat-containing C9orf72 transcripts, RNA foci, and DPRs, without altering total C9orf72 protein levels. Tissue ASO exposures were in the range of 10 mg/g of tissue in both the cortex and the spinal cord. Conclusions: These results suggest that preferential targeting of repeat-containing transcripts using stereopure ASOs may be a viable therapeutic approach for the treatment of ALS and FTD. Study Supported by: Wave Life Sciences Ltd. Disclosure: Dr. Liu has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Dodart has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Tran has nothing to disclose. Dr. Berkovitch has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Byrne has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Durbin has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Iwamoto has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Jang has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Kandasamy has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Mohapatra has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Yang has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Yin has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Zhang has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Zhong has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Vargeese has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Wave Life Sciences Ltd. Dr. Brown has nothing to disclose.