Targeting RNA-Mediated Toxicity in C9orf72 ALS and/or FTD by RNAi-Based Gene Therapy

Abstract

A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 ( C9orf72 ) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti- C9orf72 -targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts.

Non-coding RNAs in Nervous System Development and Disease

“…A new approach that has been employed to knock-down these transcripts is the use of artificial miRNAs (miC). Notably, Martier et al (2019) proved, in vivo, the delivery and efficacy of AAV5-miC in cortex and hippocampal neurons of Tg(C9orf72_3) ALS mouse model thus providing a proof of concept for the use of this strategy in the treatment of ALS and FTD.…”

“…A promising approach to circumvent the BBB and the BSCB is the delivery of therapeutic molecules directly to CNS through intrathecal injection (IT). Indeed, ASOs or AAV based molecules that have been IT administrated through intracerebroventricular (ICV) injection in rodent models and non-human primates, showed a widespread distribution in brain and spinal cord indicating the feasibility of this approach in targeting tissues mostly affected in neurodegenerative diseases (DeVos and Miller, 2013;Rigo et al, 2014;Biferi et al, 2017;Casaca-Carreira et al, 2017;Schoch and Miller, 2017;Martier et al, 2019). More importantly, pre-clinical and clinical trials involving IT/ICV delivery of ASOs against disease-associated transcripts (SMN, SOD1, and C9ORF72) have demonstrated the effectiveness and tolerability of this approach (Miller et al, 2013;Finkel et al, 2016;Cappella et al, 2019;Neil and Bisaccia, 2019).…”

Insights into disease mechanisms and potential therapeutics for C9orf72-related amyotrophic lateral sclerosis/frontotemporal dementia

“…Despite the unclear pathogenic role of RNA-mediated toxicity, a therapy targeting the repeat-containing transcripts could potentially reduce RNA foci (Hu et al, 2015;Martier et al, 2019a), alleviating disease progression.…”

“…AAV5-delivered miRNAs engineered to target C9orf72 decrease the abundance of RNA foci in the cytoplasm and the nucleus of iPSC-derived MNs and in mice (Martier et al, 2019a(Martier et al, , 2019b. Altogether, these findings highlight the feasibility of RNAi-based approaches as a therapeutic strategy for C9-ALS.…”

The Development of C9orf72-Related Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Disorders

“…For example, after intravenous administration, AAV9 has the ability to cross the blood–brain barrier and tends to transduce glial cells while AVV6 preferentially transduces neurons ( San Sebastian et al, 2013 ; Borel et al, 2014 ; Dirren et al, 2015 ). Evidences showed that AAV5 can transduce different types of neuronal cells and AAV5-delivered artificial RNAi can reduce the accumulation of repeat-containing C9orf72 transcripts ( Martier et al, 2019 ). However, non-allele-specific RNAi strategies may lead to excessive silencing of normal alleles.…”