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E - Disease Models and Clinical Applications -> Neurologic Diseases

1224: AAV-9 Mediated Delivery of RNA Targeting Systems Eliminates Hexanucleotide Repeat Expansions in C9ORF72 ALS/FTD Models

Type: Oral Abstract Session

Presentation Details
Session Title: Breakthroughs in Neuromuscular and Hearing Disorders
Location: Room 201
Start Time: 5/19/2022 10:45
End Time: 5/19/2022 11:00

A GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the first intron of C9ORF72 gene is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (c9ALS/FTD). Bidirectional transcription at the C9ORF72 repeat locus produces both sense G4C2 and antisense C4G2 containing transcripts. Studies to date have elucidated multiple pathogenic mechanisms including RNA gain-of-function of both sense and antisense HREs transcripts leading to formation of nuclear RNA foci and aggregating dipeptide repeat proteins generated by repeat-associated non-AUG (RAN) translation of both sense and antisense HREs, and a loss-of-function due to haploinsufficiency of the C9ORF72 protein. However, it remains unclear how much C9ORF72 protein loss contributes to neuronal death. To explore therapeutic targeting of both sense and antisense HREs, we engineered novel CRISPR/Cas13d based RNA targeting systems with 2 guide RNAs targeting G4C2 and C4G2-containing-transcripts that can be packaged in a single AAV genome. Notably, qPCR and RNA-FISH analyses of cells transfected with G4C2 and C4G2 reporters showed efficient elimination of HREs. Furthermore, we successfully packaged a G4C2 targeting guide RNA with two different orthologues of CRISPR/Cas13d into AAV9 with high yields. These AAV9-packaged G4C2 targeting CRISPR/Cas13ds showed no overt safety concerns in wildtype mice at 8 weeks post-subpial delivery. Importantly, these constructs reduced the G4C2-HRE containing isoforms of C9ORF72 in both cultured neonatal cortical neurons and in the spinal cord of transgenic C9ORF72 mice containing 500- G4C2 repeats following subpial delivery, while largely preserving total C9ORF72 transcript (transcripts from both alleles and including normal non-expanded isoforms) levels. In summary, we show a novel approach that can effectively target both sense and antisense HREs in c9ALS/FTD with a single product.

Jeannie Chew, Takahiro Tadokoro, Claire Geddes, Greg Nachtrab, Haydee Gutierrez, Ronald Torres, Na Li, Hank Bradford, Daniela Roth, Dan Gibbs, Ranjan Batra, John Leonard

Locanabio, San Diego, CA
 J. Chew: None.

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