738. Engineered MicroRNA Silences C9ORF72 Variants in BAC Transgenic Mouse

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult-onset neurodegenerative disease that affects upper and lower motor neurons causing progressive muscle weakening. Respiratory failure is ultimately the cause of death approximately 2-5 years after symptom onset. The recent discovery of an expanded hexanucleotide repeat located in chromosome 9 open reading frame 72(C9ORF72) now accounts for the majority of familial ALS cases as well as frontotemporal dementia (FTD) cases. Analysis of patient brain samples have shown that in the presence of the expansion, C9ORF72 mRNA is reduced and RNA sequences complimentary to the expansion aggregate into nuclear foci. Interestingly this RNA can translate by non-ATG repeat associated (RAN) translation into long dipeptide chains. These findings have led to the three following hypothesis on the pathogenies of C9ORF72: 1) An RNA gain-of-function, in which the expanded RNA foci sequester RNA binding and/or splicing proteins. 2) RAN translation from the repeat expansion generates inclusions of toxic poly-dipeptide proteins. 3) Haploinsufficiency, where decreased levels of mRNA lead to insufficient gene product. Our group has generated mice containing a bacterial artificial chromosome (BAC) composed of exons 1-6 of the human C9ORF72 gene and a 500 repeat hexanucleotide expansion. This mouse model recapitulates the major histopathological features seen in human ALS/FTD patients such as: lower levels of C9ORF72 mRNA, RNA nuclear foci, and the RAN translation products. We have used this mouse as a platform in which to test RNAi therapeutic strategies. To this end we have designed artificial microRNAs that target the human C9ORF72 gene with the purpose of decreasing the mRNA levels to determine if we can reduce the toxic RNA foci and/or RNA dipeptide proteins. We packaged one of the microRNAs into a recombinant adeno-associated virus (rAAV) serotype 9 to use with primary neuron cultures and in vivo experiments. Silencing was initially validated in primary cortical neurons from the C9ORF72 mouse model. Our results suggest that AAV9-mediated microRNA not only reduced the mRNA levels of C9ORF72 but also decreased the presence of the most abundant poly dipeptide (GP). Currently, experiments are underway to silence C9orf72 in vivo via intra cranial ventricular (ICV) injection of this microRNA in C9ORF72 transgenic pups and the results will be presented at the meeting.