INVESTIGATING THE SPECIFICITY OF RNAi MOLECULES IN HUMAN GENE THERAPY FOR SOD1-LINKED FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS

20% of familial amyotrophic lateral sclerosis (fALS) cases are caused by mutations in the gene encoding the cytosolic protein human Cu/Zn superoxide dismutase 1 (hSOD1). RNA interference (RNAi) technology offers the therapeutic potential for the treatment of SOD-linked fALS by reducing the burden of pathogenic mutant SOD1 protein. Translation of this gene therapy strategy to the clinic requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to determine treatment efficacy, successful target gene reduction, and correct dosing. Using self-complementary adeno-associated virus serotype 9 (scAAV9) to deliver RNAi against hSOD1 in the SOD1G93A mouse model, the work presented in this thesis demonstrates that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88%, and prolonged the median survival of SOD1G93A mice by up to 42%. Using a panel of purposefully designed RNAi constructs cloned into the scAAV9 backbone this is, to our knowledge, the first study to demonstrate no significant in vitro off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. This study also reports the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, this data provides further confidence in the safety of the clinical therapeutic vector.