HEREDITARY NEUROPATHIES & ALS: P.110 Development and characterization of in vitro models to test the efficiency of gene therapy approaches in SOD1-linked Amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes skeletal muscle (SM) paralysis. Familial forms of ALS are caused by mutations in superoxide dismutase-1 (SOD1) gene, in 20% of cases. A therapeutic approach for SOD1-linked ALS is to suppress mutant SOD1 mRNA and protein in the affected tissues. We have previously shown that an adeno-associated virus serotype rh10 (AAV10) vector mediating exon skipping of the human SOD1 (hSOD1) pre-mRNA significantly reduces mutant SOD1 expression in SOD1G93A-ALS mice (Biferi et al., 2017). Viral particles were delivered through combined intra-cerebroventricular and systemic injections, efficiently reducing hSOD1 levels in MNs and SM. This approach prolonged survival and restored neuromuscular functions in vivo. In order to understand the tissue-specific contribution to the disease and to characterize the effects of the gene therapy approach in the human pathological context, we have generated in vitro models from patient's dermal fibroblasts (expressing SOD1 mutations: A4V, L144F and G93D). Primary fibroblasts were immortalized and trans-differentiated into myoblasts upon conditional MyoD over-expression, using established protocol. We analyzed SOD1 mRNA and protein expression under physiological condition or upon proteasome inhibition in fibroblasts and differentiated myotubes. We observed a statistical increase of hSOD1 mRNA and protein in patient cells relative to controls, after treatment with the proteasome inhibitor MG132. Proliferation and differentiation properties in myotubes were impaired in SOD1-mutant cells, with more striking effects in the A4V cell line (determining an aggressive disease progression in patients). We are currently testing the AAV-10 mediated gene therapy effects in these in vitro models. Our results will contribute to develop in vitro cellular models to investigate ALS physiopathology and gene therapy approaches.