DUAL MODULATION OF PATHOGENIC AND NEUROPROTECTIVE PATHWAYS IN ALS USING A CHIMERIC SINEUP-BASED RNA CONSTRUCT

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the selective loss of motor neurons, for which effective disease-modifying therapies remain limited. Pathogenic variants in the Superoxide Dismutase 1 (SOD1) gene associated with toxic gain-of-function mechanisms account for approximately 15-20% of familial ALS cases and 2-4% of sporadic cases. In parallel, glial cell line-derived neurotrophic factor (GDNF) has been shown to promote motor neuron survival in both in vitro and in vivo models of neurodegeneration. In this project, we developed a novel chimeric RNA construct that combines two complementary therapeutic strategies: an artificial microRNA built upon a pri-miR155 scaffold designed to downregulate SOD1 expression and a SINEUP RNA engineered to enhance GDNF translation. The expression cassette was cloned into the 3’-UTR of a GFP expressed by a pAAV vector under a Pol-II promoter. As assessed by qPCR and western blot analyses, this construct effectively decreases SOD1 levels while concomitantly increasing GDNF expression in murine P19 and Neuro-2a cell lines. The next phase of the project will focus on in vivo evaluation of the construct, including its biodistribution, safety, and therapeutic efficacy in WT and ALS mouse models. By simultaneously reducing a key pathogenic driver and enhancing a neuroprotective factor, this approach aims to restore a more favorable cellular environment for motor neurons’ survival. Overall, this work provides a proof-of-concept for chimeric RNA constructs that integrate gene silencing and translational enhancement within a single molecular framework, with potential applicability to ALS and other neurodegenerative disorders.