Abstract Spinal muscular atrophy (SMA) is a devastating neuromuscular disease and a leading genetic cause of infantile death worldwide. Despite exciting progress in the neuromuscular field that has resulted in novel therapies, there remains no permanent cure for SMA. Therefore, developing a permanent treatment that treats the underlying genetic perturbation and all systemic manifestations of this disease would transform patients’ life. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene. An important modifier of SMA severity is the number of copies of a paralogous gene SMN2. The sequence of SMN2 mainly differs from SMN1 by a C•G-to-T•A transition in exon 7 (“C6T”), which causes the skipping of exon 7 in most SMN2 transcripts due to alternative splicing. While previous studies have demonstrated that antisense oligonucleotides (ASOs) or small molecules targeted to SMN2 gene can transiently increase full-length SMN protein expression, this approach requires repeated dosing and has incomplete penetrance that poses challenges for patients. Therefore, I am developing optimized base editors (BEs) that enable precise genetic alterations within defined regions of the SMN2 gene. In this K01 proposal, I plan to extend this work by optimizing the most optimal genome editing approaches and investigating the use of adeno-associated viruses (AAVs) to deliver these novel editing technologies in SMA mouse models. In principle, this will create a lasting single-dose permanent treatment for SMA with several advantages compared to targeting mutations in the SMN1 gene or exogenous gene replacement. The success of this proposal will provide solutions to questions at the intersection of the fields of genome editing and neurogenetics, with broad implications for the entire neuromuscular scientific community. My primary mentor in this K01 proposal is Dr. Benjamin Kleinstiver, an expert in the optimization of CRISPR technologies, and my co- mentors are Dr. Rashmi Kothary and Dr. Kathryn Swoboda, world leaders in SMA-related studies. Career development and training activities will include attendance to local meetings and seminars, attendance at national and international conferences, scientific writing, mentoring a small group in the laboratory, and managing collaborations with other academic groups and companies. The K01 Award would be a perfect opportunity to prepare me for the transition to an independent career by developing important career aspects and improving my knowledge in gene editing to establish my research program, so that I can address high- impact questions related to SMA and other neuromuscular diseases . M y long-term goals are to lead a translational research team to address fundamental questions related to neuromuscular physiology, diseases, and disorders. The convergence between physiology, genome editing, and therapeutics is where I envision my independent research laboratory will make an impact on human health.

Narrative Despite all progress in the neuromuscular field, there is no permanent cure for spinal muscular atrophy (SMA), and all current available therapies have important limitations. This study will develop strategies to permanently edit the SMN2 gene to produce full-length Survival Motor Neuron (SMN) protein by applying CRISPR technologies. The study will provide pre-clinical evidence towards the development of a lasting single-dose genetic treatment for SMA.