Mutations in the DYSF gene encoding the muscle membrane protein dysferlin causes Limb-Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi Myopathy (MM). These diseases are characterized by locomoter muscle weakness and wasting that is hastened by childhood and adolescent physical activity, sports and exercise. We have previously shown that dysferlin mediates the repair of myofiber membrane injuries by enabling rapid, injury-triggered secretion of the enzyme acid sphingomyelinase (ASM) from the injured myofiber – a process required for repair. Lack of Dysferlin in LGMD2B/MM delays and reduces injury-triggered ASM secretion upon muscle cell injury, thereby hindering repair of injuries incurred through daily activity and exercise. While there are currently no treatments available for LGMD2B, we have previously conducted a short-term, proof-of-concept study to improve myofiber repair in LGMD2B by stimulating hepatic production of circulating ASM protein via ASM gene transfer (ASM-AAV). However, progression to human trials requires vector optimization, assessment of short and long-term efficacy, and quantification of gene transfer efficiency in advance of pre-clinical testing. Here, we conducted systematic SMPD1 (ASM gene) cDNA optimization to enhance ASM enzymatic activity that may lower the required viral dose in-vivo. Codon optimization (modification #1 – MOD1) in-tandem with removal of c-terminal Cystine (MOD3) increased ASM secretion from infected liver cells in-vitro (vs. first-generation proof-of-concept cDNA) by ~50%. However, upon assessing the ASM sphingomyelin hydrolysis capacity/activity of mutated cDNAs, MOD3 exhibited striking 8-fold higher enzymatic activity (vs. 1-st generation), 2.5-fold higher (vs. MOD1), activity that was dependent upon exogenous zinc. Lastly, we tested the capacity of MOD3-infected liver cell supernatant to improve PM repair of LGMD2B patient myoblasts, and find a nearly 6-fold lower required protein levels to improve repair (vs. our 1-st generation proof-of-concept cDNA). We have thus identified an optimized ASM gene modification that produces ASM protein that is pre-cleaved of its C-terminal end, rendering it constitutively active upon cellular secretion, and necessitating significantly less circulating protein levels to improve PM repair in LGMD2B. Subsequent studies will test the capacity of this newly modified ASM cDNA to improve LGMD2B muscle health and function in-vivo, when packaged within liver-targeting AAV vectors.