Limb Girdle Muscular Dystrophy (LGMD) refers to a group of disorders characterized by progressive wasting and weakness of pelvic and shoulder girdle muscles. Patients commonly require wheelchair assistance, and individuals with some forms of LGMD may have cardiac and respiratory muscle involvement. Modest improvements in a limited set of muscles may dramatically improve patients’ quality of life, but currently no effective treatment exists. There are 8 dominant forms of LGMD (LGMD1) that would benefit from gene therapy strategies to reduce their pathogenic alleles. We previously showed the first RNAi-based pre-clinical treatment for LGMD1A, which is caused by dominant mutations in one allele of the myotilin (MYOT) gene. Our strategy involved delivering MYOT targeted artificial microRNAs (miMYOT) to muscles of the T57I transgenic mouse model of LGMD1A using AAV6 vectors. Both 3 months (short-term) and 9 months (long-term) time points showed significant reduction of mutant myotilin mRNA and soluble protein expression. MYOT gene silencing resulted in histological improvements that were accompanied by significant functional correction including increased muscle weight and improved specific force. While the initial study showed proof-of-principle for this therapy, translation of miMYOT requires additional work. Mouse knockout studies suggested MYOT is a non-essential gene, as animals lacking MYOT develop normally. In the case of LGMD1A this would suggest the mutation is harmful but complete loss would be tolerated. However, we do not currently know if haploinsufficiency of wild-type MYOT is tolerated in other organisms, including humans. To address this question, we delivered morpholinos or AAV.miRNAs to multiple species. We found knockdown of endogenous MYOT to be safe in developing Xenopus, adult mice and non-human primates, suggesting that a non-allele-specific silencing approach for MYOT would be tolerated in LGMD1A-targeted treatments. We also performed dose escalations to assess the safety parameters of AAV.miMYOT vectors and utilized microarray data to identify potential biomarkers for clinical outcome measures. We then validated that some of our lead biomarkers were significantly normalized in miMYOT-treated T57I mice. Together these data provide additional data to support the further translation of miMYOT therapy for LGMD1A.