Gene editing offers a solution for permanent DMD gene reframing as a potential treatment for Duchenne muscular dystrophy, a fatal muscle wasting disease. GEN6050X is an intravenous dual AAV product containing two AAV9 drug products, one encodes muscle-specific promoter-driven Targeted-AID mediated mutagenesis induced cytosine base editor (TAM CBE) and one carries encodes 3 copies of hE50 sgRNA targeting 5’SS of DMD IVS50 to induce DMD exon 50 skipping. In addition, the sgRNA vector carries a human ACTG1 gene, which can bind to de novo restored dystrophin to rapidly facilitate costamere and Dystroglycan-associated complex remodeling and provide a synergistic therapeutic effect with TAM CBE. The vectors will be manufactured and released separately, necessitating potency assays for each vector and final product GEN6050X.
Here we demonstrated initial potency feasibility of GNE6050X based on therapeutic mechanism of GEN6050X. GEN6050X was diluted in gradient (MOI) and infected iPSC DMD del51-53 derived myotube for 7 days, the exon50 skipping and transcription of the exogenous ACTG1 were quantified using qPCR. The specificity, linearity, accuracy and precision were assessed. For specificity, only GEN6050X can induce exon50 skipping and ACTG1 transcripts. As for the linearity, both exon skipping efficiency and the copy number of ACTG1 transcripts exhibit linear relationship with the MOI of GEN6050X. For the accuracy and precision, six replicate experiments were performed, and the data were analyzed, the coefficient of variation (CV) was within the appropriate range, and the consistency was also excellent and met the standard requirement. These results indicate that our potency assay is reliable, which is essential for ensuring the quality and efficacy of GEN6050X in each production.
Collectively, we have performed the initial potency testing for GEN6050X, which is essential for cGMP batch GEN6050X release.