DMD is a progressive muscle-wasting disease caused by mutations in the gene encoding the dystrophin protein. Exon 50 skipping can target 4% of DMD patients. Until now, there is no drug for those patients. GEN6050X is an intravenously administered DMD exon 50 skipping base editing drug. The exon 50 skipping mechanism of GEN6050X is to modify the IVS50 5’SS in the human DMD gene and result in exon 50 skipping at mRNA level. For exon 50 skipping amenable DMD patients, exon 50 skipping generates a truncated yet in-framed dystrophin protein. Dose dependent target site editing, exon 50 skipping and dystrophin protein restoration were observed in GEN6050X treated DMD iPSCs differentiated cardiomyocytes and myotubes. The exon skipping efficiency is highly correlated with the editing efficiency, confirmed that the exon skipping is caused by efficient DNA modification at IVS50 5’SS. Comprehensive off-target assessment was performed for GEN6050x in two different cell contexts, iPSCs differentiated myotubes and cardiomyocytes. For DNA off-target assessment: 1. In silicon prediction was performed according to hE50-sgRNA sequence homology to analyze the sgRNA-dependent potential off-target sites. 2. WGS was performed in GEN6050X treated and non-treated control sample to analyze the genomic sgRNA independent off target, each with 150X sequencing depth and 3 replicates. SNVs were analyzed with non-treated samples as reference. 3. Optical genome mapping was performed to analyze genome integration after GEN6050X treatment. Insertion, deletion, inversion and duplication were analyzed. In addition, single site deep sequencing was performed for the potential off-target sites. Detailed annotation includes 1) whether the off-target is in the promoter, 3’UTR, 5’UTR, and/or distance from the nearest coding region in the genome; 2) how these off-target edits can impact cell function was performed for the validated sites. For RNA off-target assessment: Deep RNA sequencing was performed in GEN6050X treated and non-treated control sample, each with 3 replicates. The overall base substitution and conversion was calculated. To assess long term off-target risk in highly distributed non-target tissues in vivo, samples in cynomolgus monkeys of single-dose toxicity study with 26-week period were collected for editing analysis. Detail data will be presented on site.
Collectively, with those comprehensive off-target evaluations, no high-risk DNA or RNA off-target sites were identified.