A Transformative Base Editing Drug for DMD Therapy


Pre-Clinical Research

Poster Number: S21


Li Cheng, PhD, Suzhou GenAssist Therapeutics Co., Ltd, Tiangang Yuan, MD, Suzhou GenAssist Therapeutics Co., Ltd, Jingsheng Li, MD, Suzhou GenAssist Therapeutics Co., Ltd, Qiuyu Cao, MD, Suzhou GenAssist Therapeutics Co., Ltd, Guangyuan Xiao, MD, PhD, Suzhou GenAssist Therapeutics Co., Ltd, Fengpeng Li, PhD, Suzhou GenAssist Therapeutics Co., Ltd, Yi Dai, MD, PhD, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Chunyan He, PhD, Suzhou GenAssist Therapeutics Co., Ltd

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 with large deletions. Until now, there is no drug for those patients. Without causing double-stranded breaks, base editors are considered as safe editing tools for genetic diseases.
GEN6050 is a DMD exon 50 skipping base editing drug containing two AAV9 vectors, ss.AAV9.oTAM (optimized Targeted AID-mediated Mutagenesis) and ss.AAV9.hE50-sgRNA. GEN6050 target the IVS50 5’SS in the human DMD gene and result in exon 50 skipping at mRNA level, generate a truncated yet in-framed dystrophin protein for exon 50 skipping amenable DMD patients.
We aim to assess the efficacy and off-targets of GEN6050 in normal and exon 50 skipping amenable DMD iPSCs (del 51-53) derived cardiomyocytes/myotubes.
First, the editing window of GEN6050 was defined as 10 bp upstream to 30 bp downstream of hE50-sgRNA. The exon skipping is highly correlated with the editing efficiency, confirmed the exon skipping is caused by efficient DNA modification at IVS50 5’SS.
High exon skipping were observed at the MOI of 1E6 in DMD iPSC derived cardiomyocytes (80%) or myotubes (90%). Up to 40% (MOI=1E6) dystrophin protein immunofluorescence restoration were observed in DMD iPSC derived cardiomyocytes/myotubes. These indicate that GEN6050 has the therapeutical potential for exon 50 skipping amenable DMD patients.
To assess off-target of GEN6050, we identified 167 Genomic rearrangements, 38709 SNV and 1704 inser/del site through WGS data analysis in GEN6050-treated myotubes with high on-target editing efficiency using non-treated sample as reference; Simultaneously, 134,849 (mismatch <= 4 and <=3 DNA bulge) off-target sites were identified via in silicon prediction by Cas-Offinder. Then, the overlapped sites from WGS and in silicon prediction was selected as potential off-target sites which were validated by target-NGS. No potential off-targets were found in treated normal iPSC myotubes. Conclusion In summary, GEN6050 leverages base editing activities to restore the Dystrophin protein in DMD iPSC derived cardiomyocytes/myotubes with low to non-detectable off-target risk. Our results indicate that GEN6050 can provide a potential cure for DMD exon 50 skipping amenable patients.