No abstract

Rationale: Genetic editing has shown great potential for the treatment of human hereditary disorders via the elimination of mutations in embryos. However, the efficiency and safety of germline gene editing are not well understood. Objective: We aimed to examine the preclinical efficacy/safety of embryonic base editing in a mouse model of hypertrophic cardiomyopathy (HCM) using a novel adenine base editor (ABE) platform. Methods and Results: Here, we described the use of an ABEmax-NG to directly correct the pathogenic R404Q/+ mutation (Myh6 c.1211C>T) in embryos for a mouse model of HCM, increasing the number of wild-type embryos for in vitro fertilization. Delivery of the ABEmax-NG mRNA to embryos from R404Q/+ HCM mice resulted in 62.5-70.8% correction of the Myh6 c.1211C>T, reducing the level of mutant RNA and eliminating HCM in the post-natal mice as well as their offspring. In addition, the same sgRNA was also used to target an intronic locus (TGG PAM) with an overall editing rate of 86.7%, thus confirming that ABEmax-NG can efficiently edit target loci with different PAMs (NG) and genomic distribution in vivo. Compared with CRISPR/ssODN-mediated correction, ABEmax-NG displayed a much higher correction rate without introducing indels. DNA and RNA off-target analysis did not detect off-target editing in treated embryos and founder mice. In utero injection of adeno-associated virus 9 (AAV9) encoding the ABEmax-NG also resulted in around 25.3% correction of the pathogenic mutation and reduced of mutant RNA, thereby indicating ABEmax-NG has the potential to correct the HCM mutation in vivo. Conclusions: We developed an ABEmax-NG system, which efficiently corrected a pathogenic Myh6 HCM mutation in mouse embryos without off target lesions, thus safely eliminating HCM in derived mice and their progeny.