Efficient correction of a hypertrophic cardiomyopathy mutation in mouse embryos by ABEmax-NG (Mouse). Mus musculus

Genetic editing has shown great potential for the treatment of human hereditary disorders via the elimination of mutations in embryos for implantation via in vitro fertilization (IVF). However, the efficiency and safety of germline gene editing are not well understood. We aimed to examine the preclinical efficacy/safety of zygotic base editing in a mouse model of hypertrophic cardiomyopathy (HCM) using a novel adenine base editor (ABE) platform. To correct the heterozygous alpha-MHC R404Q mutation, we developed an ABEmax-NG system. To identify the cardiac transcriptional responses in mice who underwent genetic editing for the Myh6 mutation, we performed RNA-sequencing (RNA-Seq) analysis on 8-week mice heart, which contain WT(BE_W, N=3),R404Q/+ (Mutation, N=3) and Correction (BE_C, N=3). Heat maps showed that ABEmax-NG corrected mice and wild-type animals had similar transcriptome profiling. Genes associated with energy metabolism in HCM and extracellular matrix remodeling were found to be corrected in gene edited heart tissue. The injection of base-edited mRNA together with sgRNA is capable of correcting mutant bases in genomic DNA, with a potential for off-target effects in DNA or RNA. To assess the sgRNA-dependent DNA off-target effect of ABEmax-NG, we performed whole gene sequencing on 8-week mice heart(WT, N=1; Correction, N=1). Our results indicated that no indels or off-target conversions were induced in the genomic DNA of corrected mice by ABEmax-NG at off-target sites. To determine RNA edits, we performed an RNA-seq analysis of the early embryos, which contained 2-cell embryos(Control, n=2; ABEmax-NG, n=2), 8-cell embryos(Control,n=3; ABEmax-NG, n=3) and Blastocyst (Control,n=2; ABEmax-NG, n=2). And data showed that our ABEmax-NG system also induced slight RNA edits in the 8-cell stage, which were then eliminated as the embryos developed. We developed an ABEmax-NG system, which efficiently corrected a pathogenic Myh6 mutation in mouse zygotes without off target lesions, thus safely eliminating HCM in the derived mice and in their offspring.