Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG

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.

Rare Genetic Variants Associated With Myocardial Fibrosis: Multi-Ethnic Study of Atherosclerosis

“…Early preventative care in these carriers coupled with effective control of comorbidities may attenuate the development of HF ( 14 ). Moreover, with the discovery of novel and promising therapeutic approaches, including sarcomere protein inhibitors ( 15 ) and base editors ( 16 ), carriers of pathogenic variants in CM-related genes can be treated, if not cured.…”

“…However, whether rare pathogenic/likely pathogenic (P/LP) variants in CM genes are associated with myocardial fibrosis, a predecessor of evident morphologic changes in CM, is unknown. Early identification of pathogenic variants in CM-related genes not only would call for a stricter surveillance in CM prevention and progression in proband and relatives ( 14 ), but also identifies the target population for the novel yet promising treatment methods ( 15 , 16 ).…”

Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

“…Previous studies have demonstrated the use of base editors to create or correct specific mutations in different genomic loci in various genetic disorders (28,(36)(37)(38). Most studies in β hemoglobinopathies have demonstrated the therapeutic up-regulation of fetal globin expression by introducing HPFH-like mutations or by disruption of fetal globin repressors using base editors to compensate for the deficient β-globin (23).…”

CRISPR Modeling and Correction of Cardiovascular Disease

“…147 In another proof-of-concept study, a mouse model for genetic HCM, carrying a pathogenic MYH6 R404Q/+ (c1211 C>T) mutation, was corrected by ABE in mouse zygotes. 148 Germline injection of ABEmax-NG mRNA and sgRNA base editing components resulted in highly efficient correction of the point mutation, reducing the level of mutant mRNA and eliminating HCM in the postnatal mice and their progeny. 148 In utero gene editing by the AAV9-ABEmax-NG split system in embryos resulted in correction in the heart.…”

“…148 Germline injection of ABEmax-NG mRNA and sgRNA base editing components resulted in highly efficient correction of the point mutation, reducing the level of mutant mRNA and eliminating HCM in the postnatal mice and their progeny. 148 In utero gene editing by the AAV9-ABEmax-NG split system in embryos resulted in correction in the heart. 148 In utero ABE was also shown to correct a lethal lysosomal storage disease, resulting in improved survival and amelioration of abnormalities in several organs including liver, heart, and musculoskeletal organs.…”