Pooja Nair1,2, Jennifer Ataam Arthur3, and Ioannis Karakikes1,2
1. Department of Cardiothoracic Surgery; 2. Stanford Cardiovascular Institute; 3. Institute for Immunity, Transplantation, and Infection, Stanford Medicine, Stanford, CA
Rationale: The recently developed CRISPR–Cas-based genome editing holds great promise for targeting genetic disorders, such as cardiomyopathies. Adenine base editors (ABE) enable efficient adenine-to-guanine base (A∙T to G∙C) conversion in post-mitotic cells independent of dsDNA break formation and homology-directed repair (HDR). Familial dilated cardiomyopathy arising from C-to-T point mutations, such as TNNT2R173W, can potentially be corrected by an ABE base editing system in vivo.
Objective: As precise correction of disease-causing mutations in adult tissues in vivo is challenging, we are establishing a versatile adeno-associated viral (AAV) platform for ABE-dependent base editing in adult animals.
Methods and Results: We engineered a dual trans-splicing AAV vector system encoding the newly described xCas9 and SpCas9-NG variants that recognize a wider range of protospacer adjacent motif (PAM) sequences that is compatible with gRNA-directed targeting of the TNNT2R173W mutation. This system allows splitting of the fusion ABE-Cas9 protein into two parts, thereby circumventing the limited cargo capacity of AAV vectors. Combined with an AAV vector expressing a targeting gRNA, a three-vector base editing system was developed and validated in vitro. In silico analysis of the TNNT2R173W site has shown that it is amenable to adenine base editing with multiple gRNAs. We are currently testing this system in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring the TNNT2R173W mutation in vitro, and in a transgenic mouse model of dilated cardiomyopathy carrying the same mutation in vivo.
Conclusions: The therapeutic potential offered by this AAV-ABE system holds promise in future testing on transgenic mice models of dilated cardiomyopathy. Our system addresses two key challenges in therapeutic base editing, namely target recognition and in vivo delivery of the large ABE-Cas9 fusion gene, by introducing two engineered AAV-ABE variants with relaxed PAM recognition and a modified split-AAV platform respectively.