Development of Highly Efficient Dual-AAV Split Adenosine Base Editor for In Vivo Gene Therapy

Abstract

The adenosine base editor (ABE) is able to catalyze A•T to C•G conversion efficiently and precisely in vivo, representing a new method for gene therapy. Adeno associated virus (AAV) is a well-studied vector for gene delivery in vivo. However, due to the limited loading capacity of AAV vector (≈4800 bp), it is difficult to package ABE (≈5400 bp) into a single AAV. To tackle this problem, ABE can be split into two smaller parts through intein-mediated protein trans-splicing. Here, 14 different split sites of nCas9 (Cas9 nickase) in combination with three different inteins (Mxe, Npu, and Rma) are screened through a GFP-based reporter system to identify novel split-ABEs. After infecting HEK293T and HeLa cells with dual AAVs, two split-ABEs (split-ABE-Rma573 and split-ABE-Rma674) that can edit the target gene efficiently are identified. Furthermore, these dual-AAV split-ABEs can effectively disrupt the splicing acceptor of PCSK9 in mouse liver and the splicing donor of NR2E3 in mouse retina through AI-MAST strategy. This study provides two new split-ABEs to investigate gene function in vivo and in gene therapy, representing a new method to treat diseases by precisely repairing point mutations or inactivating genes through the AI-MAST strategy.