Optimized dual-AAV base editor delivery system with enhanced editing efficiency and virion production titer

Abstract

Base editors (BEs) are a promising tool for precise base conversion in human cells and animals, while the adeno-associated virus (AAV) is the major vector for human gene therapy. However, the size of the DNA cassette required for BE expression exceeds the 4.7 kb packing capacity of the AAV vector, making dual-AAV approaches based on trans-splicing intein necessary. Even with this approach, current split DNA cassettes are still larger than the AAV packing limit, posing a challenge for cellular production of AAV. Moreover, some split strategies yield variable editing results and target coverage. To address these limitations, 25 different split sets for BE4max and A3A-BE4max were tested at two target sites respectively, with splitting sites ranging from 493rd to 517th amino acids on the Cas9 peptide. Fortunately, the best Cas9 split site was identified between His511 and Ser512 and the arrangement of the AAV expression cassette was further manipulated to create evenly distributed CBE and ABE intein systems within 4.7 kb. These novel dual-AAV systems, designated 4.6AAV-CBE and 4.7AAV-ABE, were found to have base editing efficiencies similar to wild-type BEs, with a narrower editing window than the current 573 split system. Notably, 4.6AAV-CBE yield a higher AAV production titer, up to 2.1-fold in AAV-N and 1.5-fold in AAV-C, compared to the split-573BE system, likely due to the reduction of DNA cassette size within the AAV packaging capacity. Moreover, after packaging and infecting cells with AAV-N and AAV-C at the same volume and number of cells, the multiplicities of infection (MOI) and editing efficiency of 4.6 AAV-CBE were both higher than those of the split-573BE system. This study present advanced dual-AAV systems for ABE and CBE delivery, establishing a basis for safe and efficient BE therapies.