mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

Abstract

Large genes including several CRISPR-Cas modules, such as gene activators (CRISPRa), require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, intravitreal supplementation of ABCA4 via REVeRT improves retinal degeneration and function in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.