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

Abstract

AbstractLarge genes including several CRISPR-Cas modules, such as gene activators (CRISPRa), require dual adeno-associated viral (AAV) vectors for an efficientin vivodelivery 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 onreconstitutionvia mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerousin vitromodels, in human organoids, andin 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 ofABCA4via 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.