Department of Pharmacy - Center for Drug Research, LMU Munich, Munich, 81377, Germany.
ViGeneron GmbH, Planegg, 82152, Germany.
Nat Commun. 2023 Oct 18;14(1):6578. doi: 10.1038/s41467-023-42386-0.
Large genes including several CRISPR-Cas modules like 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, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.
大型基因包括几个 CRISPR-Cas 模块，如基因激活剂（CRISPRa），需要双腺相关病毒（AAV）载体才能实现高效的体内传递和表达。目前的双 AAV 载体方法存在重要的局限性，例如，重组效率低、产生异源蛋白或在分裂位点选择方面灵活性低。在这里，我们提出了一种基于 mRNA 反式剪接（REVeRT）重组的双 AAV 载体技术。REVeRT 在分裂位点选择方面具有灵活性，可以在许多体外模型、人类类器官和体内高效重组不同的分裂基因。此外，REVeRT 可以在不同的给药途径下，通过单重或多重方式，在各种小鼠组织和器官中针对基因的 CRISPRa 模块进行功能重组。最后，REVeRT 能够在 Stargardt 病小鼠模型中通过玻璃体内注射来重组全长 ABCA4。由于其灵活性和效率，REVeRT 在基础研究和临床应用中具有巨大的潜力。
