Halting Recombinant Adeno-Associated Virus Transgene Expression Using mRNA-Lipid Nanoparticle-Delivered Meganucleases

Abstract

Recombinant adeno-associated virus (rAAV) vectors are increasingly preferred for in vivo gene therapy due to their broad tropism, low immunogenicity, and sustained transgene expression. Nevertheless, in cases of adverse reactions to these expressions, a method to suppress or permanently halt rAAV transgene activity could significantly enhance the safety of these vectors. To address this need, we employed meganucleases—highly specific DNA endonucleases with long recognition sequences. By placing meganuclease target sites within rAAV transgenes, we created a system in which targeted cleavage leads to controlled disruption of transgene expression. Utilizing a luciferase assay, we screened various meganucleases and identified I-AniI-Y2, I-BmoI, and I-PpoI as prime candidates due to their high cleavage efficiencies. By strategically placing multiple meganuclease target sequences within introns, as well as in the 5′ and 3′ untranslated regions (UTRs) of transgenes, we significantly enhanced the cleavage efficiency of these meganucleases, ensuring robust and targeted suppression of transgene expression. Finally, we employed an mRNA-loaded lipid nanoparticledelivery system to demonstrate the ability of meganucleases to robustly inhibit rAAV-mediated transgene expression in vitro. Our findings underscore the potential of meganucleases as a viable safety mechanism in rAAV gene therapies, marking a significant advance toward safer long-term gene therapy approaches.