Tavora Rubens, Zhang Lizhou, Tran Mai H, Li Hao, O'Hagan Dan, Pan Andi, Barrett Lorenzo, Jablonski Joseph A, Mediouni Sonia, Lopez Alexander, Comella Zachary, Bailey Charles, Choe Hyeryun, Farzan Michael, Valente Susana T
Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, Florida, USA.
The Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, Florida, USA.
Hum Gene Ther. 2025 Jun;36(11-12):870-883. doi: 10.1089/hum.2025.011. Epub 2025 May 12.
Recombinant adeno-associated virus (rAAV) vectors are increasingly preferred for 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 . 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.
重组腺相关病毒（rAAV）载体因其广泛的嗜性、低免疫原性和持续的转基因表达，在基因治疗中越来越受到青睐。然而，在这些表达出现不良反应的情况下，一种抑制或永久终止rAAV转基因活性的方法可以显著提高这些载体的安全性。为满足这一需求，我们采用了具有长识别序列的高度特异性DNA内切酶——归巢核酸酶。通过将归巢核酸酶靶位点置于rAAV转基因内，我们创建了一个系统，其中靶向切割导致转基因表达的可控破坏。利用荧光素酶测定法，我们筛选了各种归巢核酸酶，并确定I-AniI-Y2、I-BmoI和I-PpoI因其高切割效率而成为主要候选者。通过在基因内以及转基因的5'和3'非翻译区（UTR）中策略性地放置多个归巢核酸酶靶序列，我们显著提高了这些归巢核酸酶的切割效率，确保了对转基因表达的有力且靶向性抑制。最后，我们采用了载有mRNA的脂质纳米颗粒递送系统，以证明归巢核酸酶有力抑制rAAV介导的转基因表达的能力。我们的研究结果强调了归巢核酸酶作为rAAV基因治疗中一种可行的安全机制的潜力，标志着朝着更安全的长期基因治疗方法迈出了重要一步。
