Estes Brett J G, Gandhi Nisha, Von Stetina Jessica R, Paudel Dev, Nan Angela X, Amin Parth, Rose Joshua, Wu Shuai, Zheng Kangni, Zhang Yijun, Cochrane Jesse C, Finn Jonathan D, Xie Jenny
Tome Biosciences, Watertown, MA 02472, USA.
Mol Ther Methods Clin Dev. 2025 May 14;33(2):101490. doi: 10.1016/j.omtm.2025.101490. eCollection 2025 Jun 12.
Recent advancements in gene insertion have shifted from DNA-repair-dependent mechanisms to more precise approaches, enhancing safety and predictability for editing outcomes. Integrase-mediated programmable genomic integration (I-PGI) utilizes a DNA cargo to insert transgenes in a targeted, unidirectional manner. , where nuclear delivery of DNA is challenging, adeno-associated virus (AAV) can act as the cargo vector. Although I-PGI does not require DNA double-strand breaks (DSBs) for activity, linear cargo, like AAV, stimulates DNA end-joining activity after integration. To mitigate potential risks from DSBs, we developed two circular AAV cargos capable of seamless gene insertion in non-dividing cells. We first harnessed the orthogonal property of large serine integrases to produce circle-AAV (cAAV) from linear viral genomes in cells. cAAV demonstrated seamless cargo integration in primary human hepatocytes (PHHs) and robust DSB-free insertion structures . We then investigated the delivery of a packaged circular AAV cargo (AAV.AD), which eliminates the need for enzymatic manipulation in the cell. AAV.AD exhibited functional seamless gene insertion in PHHs and showed cargo efficacy . Together, these findings provide evidence of DSB-free programmable genomic integration using integrase and AAV cargo, addressing a previously unrecognized challenge in the field.
基因插入技术的最新进展已从依赖DNA修复的机制转向更精确的方法，提高了编辑结果的安全性和可预测性。整合酶介导的可编程基因组整合（I-PGI）利用DNA载体以靶向、单向的方式插入转基因。在DNA核递送具有挑战性的情况下，腺相关病毒（AAV）可作为载体。虽然I-PGI的活性不需要DNA双链断裂（DSB），但线性载体（如AAV）在整合后会刺激DNA末端连接活性。为了降低DSB带来的潜在风险，我们开发了两种能够在非分裂细胞中无缝插入基因的环状AAV载体。我们首先利用大丝氨酸整合酶的正交特性在细胞中从线性病毒基因组产生环状AAV（cAAV）。cAAV在原代人肝细胞（PHH）中显示出无缝载体整合以及强大的无DSB插入结构。然后，我们研究了一种包装好的环状AAV载体（AAV.AD）的递送，这种载体消除了细胞内酶促操作的需要。AAV.AD在PHH中表现出功能性无缝基因插入并显示出载体功效。总之，这些发现提供了使用整合酶和AAV载体进行无DSB可编程基因组整合的证据，解决了该领域之前未被认识到的挑战。
