Boucher Jonathan D, Stranford Devin M, Edelstein Hailey I, Tullman-Ercek Danielle, Kamat Neha P, Leonard Joshua N
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.
Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, Illinois 60208, United States.
bioRxiv. 2025 Jul 4:2025.07.02.662894. doi: 10.1101/2025.07.02.662894.
Adeno-associated virus (AAV) gene therapies have achieved clinical success, with multiple products reaching regulatory approval. Encapsulation of AAV vectors within engineered extracellular vesicles (EVs) is an emerging strategy which could help overcome challenges including pre-existing anti-capsid immunity and the need for controlling targeting and tropism. To guide the development of EV-AAV technologies, we developed an assay for quantifying and controlling for the contribution of pseudotransduction to evaluations of EV-AAV-mediated gene delivery. We developed an AAV vector that switches its transgene output from one reporter to another when acted upon by Cre recombinase expressed in a recipient cell. Using this platform, we investigated EV-AAV transduction as a function of various engineered EV surface modifications. For actively endocytic cells (HEK293FTs), modifications that enhance EV uptake and membrane fusion influence protein delivery but not gene delivery. Conversely, in less endocytic Jurkat T cells, modifications enhancing EV uptake improved both protein and gene delivery. These conclusions held across multiple AAV serotypes. Our results resolve apparent conflicts in prior reports and suggest that effects of enhancing uptake and membrane fusion of EV-AAV vectors are recipient cell type specific. The methods developed here unambiguously dissect EV-AAV transduction mechanisms and can guide future bioengineering of EV-AAV vectors.
腺相关病毒（AAV）基因疗法已取得临床成功，多种产品已获得监管批准。将AAV载体封装在工程化细胞外囊泡（EV）中是一种新兴策略，有助于克服包括预先存在的抗衣壳免疫以及控制靶向性和嗜性等挑战。为指导EV-AAV技术的发展，我们开发了一种检测方法，用于量化和控制假转导对EV-AAV介导的基因传递评估的贡献。我们开发了一种AAV载体，当受到受体细胞中表达的Cre重组酶作用时，其转基因输出会从一种报告基因切换到另一种报告基因。利用这个平台，我们研究了作为各种工程化EV表面修饰函数的EV-AAV转导。对于主动内吞细胞（HEK293FT细胞），增强EV摄取和膜融合的修饰会影响蛋白质传递，但不会影响基因传递。相反，在较少内吞的Jurkat T细胞中，增强EV摄取的修饰改善了蛋白质和基因传递。这些结论适用于多种AAV血清型。我们的结果解决了先前报告中明显的矛盾，并表明增强EV-AAV载体摄取和膜融合的效果是受体细胞类型特异性的。这里开发的方法明确剖析了EV-AAV转导机制，并可指导未来EV-AAV载体的生物工程。
