Hoffmann Mareike Daniela, Sorensen Ryan James, Extross Ajay, He Yungui, Schmidt Daniel
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455, United States.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, United States.
ACS Nano. 2025 Apr 1;19(12):12308-12322. doi: 10.1021/acsnano.5c01498. Epub 2025 Mar 21.
Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy, enabling the delivery of therapeutic DNA to target cells. However, the potential of AAV to deliver protein payloads has been unexplored. In this study, we engineered a protein carrier AAV (pcAAV) to package and deliver proteins by inserting binding domains on the interior capsid surface. These binding domains mediate the packaging of specific target proteins through interaction with cognate peptides or protein tags during the capsid assembly process. We demonstrate the packaging of multiple proteins, including green fluorescent protein, Cas9, Cre recombinase, and the engineered peroxidase APEX2. Packaging efficiency is modulated by the binding domain insertion site, the viral protein isoform containing the binding domain, and the subcellular localization of the target protein. We show that pcAAV can enter cells and deliver the protein payload and that enzymes retain their activity after packaging. Importantly, this protein packaging capability can be translated to multiple AAV serotypes. Our work establishes AAV as a protein delivery vehicle, significantly expanding the utility of this viral vector for biomedical applications.
腺相关病毒（AAV）已成为基因治疗的主要平台，能够将治疗性DNA递送至靶细胞。然而，AAV递送蛋白质有效载荷的潜力尚未得到探索。在本研究中，我们通过在衣壳内表面插入结合结构域，构建了一种蛋白质载体AAV（pcAAV），用于包装和递送蛋白质。这些结合结构域在衣壳组装过程中通过与同源肽或蛋白质标签相互作用，介导特定靶蛋白的包装。我们展示了多种蛋白质的包装，包括绿色荧光蛋白、Cas9、Cre重组酶和工程化过氧化物酶APEX2。包装效率受结合结构域插入位点、包含结合结构域的病毒蛋白异构体以及靶蛋白的亚细胞定位的调节。我们表明，pcAAV可以进入细胞并递送蛋白质有效载荷，并且酶在包装后仍保持其活性。重要的是，这种蛋白质包装能力可以转化到多种AAV血清型。我们的工作将AAV确立为一种蛋白质递送载体，显著扩展了这种病毒载体在生物医学应用中的效用。
