Hoffmann Mareike D, Sorensen Ryan J, Extross Ajay, He Yungui, Schmidt Daniel
Department of Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, MN, 55455, USA.
Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, United States.
bioRxiv. 2024 Aug 14:2024.08.14.607995. doi: 10.1101/2024.08.14.607995.
AAV is widely used for efficient delivery of DNA payloads. The extent to which the AAV capsid can be used to deliver a protein payload is unexplored. Here, we report engineered AAV capsids that directly package proteins - Protein Carrier AAV (pcAAV). Nanobodies inserted into the interior of the capsid mediate packaging of a cognate protein, including Green Fluorescent Protein (GFP), Cas9, Cre recombinase, and the engineered peroxidase APEX2. We show that protein packaging efficiency is affected by the nanobody insertion position, the capsid protein isoform into which the nanobody is inserted, and the subcellular localization of the packaged protein during recombinant AAV capsid production; each of these factors can be rationally engineered to optimize protein packaging efficiency. We demonstrate that proteins packaged within pcAAV retain their enzymatic activity and that pcAAV can bind and enter the cell to deliver the protein payload. Establishing pcAAV as a protein delivery platform may expand the utility of AAV as a therapeutic and research tool.
腺相关病毒（AAV）被广泛用于高效递送DNA载荷。AAV衣壳用于递送蛋白质载荷的程度尚未得到探索。在此，我们报告了直接包装蛋白质的工程化AAV衣壳——蛋白质载体AAV（pcAAV）。插入衣壳内部的纳米抗体介导同源蛋白质的包装，包括绿色荧光蛋白（GFP）、Cas9、Cre重组酶和工程化过氧化物酶APEX2。我们表明，蛋白质包装效率受纳米抗体插入位置、纳米抗体插入的衣壳蛋白异构体以及重组AAV衣壳生产过程中包装蛋白的亚细胞定位影响；这些因素中的每一个都可以通过合理设计来优化蛋白质包装效率。我们证明，包装在pcAAV内的蛋白质保留其酶活性，并且pcAAV可以结合并进入细胞以递送蛋白质载荷。将pcAAV确立为蛋白质递送平台可能会扩大AAV作为治疗和研究工具的用途。
