Department of Infectious Diseases/Virology, BioQuant Center, Heidelberg University Hospital, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany; German Center for Infection Research (DZIF), Heidelberg, Germany.
Department of Infectious Diseases/Virology, BioQuant Center, Heidelberg University Hospital, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany; Cluster of Excellence CellNetworks, 69120 Heidelberg, Germany.
Mol Ther. 2020 Apr 8;28(4):1016-1032. doi: 10.1016/j.ymthe.2020.02.009. Epub 2020 Feb 13.
Display of short peptides on the surface of adeno-associated viruses (AAVs) is a powerful technology for the generation of gene therapy vectors with altered cell specificities and/or transduction efficiencies. Following its extensive prior use in the best characterized AAV serotype 2 (AAV2), recent reports also indicate the potential of other AAV isolates as scaffolds for peptide display. In this study, we systematically explored the respective capacities of 13 different AAV capsid variants to tolerate 27 peptides inserted on the surface followed by production of reporter-encoding vectors. Single-round screening in pre-arrayed 96-well plates permitted rapid and simple identification of superior vectors in >90 cell types, including T cells and primary cells. Notably, vector performance depended not only on the combination of capsid, peptide, and cell type, but also on the position of the inserted peptide and the nature of flanking residues. For optimal data availability and accessibility, all results were assembled in a searchable online database offering multiple output styles. Finally, we established a reverse-transduction pipeline based on vector pre-spotting in 96- or 384-well plates that facilitates high-throughput library panning. Our comprehensive illustration of the vast potential of alternative AAV capsids for peptide display should accelerate their in vivo screening and application as unique gene therapy vectors.
腺相关病毒（AAV）表面展示短肽是一种强大的技术，可用于生成改变细胞特异性和/或转导效率的基因治疗载体。在广泛应用于特征最明显的 AAV 血清型 2（AAV2）之后，最近的报告还表明其他 AAV 分离株作为肽展示支架的潜力。在这项研究中，我们系统地研究了 13 种不同的 AAV 衣壳变体各自容纳 27 个表面插入肽的能力，随后生产了报告基因编码载体。在预先排列的 96 孔板中的单轮筛选允许在 >90 种细胞类型（包括 T 细胞和原代细胞）中快速、简单地识别出优越的载体。值得注意的是，载体性能不仅取决于衣壳、肽和细胞类型的组合，还取决于插入肽的位置和侧翼残基的性质。为了获得最佳的数据可用性和可访问性，我们将所有结果组装到一个可搜索的在线数据库中，该数据库提供多种输出样式。最后，我们建立了基于 96 孔或 384 孔板中载体预点样的反向转导管道，该管道可促进高通量文库筛选。我们对替代 AAV 衣壳在肽展示方面的巨大潜力的全面说明应加速它们在体内的筛选和应用，作为独特的基因治疗载体。
