One of the biggest challenges for gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability, and gene transfer rates. , up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and gene therapy options.

中文翻译：

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使用针对 CD8 的 DART-AAV 进行 T 细胞特异性体内基因递送


基因治疗面临的最大挑战之一是载体介导高度选择性的基因转移到特定的治疗相关细胞群中。在这里，我们展示了 DARPin 靶向 AAV (DART-AAV)，其显示了人类和小鼠 CD8 特异性的 DARPins。将 DARPins 插入 AAV2 和 AAV6 衣壳蛋白 1 (VP1) 的 GH2/GH3 环中，可对 CD8 阳性 T 细胞产生高选择性，且基因递送活性未受影响。值得注意的是，衣壳核心结构未改变，可检测到突出的 DARPins。在复杂的原代细胞混合物中，包括供体血液或小鼠全身注射，CD8靶向AAV在选择性、靶细胞活力和基因转移率方面远远优于未修饰的AAV2和AAV6。 ，将单一载体注射到条件化人源化或免疫活性小鼠体内后，高达 80% 的活化 CD8+ T 细胞被击中。虽然基因转移率在非激活条件下显着下降，但在将 Cre 递送至指示小鼠后，CD8+ T 细胞中的基因组选择性修饰仍然可检测到。在这两种小鼠模型中，对 CD8+ T 细胞的选择性接近绝对，并且从肝脏中异常脱靶。这里描述的 CD8-AAV 扩展了免疫学研究和基因治疗选择的策略。