One of the biggest challenges for in vivo 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. In vivo, 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 in vivo gene therapy options.

中文翻译：

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使用靶向 CD8 的 DART-AAV 进行 T 细胞特异性体内基因递送


体内 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确定的治疗相关细胞群中。在这里，我们介绍了 DARPin 靶向 AAV （DART-AAV），它们显示了对人和小鼠 CD8 具有特异性的 DARPin。将 DARPins 插入 AAV2 和 AAV6 衣壳蛋白 1 （VP1） 的 GH2/GH3 环中，导致对 CD8 阳性 T 细胞具有高选择性，基因递送活性未受损。值得注意的是，衣壳核心结构没有改变，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，靶向 CD8 的 AAV 在选择性、靶细胞活力和基因转移速率方面远远优于未修饰的 AAV2 和 AAV6。在 体内，高达 80% 的活化 CD8 + T 细胞在将单个载体注射到条件人源化或免疫功能正常的小鼠中时受到打击。虽然在未活化条件下基因转移速率显着降低，但在 Cre 递送到指示小鼠中时，仍然可以检测到 CD8+ T 细胞中的选择性基因组修饰。在两种小鼠模型中，CD8+ T 细胞的选择性接近绝对，并且肝脏脱位异常。此处描述的 CD8-AAV 扩展了免疫学研究和 体内基因治疗选择的策略。