Muhammed Burak Demircan, Luca J Zinser, Alexander Michels, Mar Guaza-Lasheras, Fabian John, Johanna M Gorol, Samuel A Theuerkauf, Dorothee M Günther, Dirk Grimm, Florian R Greten, Petr Chlanda, Frederic B Thalheimer, Christian J Buchholz
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引用次数: 0
摘要
体内基因治疗面临的最大挑战之一是如何将高选择性基因转移到确定的治疗相关细胞群中。在这里,我们展示了DARPin靶向AAVs(DART-AAVs),它显示了特异于人类和小鼠CD8的DARPin。将 DARPins 插入 AAV2 和 AAV6 的囊膜蛋白 1 (VP1) 的 GH2/GH3 环后,CD8 阳性 T 细胞具有高度选择性,基因递送活性不受影响。值得注意的是,AAV2 和 AAV6 的噬菌体核心结构没有改变,而且还能检测到突出的 DARPins。在复杂的原代细胞混合物(包括供体血液或小鼠全身注射)中,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。在体内,向条件化人源化小鼠或免疫功能健全的小鼠注射一次载体后,高达 80% 的活化 CD8+ T 细胞被击中。虽然基因转移率在非活化条件下明显下降,但将 Cre 运送到指示性小鼠体内后,仍可检测到 CD8+ T 细胞中选择性的基因组修饰。在这两种小鼠模型中,CD8+ T 细胞的选择性接近绝对值,而且能从肝脏中脱靶。本文描述的 CD8-AAVs 扩展了免疫学研究和体内基因治疗的策略。
T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.
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.
期刊介绍:
Molecular Therapy is the leading journal for research in gene transfer, vector development, stem cell manipulation, and therapeutic interventions. It covers a broad spectrum of topics including genetic and acquired disease correction, vaccine development, pre-clinical validation, safety/efficacy studies, and clinical trials. With a focus on advancing genetics, medicine, and biotechnology, Molecular Therapy publishes peer-reviewed research, reviews, and commentaries to showcase the latest advancements in the field. With an impressive impact factor of 12.4 in 2022, it continues to attract top-tier contributions.