Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Paul F. Glenn Center for the Biology of Aging, Harvard Medical School, Boston, MA 02115, USA.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Cell Rep. 2019 Apr 23;27(4):1254-1264.e7. doi: 10.1016/j.celrep.2019.03.105.
In vivo delivery of genome-modifying enzymes holds significant promise for therapeutic applications and functional genetic screening. Delivery to endogenous tissue stem cells, which provide an enduring source of cell replacement during homeostasis and regeneration, is of particular interest. Here, we use a sensitive Cre/lox fluorescent reporter system to test the efficiency of genome modification following in vivo transduction by adeno-associated viruses (AAVs) in tissue stem and progenitor cells. We combine immunophenotypic analyses with in vitro and in vivo assays of stem cell function to reveal effective targeting of skeletal muscle satellite cells, mesenchymal progenitors, hematopoietic stem cells, and dermal cell subsets using multiple AAV serotypes. Genome modification rates achieved through this system reached >60%, and modified cells retained key functional properties. This study establishes a powerful platform to genetically alter tissue progenitors within their physiological niche while preserving their native stem cell properties and regulatory interactions.
体内递送基因修饰酶在治疗应用和功能基因筛选方面具有重要的应用前景。将其递送到内源性组织干细胞中尤其具有吸引力，这些干细胞在体内平衡和再生过程中提供了持久的细胞替代来源。在这里，我们使用灵敏的 Cre/lox 荧光报告系统来测试腺相关病毒（AAV）在体内转导后对组织干细胞和祖细胞进行基因组修饰的效率。我们将免疫表型分析与体外和体内干细胞功能测定相结合，使用多种 AAV 血清型有效靶向骨骼肌卫星细胞、间充质祖细胞、造血干细胞和皮肤细胞亚群。通过该系统实现的基因修饰率超过 60%，并且修饰后的细胞保留了关键的功能特性。这项研究建立了一个强大的平台，可以在保留其天然干细胞特性和调控相互作用的情况下，在生理龛位内遗传改变组织祖细胞。
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