Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations

Ex vivo CRISPR gene editing in hematopoietic stem and progenitor cells has opened new treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a novel loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. Engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to non-treated cells, with no differences in differentiation. This is the first demonstration of nontoxic delivery of the entire CRISPR payload into primary human blood progenitors

体外CRISPR基因编辑（CRISPR gene editing）应用于造血干祖细胞（hematopoietic stem and progenitor cells），已为诸多疾病开辟了全新的治疗手段。现有流程多采用电穿孔（electroporation）技术，部分场景后续还会辅以病毒转导（virus transduction）。尽管这类复杂操作可在部分基因位点实现高水平的基因编辑，但同时也会引发细胞毒性（cellular toxicity）。本团队开发了一种基于胶体金纳米颗粒（colloidal gold nanoparticles）的CRISPR纳米制剂（CRISPR nanoformulation），其搭载设计新颖，无需依赖电穿孔或病毒即可完成细胞摄取。该高度单分散的纳米制剂可规避溶酶体捕获（lysosomal entrapment），且能在不产生细胞毒性的前提下，靶向定位于原代人血液祖细胞（primary human blood progenitors）的细胞核内。针对多种具有治疗价值的基因位点，该纳米制剂介导的基因编辑可搭配不同CRISPR核酸酶（CRISPR nucleases），实现高效且持久的编辑效果。在人源化小鼠模型中，经纳米制剂处理的原代细胞的植入动力学（engraftment kinetics）优于未处理的细胞，且细胞分化过程无明显异常。本研究首次实现了将完整CRISPR载荷（CRISPR payload）无毒性递送至原代人血液祖细胞的技术验证。