Hematopoietic gene delivery, such as hematopoietic stem/progenitor cells (HSPCs), is a promising treatment for both inherited and acquired diseases, such as hemophilia. Recently, a combined strategy to achieve more than 90% transduction efficiency was documented using recombinant adeno‐associated virus serotype 6 (rAAV6) vectors. However, the mechanisms of enhanced vector transduction efficiency in hematopoietic cells are largely unknown. In this manuscript, we first reported that proteasome inhibitors, which are well‐known to facilitate rAAV intracellular trafficking in various cell types, are not effective in hematopoietic cells. From the screening of small molecules derived from traditional Chinese medicine, we demonstrated that shikonin, a potential reactive oxygen species (ROS) generator, significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, including human cord blood‐derived CD34 + HSPCs. Shikonin mainly targeted vector intracellular trafficking, instead of host cell entry or endonuclear single to double strand vector DNA transition, in a vector serotype‐dependent manner. Moreover, a ROS scavenger completely prevented the capability of shikonin to enhance rAAV6 vector‐mediated transgene expression. Taken together, these studies expand our understanding of rAAV6‐mediated transduction in hematopoietic cells and are informative for improving rAAV6‐based treatment of blood diseases.

中文翻译：

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重组腺相关病毒6型载体增强造血细胞转导的机制

造血基因传递，如造血干/祖细胞 (HSPC)，是治疗遗传性和获得性疾病（如血友病）的一种有前途的治疗方法。最近，使用重组腺相关病毒血清型 6 (rAAV6) 载体实现了 90% 以上的转导效率的联合策略被记录在案。然而，在造血细胞中增强载体转导效率的机制在很大程度上是未知的。在这份手稿中，我们首先报道了众所周知的蛋白酶体抑制剂可促进 rAAV 在各种细胞类型中的细胞内运输，但对造血细胞无效。通过筛选来自中药的小分子，我们证明了紫草素，一种潜在的活性氧 (ROS) 发生器，显着增加了造血细胞中由 rAAV6 载体介导的体外和离体转基因表达，包括人脐带血衍生的 CD34 + HSPC。Shikonin 主要以载体血清型依赖性方式靶向载体细胞内运输，而不是宿主细胞进入或核内单链到双链载体 DNA 转换。此外，ROS 清除剂完全阻止了紫草素增强 rAAV6 载体介导的转基因表达的能力。总之，这些研究扩展了我们对 rAAV6 介导的造血细胞转导的理解，并为改善基于 rAAV6 的血液疾病治疗提供了信息。而不是宿主细胞进入或核内单链载体 DNA 转换，以载体血清型依赖的方式。此外，ROS 清除剂完全阻止了紫草素增强 rAAV6 载体介导的转基因表达的能力。总之，这些研究扩展了我们对 rAAV6 介导的造血细胞转导的理解，并为改善基于 rAAV6 的血液疾病治疗提供了信息。而不是宿主细胞进入或核内单链载体 DNA 转换，以载体血清型依赖的方式。此外，ROS 清除剂完全阻止了紫草素增强 rAAV6 载体介导的转基因表达的能力。总之，这些研究扩展了我们对 rAAV6 介导的造血细胞转导的理解，并为改善基于 rAAV6 的血液疾病治疗提供了信息。