Mechanism for enhanced transduction of hematopoietic cells by recombinant adeno‐associated virus serotype 6 vectors

Abstract

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.

Reactive oxygen species enhance rAAV transduction by promoting its escape from late endosomes

“…1 B, D, G, I). Similar to the previous report [ 1 ], shikonin pretreatment increased sc-rAAV2-GFP transduction in 293 T and Hela cells (Fig. 1 C, E, H, J).…”

“…Recently, several compounds extracted from Traditional Chinese medicine (TCM) have been reported to promote rAAV transduction both in vivo and in vitro, including shikonin, pristimerin, celastrol, and arsenic [ 1 , 16 , 24 ]. Shikonin was found to improve sc-rAAV6 transduction of hematopoietic cells through enhanced ROS formation [ 1 ]. This study focused on exploring whether the enhancement is maintained both for ss-rAAV and sc-rAAV, as well as in different types of cells.…”

“…Recombinant adeno-associated virus (rAAV) vectors have revolutionized gene therapy due to their wide tissue tropism, non-pathogenicity, and long-term transgene expression [ 1 , 2 ]. They have shown great clinical success in several diseases such as Leber’s hereditary optic neuropathy, spinal muscular atrophy type 1, lipoprotein lipase deficiency, and hemophilia A and B [ 3 ].…”

“…Meanwhile, the efficiency of rAAV nuclear entrance was significantly elevated after H 2 O 2 treatment [ 5 ], suggesting that its intracellular trafficking is also enhanced. It was reported that shikonin, a potential ROS generator, has significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, prompting us to investigate how ROS influence the cellular fate of rAAV [ 1 ].…”

Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo

“…Western blot assays were performed as previously described. 35 Briefly, cells were harvested after the indicated treatment, rinsed by PBS, and disrupted in lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China). The total protein was determined and normalized by BCA Protein Assay Kit (P0012, Beyotime Biotechnology, Shanghai, China).…”