scAAV8-GFP

Recent studies have shown that endosomal reactive oxygen species (ROS) promote rather than inhibit viral infection. Some ROS generators, including shikonin and H₂O₂, have the potential to enhance recombinant adeno-associated virus (rAAV) transduction. To understand the effects of ROS on rAAV vector transduction, especially the rAAV subcellular distribution profile, researchers systematically explored the effects of ROS on each step of the rAAV intracellular trafficking pathway using fluorescently labeled rAAV and qPCR quantification assays. The results showed that ROS exposure promoted rAAV transduction in vivo and in vitro regardless of vector serotype or cell type. ROS treatment directed rAAV intracellular trafficking to a more efficient pathway by upregulating the expression of proteinases B and L, accelerating rAAV trafficking in late endosomes, and increasing rAAV entry into the nucleus. These data support the potential of ROS generative drugs, such as shikonin, to promote rAAV vector transduction by promoting the escape of rAAV from late endosomes and enhancing its productive trafficking to the nucleus.

Considering that the liver is an ideal target for rAAV gene therapy and that rAAV8 vectors are reported to have hepatic tropism after intravenous injection, the researchers hypothesized that shikonin could enhance rAAV8 transduction of hepatocytes. As expected, transduction of LO-2 cells by ssAAV8-GFP and scAAV8-GFP was significantly enhanced compared with controls (Figure 1B–E), and the shikonin concentration used had little effect on cell viability (Figure 1A). These results confirmed the ability of shikonin to increase rAAV transduction, with no AAV serotype and cell type dependence found.

Figure 1. Shikonin pretreatment increased ssAAV8-GFP or scAAV8-GFP transduction in LO-2 cells. (Huang X, et al., 2023)