In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. We observed that treatment with pristimerin, at as low as 1 μmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg·d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.
Bibliographical noteFunding Information:
We thank Dr. Xiao Xiao for providing pdsAAV-CB-EGFP plasmid. This work was supported in part by the Alex’s Lemonade Foundation, and Bankhead-Coley Cancer Research Program, Florida Department of Health (to CL), Public Health Service grants R01 HL-097088 and R21 EB-015684 from the National Institutes of Health, and an institutional grant from the Children’s Miracle Network (to AS, CL and GA). LW and YW were supported in part by the state-sponsored program for Graduate Students from China Scholarship Council, Government of China, and the National Natural Science Foundation of China (No. 30730114), respectively. All authors helped design the study and analyzed the data. LW, YW, YL, ZY and YZ performed the experiments. GA performed experiments regarding tyrosine-mutant rAAV vectors. AS and CL wrote the manuscript.