The DNA minor groove binding agents Hoechst 33258 and 33342 enhance recombinant adeno‐associated virus (rAAV) transgene expression

Abstract

Recombinant adeno-associated viruses (rAAV) are commonly used in pre-clinical and clinical gene transfer studies. However, the relatively slow kinetics of rAAV transgene expression complicates in vitro and in vivo experiments. 293 and COS-1 cells were transduced with rAAV2-EGFP, rAAV1-EGFP, or rAAV5-EGFP. The rAAV-EGFP expression was analyzed in the presence of Hoechst 33 258 or 33 342 as a function of time and concentration by flow cytometry and fluorescent microscope. Effects of Hoechst on cell cycle populations were determined by flow cytometry. Enhanced green fluorescent protein (EGFP) expression plasmids with or without AAV inverted terminal repeats (ITR) were constructed and gene expression by transient transfection was compared in the presence of Hoechst. We found that Hoechst 33 258 and 33 342 increase both the level and the population of EGFP gene expressing cells, transduced by several different serotypes of rAAV-EGFP. The augmentation of rAAV-EGFP expression occurs in different cell types in a concentration-dependent manner. In addition, the Hoechst 33 258 or 33 342 mediated enhancement of rAAV gene expression correlated with an increase of cells in S phase and G2/M phases of the cell cycle. Finally, gene expression from transfected ITR-containing plasmid DNA was also enhanced by Hoechst dyes. Our results revealed that two different, although related, DNA-binding drugs, Hoechst 33 258 and 33 342, accelerate the kinetics of rAAV transgene expression. These findings may provide the basis for more sensitive assessment of rAAV biological activity and also extend the applications of rAAV for in vivo gene transfer.