Retroviral transfer of human MDR1 gene to hematopoietic cells: Effects of drug selection and of transcript splicing on expression of encoded P-glycoprotein

Protection of hematopoietic cells of patients undergoing anticancer chemoth erapy by MDR1 gene transfer is currently being studied in clinical trials. From animal studies, it has been suggested that aberrant splicing. due to c ryptic donor and acceptor sites in the MDR1 cDNA could be a major reason fo r failure to obtain high-level expression of P-glycoprotein in bone marrow. We investigated effects of drug selection on protein expression levels and on splicing of MDR1 transcripts in murine bone marrow cells (BMCs) in vitr o. To this end, retroviruses were generated through an identical plasmid, p HaMDR1/A, introduced into different packaging cells. GP + E86- but not PA31 7-derived producer cells were found to express truncated in addition to ful l-length message. In BMCs transduced with GP + E86-derived viruses, both me ssages were increased after treatment with colchicine or daunomycin. Simila r results were obtained with NIH 3T3 fibroblasts. However, transduced and d rug-selected BMCs displayed the spliced transcript even if the respective P A317-derived producer cells contained no truncated RNA as detected in trans duced NIH 3T3 fibroblasts, Short-term drug selection in BMCs transduced wit h either ecotropic or amphotropic retroviruses resulted in a striking incre ase in P-glycoprotein expression. Thus, aberrant splicing failed to abrogat e P-glycoprotein expression in BMCs, We also studied a vector in which MDR1 was coexpressed with glucocerebrosidase, using an internal ribosomal entry site. Although chemoprotection was less efficient than with pHaMDR1/A, aug mentation of protein expression was observed at low selecting drug concentr ations. Our study shows that drug selection can partially compensate for in efficient transduction of hematopoietic cells, and may help to develop stra tegies by which unstable expression of transduced genes can be overcome.