Application of a human multidrug transporter (ABCG2) as selectable marker in gene therapy (Hungarian text).

摘要

Stem cell-based gene therapy is often unsuccessful because of the relatively low number of genetically modified cells with repopulating capabilities. To provide a selective advantage to the modified cells we applied the human ABCG2 protein, a resident xenobiotic transporter in stem cells, as a selectable marker. This protein is active as a homodimer, and its relatively small cDNA is an advantage in gene therapy applications.; In the present study the gene therapy application of a mutant form of ABCG2 (R482G) was investigated.; ABCG2 variants were expressed in haematopoietic stem cells alone or co-expressed with the therapeutic gene (gp91phox) of X-linked Chronic Granulomatous Disease (X-CGD) by an efficient retroviral transduction system. Transgene expression was determined by Western blotting, immunohistochemistry and flow cytometry analysis. To estimate the multidrug resistance phenotype, functional assays of ABCG2 were performed. The differentiation of the transduced cells was followed by in vitro clonogen and in vivo mouse transplantation experiments.; High proportion of transgene positive cells could be detected in the ABCG2 transduced cells, where the mutant ABCG2 protein selectively protected the cells against clinically applicable cytostatic drugs as mitoxantrone (MX) and doxorubicin. Expression of the gp91phox protein in human gp91phox knock out hematopoietic progenitor cells corrected the mutation responsible for X-CGD after MX selection. Overexpression of ABCG2 did not affect hematopoietic cell maturation both in vitro and in vivo.; We suggest that the mutant ABCG2 protein is an ideal candidate for human stem cell protection and for use as a selectable marker in gene therapy.