Fibronectin Fragment-Facilitated Retroviral Transfer of the Glutathione-S-Transferase π Gene into CD34+Cells to Protect Them Against Alkylating Agents

Abstract

To protect bone marrow cells from the toxicity of chemotherapy, a multidrug resistant gene or a dihydrofolate reductase gene has been introduced into stem cells. These genes, however, are not capable of conferring refractoriness to alkylating agents (AA), which are some of the most commonly used agents in chemotherapy regimens. In the present study, an attempt was made to endow human stem cell (CD34⁺ cells) with resistance to cyclophosphamide, a well-known AA, and adriamycin (ADM) by transducing the glutathione-S-transferase π (GST-π) gene whose product is thought to detoxify AA by conjugating them with glutathione and to remove a toxic peroxide formed by ADM. The gene transduction was carried out retrovirally with a virus titer of 1 × 10⁵ FFU/ml, employing a recombinant fibronectin fragment; transduction efficiency was extremely low without the fragment. Incubation with interleukin-6 and stem cell factor enhanced the expression of fibronectin ligands VLA4 and VLA5 on CD34⁺ cells. This enhanced expression of VLA4 and VLA5 was considered to facilitate a close contact of the CD34⁺ cell to the retroviral vector via fibronectin fragments and the subsequent transduction process. The GST-π gene-transduced CD34⁺ cells formed almost 3- and 2.5-fold more CFU-GM than neo gene-transduced CD34⁺ cells in the presence of 2.5 μg/ml of 4-hydroperoxycyclophosphamide (4-HC), an active form of cyclophosphamide, and 30 ng/ml ADM, respectively. The transfectants formed an appreciable number of colonies, even at higher concentrations of these drugs (5.0 μg/ml of 4-HC, 50 ng/ml of ADM) whereas neo gene-transduced or nontransduced CD34⁺ cells formed no colonies at all, indicating the possibility of selecting out the transfectants by exposing them to these anticancer drugs. Thus, we were able to demonstrate that transduction of the GST-π gene confers resistance to cyclophosphamide as well as to ADM, and therefore this approach can be applied clinically for high-dose chemotherapy. Human hematopoietic progenitor CD34⁺ cells were capable of being efficiently transduced with the glutathione-S-transferase π (GST-π) gene employing human fibronectin fragment (CH296). The enhanced expression of VLA-4 and 5 was considered to facilitate a close contact of the CD34⁺ cells to the retroviral vector via CH296 and subsequent transduction. The GST-π gene-transduced CD34⁺ cells showed a resistance against 4-hydroperoxicyclophosphamide (4-HC) that is not relevant to the MDR-1 gene products.