Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene

Abstract

Hematopoietic toxicity produced by most anticancer drugs limits their potential for curative therapy. We have shown previously that the human cytidine deaminase ( CD ) gene can confer drug resistance in murine bone marrow cells ( BMCs ) to the nucleoside analog, cytosine arabinoside ( ARA -C ) . In the present study, as the first objective we showed that the CD gene can also render drug resistance in BMCs to related analogs, 2 0 ,2 0 -difluorodeoxycytidine ( dFdC ) and 5 -azadeoxycytidine ( 5 -AZA -CdR ) . As a second objective, we investigated the potential of ex vivo selection with cytosine nucleoside analogs of CD -transduced BMC. The goal of this approach was to enrich the fraction of CD -transduced BMCs so as to increase the transgene expression and level of drug resistance before transplantation. This strategy may have the potential to circumvent the problem in clinical gene therapy of low level of gene transfer and adequate long -term gene expression. Using a bicistronic retroviral vector containing the CD and the green fluorescent protein ( CDiGFP ) , we transduced murine L1210 leukemic cells. All three analogs, ARA -C, dFdC, and 5 -AZA -CdR were demonstrated in vitro to enrich ( > 95% ) the population of leukemic cells expressing the GFP transgene. However, with CD -transduced primary murine BMCs cultivated at high cell density we observed that in vitro selection with ARA -C was not possible due to release of CD into the culture medium at amounts that were sufficient to inactivate the analog. The CD -containing medium produced a chemoprotective effect on mock BMCs as shown by lack of significant growth inhibition in the presence of ARA -C. However, at low cell density in a cell mixture containing CD -transduced cells, the mock BMCs showed marked drug sensitivity to ARA -C as determined by clonogenic assay. Selection with ARA -C was shown to significantly increase the CD enzyme activity in transduced BMC. These results suggest that CD gene has the potential to be a good selectable marker and a possible tool for chemoprotection in cancer gene therapy. Cancer Gene Therapy ( 2001 ) 8, 669 -676

Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells

“…This is in contrast to previous experiments performed with primary mononuclear murine bone marrow cells in which in vitro selection was prevented by the release of large amounts of CDD from CDD-transduced cells. 37 Therefore, another attractive application of CDD gene transfer may be the enrichment of genetically modified cells for gene therapy of monogenetic diseases. Here, selection strategies have been advocated for diseases caused predominantly by malfunction of hematopoietic cells in which the introduced therapeutic transgene does not confer a selective growth advantage to the transduced cells such as chronic granulomatous or Gaucher's disease.…”

Therapeutic Modulation of DNA Damage and Repair Mechanisms in Blood Cells

“…Although self-renewal would be presumably be an integral part of this process, overtime preferential differentiation beyond normal hematopoietic limits may lead to extinction of the transduced stem-cell pool (Pollok, 2003). The expression of chemoprotective gene products such as the multidrug resistance protein (MDR1) (Abonour et al, 2000;Hanania et al, 1995;Hesdorffer et al, 1998;Hildinger et al, 1998;Schiedlmeier et al, 2000), dihydrofolate reductase (DHFR) (Allay et al, 1998;Brenner et al, 1993b;Corey et al, 1990;Persons et al, 2004;Warlick et al, 2002;Williams et al, 1987), cytidine deaminase gene (Beausejour et al, 2001;Eliopoulos et al, 2002;Momparler et al, 1996;Rattmann et al, 2006) and MGMT (Cai www.intechopen.com et al, 2008;Cai et al, 2006;Cai et al, 2005;Chinnasamy et al, 1998b;Davis et al, 2000;Davis et al, 1999;Hickson et al, 1998;Jansen et al, 2001;Jansen et al, 2002;Moritz et al, 1995;Neff et al, 2003;Ragg et al, 2000;Sawai et al, 2001) have been thoroughly investigated as potential candidates to protect hematopoietic cells residing in the bone marrow. A number of retroviral vectors derived from gamma-retroviruses, lentiviruses, and foamy viruses as described above continue to be tested for delivery of drug-resistance genes to primitive hematopoietic cells.…”

Development of gene therapy in association with clinically used cytotoxic deoxynucleoside analogues

“…This result was confirmed by others, both on fibroblasts and mouse bone marrow with araC, gemcitabine and 5-aza-deoxycytidine. [68][69][70][71][72] It was also shown that the resistance was reversed by using THU. 70 In the next step, transduced hematopoietic cells were transplanted into the bone marrow of mice to show the production of CDA in blood and different tissues.…”

“…In vitro selection of transduced cells to select cells with high CDA expression before transplantation was easily obtained with murine leukemic L1210 cells and, although less easily, in murine bone marrow cells. 72 The selection was further applied to human peripheral and cord blood progenitor cells with araC and gemcitabine. 74 Finally, in vivo myeloprotection has been observed in mice treated with araC or gemcitabine after transplantation of CDAtransduced murine bone marrow cells.…”