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Objective: Retroviral vectors represent one of the most robust technologies for in vivo expression of heterologous gene sequences and are still the most commonly used vectors in clinical gene therapy trials. The production of high titer retroviral preparations, however, can be a problematic procedure for certain constructs. Methods: GALV- or RD114-pseudotyped retroviral particles carrying selectable fluorescence markers or drug resistance genes, such as the green fluorescent protein (GFP) or the O6-methylguanine-DNA-methyltransferase (MGMT) mutants, were used to stably transduce Phoenix-(FNX-)eco cells. Thereafter, a polyclonal population of producer cells was generated by enriching cells with high marker gene expression. In addition, single producer clones were selected by limiting dilution. Results: Retroviral titers were increased 1–2 logs by enriching for a polyclonal population of producer cells, and selection of single producer clones allowed another 1- to 2-log increase in titers. Using this method, reproducibly high titer viral preparations allowing efficient transduction of hematopoietic stem cells were generated. Conclusion: A reliable and time-effective method to generate stable high titer producer cells based on the FNX-cell line for problematic retroviral vector constructs is described. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The overexpression of mutant forms of O6-methylguanine-DNA-methyltransferase (MGMT), resistant to the MGMT inhibitor O6-benzylguanine (BG), protects hematopoietic cells from the toxicity of combined BG plus O6-alkylating agent chemotherapy. To evaluate the feasibility of this approach for clinically relevant O6-alkylating agents, combined therapy with BG and two chloroethylnitrosourea-type drugs, ACNU or BCNU, or the triazene derivative temozolomide (TMZ) was investigated in a murine bone marrow transplant model allowing transgenic expression of the highly BG-resistant MGMTP140K mutant. Whereas 20/20 control animals transplanted with nontransduced cells died of progressive myelosuppression during therapy, nearly all animals transplanted with MGMTP140K-transduced cells survived treatment with BG/ACNU (12/15), BG/TMZ (10/10), or BG/BCNU (5/5). In surviving animals, hematological parameters improved during chemotherapy and pretreatment levels were reestablished during or shortly after therapy. All animals showed enrichment of transgenic granulocytes (range: 15- to 101-fold) and lymphocytes (range: 16- to 55-fold) in peripheral blood, bone marrow, and spleen. No significant differences were observed between individual treatment groups. Serial transplants demonstrated protection in secondary recipients and confirmed the transduction of transplantable stem cells. Thus, these data demonstrate efficient protection from hematotoxicity and substantial enrichment of transgenic cells following MGMTP140K gene transfer and treatment with different O6-alkylating drugs. Cancer Gene Therapy (2002) 9, 737–746 doi:10.1038/sj.cgt.7700490 [ABSTRACT FROM AUTHOR]

Abstract: Overexpression of O6-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O6-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O6-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8–12 µg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer. [ABSTRACT FROM AUTHOR]

Gene transfer of drug resistance (CTX-R) genes can be used to protect the hematopoietic system from the toxicity of anticancer chemotherapy and this concept recently has been proven by overexpression of a mutant O6-methylguaninemethyltransferase in the hematopoietic system of glioblastoma patients treated with temozolomide. Given its protection capacity against such relevant drugs as cytosine arabinoside (ara-C), gemcitabine, decitabine, or azacytidine and the highly hematopoiesis-specific toxicity profile of several of these agents, cytidine deaminase (CDD) represents another interesting candidate CTX-R gene and our group recently has established the myeloprotective capacity of CDD gene transfer in a number of murine transplant studies. Clinically, CDD overexpression appears particularly suited to optimize treatment strategies for acute leukemias and myelodysplasias given the efficacy of ara-C (and to a lesser degree decitabine and azacytidine) in these disease entities. This article will review the current state of the art with regard to CDD gene transfer and point out potential scenarios for a clinical application of this strategy. In addition, risks and potential side effects associated with this approach as well as strategies to overcome these problems will be highlighted. [ABSTRACT FROM AUTHOR]

: ObjectiveWhile retrovirally mediated gene transfer of dihydrofolate reductase mutants (mutDHFR) has convincingly been demonstrated to confer methotrexate (MTX) resistance to murine hematopoietic cells, clinical application of this technology will require high efficacy in human cells. Therefore, we investigated retroviral constructs expressing various point mutants of human DHFR for their ability to confer MTX resistance to human clonogenic progenitor cells (CFU-C) and to allow for in vitro selection of transduced CFU-C.: MethodsPrimary human hematopoietic cells were retrovirally transduced using MMLV- and SFFV/MESV-based vectors expressing DHFRSer31, DHFRPhe22/Ser31, or DHFRTyr22/Gly31. MTX resistance of unselected and in vitro–selected CFU-C was determined using MTX-supplemented methylcellulose cultures and gene transfer efficiency was assesed by single–colony PCR analysis.: ResultsWhile less than 1% mock-transduced CFU-C survived the presence of ≥5×10−8 M MTX, MMLV- and SFFV/MESV-based vectors expressing DHFRSer31 significantly protected CFU-C from MTX at doses ranging from 2.5 to 30×10−8 M. Vectors expressing DHFRPhe22/Ser31 or DHFRTyr22/Gly31 were even more protective and MTX-resistant CFU-C were observed up to 1×10−5 M MTX. Three-day suspension cultures in the presence of 10–20×10−8 M MTX resulted in significant selection of mutDHFR-transduced CFU-C. The percentage of CFU-C resistant to 10×10−8 M MTX increased fourfold to 20-fold and provirus-containing CFU-C increased from 27% to 79–100%.: ConclusionGene transfer of DHFR using suitable retroviral backbones and DHFR mutants significantly increases MTX resistance of human CFU-C and allows efficient in vitro selection of transduced cells using a short-term selection procedure. [Copyright &y& Elsevier]