MDR1 Gene Transfer Research Articles

Optimization of multidrug resistance 1 gene transfer confers chemoprotection to human hematopoietic cells engrafted in immunodeficient mice

To investigate whether an optimization of MDR1 gene transfer protocol would result in stable hematopoietic stem cell (HSC) engraftment and myeloprotection in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice after paclitaxel chemotherapy. We transplanted freshly isolated CD34+ cells or MDR1-transduced CD34+ cells derived from human umbilical cord blood (UCB) into sublethally irradiated NOD/SCID mice. Twenty-eight days after transplantation, mice received paclitaxel chemotherapy and peripheral blood (PB) was collected for analysis of WBC, RBC and PLT counts once every week. We found that MDR1-transduced human hematopoietic cells could facilitate hematopoietic recovery and completely reconstitute hematopoiesis in mice as well as freshly isolated CD34+ cells. Mice transplanted with MDR1-transduced human hematopoietic cells were protected from paclitaxel chemotherapy with higher survival rate and higher level of WBC counts and RBC counts compared with mice transplanted with untransduced HSCs. We also demonstrated that hematopoietic cells transduced with MDR1 gene were enriched in vivo after paclitaxel chemotherapy determined by the higher percentage of human Rh-123(dull) CD45+ cells in bone marrow of mice. Our results demonstrated successful chemoprotection against myelosuppression in mice by MDR1-transduced repopulating human hematopoietic cells with an optimized transduction protocol.

Gene therapy for breast cancer. — review of clinical gene therapy trials for breast cancer and mdr1 gene therapy trial in cancer institute hospital

Gene therapy for advanced breast cancer is anticipated to be a useful therapeutic approach. Strategies in ongoing clinical protocols can be divided into four groups: (1) suppression of oncogenes or transfer of tumor-suppressor genes; (2) enhancement of immunological response; (3) transfer of suicide genes; (4) protection of bone marrow using drug resistance genes. We have started a clinical study of multidrug resistance (MDR1) gene therapy. Advanced breast cancer patients received high dose chemotherapy and autologous peripheral blood stem cell transplantation(PBSCT)with MDR1-transduced hematopoietic cells, and then were treated with docetaxel. Two patients have been treated so far, and in vivo enrichment of MDR1-transduced cells with docetaxel treatment has been seen. Both patients are in complete remission and had no apparent adverse effects from the MDR1 gene transfer.

Quantitative Real Time PCR Analysis of MDR1 Transduced Rhesus Macaque Hematopoietic Stem Cells Shows No Indication for Proliferative Advantage after Long-Term Follow-Up.

Introduction: It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral MDR1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analyses on the clonal level and a long-term follow-up in a model with more direct relevance to human HSC biology were lacking.Methods: In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were transduced either with a MDR1 gene-retroviral vector (HaMDR1-vector) or a NeoR-retroviral vector (G1Na-vector). After autologous transplantation granulocytes and mononuclear cells of each animal were obtained at different time points and analysed by using a highly sensitive and specific ligation-mediated PCR (LM-PCR) and by quantitative real time PCR.Results: In monkey M120 73 different cell populations (clones) were detected between 8 weeks and 4 years after transplantation; in monkey M038 49 clones could be identified between 16 weeks and 4 years after transplantation. Remarkably, 99 clones descend from the G1Na-vector, whereas only 23 clones descend from the HaMDR1-vector. Quantitative real time PCR analyses support these data: in both animals, the mean proportion of the G1Na-vector and the HaMDR1-vector, respectively, is 2,46%±0,014% and 0,455%±0,003%. Furthermore, single G1Na clones identified with LM-PCR 4 years after transplantation were quantified, showing a copy number between 1.43±0,52 and 7.1±0.34, according to 0.001%±0.0003% and 0.004%±0.0003% of the granulocytes fraction. In the MNC fraction, the G1Na clones were not detectable. At the same time point, 2 HaMDR1 clones were quantified, one in the granulocyte and one in the MNC fraction, respectively. Here, the copy numbers were 2.87±0.56 and 3.52±1.11, according to 0.002%±0.0003% and 0.002%±0.0008% of the granulocyte and MNC fraction, respectively.Conclusions: We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

New Aspects on the Safety of Multidrug-Resistance 1 Gene Transfers: No Indication for Clonal Dominance after Long-Term Follow-Up in a Primate Transplantation Model.

It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral multidrug-resistance 1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analysis on the clonal level were lacking.One method to study the in vivo behaviour of repopulating progenitor and stem cells is marking the cells with replication-incompetent retroviral vectors that integrate into identifiable host DNA sequences, thus allowing the tracking of cell progeny based on unique proviral insertion sites.In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were split into two aliquots and transduced either with a multidrug-resistance 1 gene-retroviral vector based on the Harvey murine sarcoma virus (HaMDR1-vector) or a NeoR-retroviral vector based on the Moloney murine leukaemia virus (G1Na-vector). After autologous retransplantation, DNA from blood and bone marrow was collected at different time points in a period of 4 years and the animals are still alive.By using a highly sensitive and specific ligation-mediated polymerase chain reaction (LM-PCR) followed by sequencing of vector integration sites, we found in animal M120 32 different contributing hematopoietic clones 8 weeks and 50 weeks after transplantation and in animal M038 17 clones 58 weeks after transplantation. Based on the difference between the sequences of the HaMDR1-LTR and the G1Na-LTR, the clones can be allocated definitely to one of the two vectors. Remarkably, 36 clones descend from the G1Na-vector, whereas only 13 clones descend from the HaMDR1-vector.We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells.

We describe the functional analysis of a novel retroviral vector, SF91m3, which was designed for improved expression of the in vivo selectable marker, multidrug resistance 1 gene (MDR1), in hematopoietic cells. SF91m3 combines several promising features. The vector backbone lacks viral coding sequences and AUG-start codons 5' of the MDR1 cDNA. A point mutation of a cryptic splice acceptor of the MDR1 cDNA increases the probability of transferring an intact provirus. The titer of a PG13 packaging cell clone containing a single proviral integration is high (>2 x 10(6) particles/ml from frozen stocks of serum-free vector harvests). Human hematopoietic cells transduced with SF91m3 reliably express MDR1 before and after passage through NOD/SCID mice, as shown by quantitative PCR and efflux assays with rhodamine 123 or Hoechst 33342. Finally, SF91m3 mediates resistance to escalated doses of cytotoxic agents, as shown by survival and differentiation of transduced colony-forming cells in the presence of colchicine at 48 ng/ml (>10 x IC(50)). Thus, SF91m3 may represent an interesting candidate for future trials addressing the safety and utility of MDR1 gene transfer; moreover, this study demonstrates that sequence alterations improving post-transcriptional processing of retroviral vectors have a substantial impact for gene expression in hematopoietic cells.

The effects of dose, route of administration, drug scheduling and MDR-1 gene transfer on the genotoxicity of etoposide in bone marrow.

We have used the bone marrow micronucleus assay (BMMN) as a measure of clastogenicity, in response to etoposide exposure in murine bone marrow. Oral delivery of etoposide resulted in a reduced number of micronucleated polychromatic erythrocytes (MPE) relative to the same dose delivered intraperitoneally (P < 0.001). Daily fractionation of the oral schedule of etoposide led to a more than six-fold increase in cumulative MPE frequency over that observed with the same total, unfractionated dose, with the potency of the response increasing with serial exposure (r = 0.79). Retrovirally-mediated expression of MDR1 in murine bone marrow resulted in partial protection against the clastogenic activity of etoposide relative to mock transduced control mice. The model system developed has indicated a variety of factors able to influence the genotoxicity of etoposide. It should now be possible to further exploit this model in order to define other factors governing haemopoietic sensitivity to etoposide.

Chemoprotection of hematopoietic cells by a mutant P-glycoprotein resistant to a potent chemosensitizer of multidrug-resistant cancers.

Cancers are frequently chemoresistant because of overexpression of P-glycoprotein. Two different approaches to improve cancer treatment are currently being investigated in clinical trials: inhibition of P-glycoprotein function by reversing agents, and alleviation of leukocytopenia by MDR1 gene transfer to normal bone marrow of patients. We report here that retroviral vectors encoding a mutant P-glycoprotein (MDR1-F983A) protect hematopoietic cells from anticancer drugs even in the presence of trans-(E)-flupentixol, an inhibitor of P-glycoprotein. Transfer of either mutant or wild-type MDR1 to K562 erythroleukemia cells or primary murine bone marrow resulted in reduced accumulation of daunomycin and vinblastine because of increased drug efflux.trans-(E)-Flupentixol at concentrations up to 10 microM failed to reverse drug efflux mediated by the product of the mutant MDR1 while wild-type P-glycoprotein was inhibited. In the presence of 2 microM trans-(E)-flupentixol chemoresistance to daunomycin was circumvented only in K562 cells transduced with wild-type, but not with mutant, MDR1. Moreover, drug resistance of KB-8-5 epidermoid cancer cells, which express the wild-type MDR1 gene at levels comparable to clinical specimens from multidrug-resistant cancers, was fully overcome in the presence of trans-(E)-flupentixol. Vectors expressing mutant P-glycoprotein may help improve chemotherapy by allowing safe dose intensification under conditions in which multidrug-resistant cancers are rendered drug sensitive by reversing agents.

Effects of several cytokine combinations on retrovirus-mediated human MDR1 gene transfer into bone marrow hematopoietic cells.

Annals of the New York Academy of SciencesVolume 872, Issue 1 p. 391-394 Effects of Several Cytokine Combinations on Retrovirus-Mediated Human MDR1 Gene Transfer into Bone Marrow Hematopoietic Cells S. ZHOU, Corresponding Author S. ZHOU Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, China Correspondence author: S. Zhou, Department of Cell Biology, Institute of Basic Medical Sciences, 27 Taiping Road, Beijing 100850, P. R. China. Phone, (010)-66931320; fax, (010)-68213039; e-mail, s.zhou@cenpok.netSearch for more papers by this authorY. TONG, Y. TONG Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, ChinaSearch for more papers by this authorP. H. TANG, P. H. TANG Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, ChinaSearch for more papers by this authorN. MAO, N. MAO Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, ChinaSearch for more papers by this author S. ZHOU, Corresponding Author S. ZHOU Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, China Correspondence author: S. Zhou, Department of Cell Biology, Institute of Basic Medical Sciences, 27 Taiping Road, Beijing 100850, P. R. China. Phone, (010)-66931320; fax, (010)-68213039; e-mail, s.zhou@cenpok.netSearch for more papers by this authorY. TONG, Y. TONG Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, ChinaSearch for more papers by this authorP. H. TANG, P. H. TANG Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, ChinaSearch for more papers by this authorN. MAO, N. MAO Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, ChinaSearch for more papers by this author First published: 06 February 2006 https://doi.org/10.1111/j.1749-6632.1999.tb08484.xCitations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume872, Issue1HEMATOPOIETIC STEM CELLS BIOLOGY AND TRANSPLANTATIONApril 1999Pages 391-394 RelatedInformation

Retrovirus-mediated gene transfer of the multidrug resistance-associated protein (MRP) cDNA protects cells from chemotherapeutic agents.

Transduction of hematopoietic progenitors with a multidrug resistance gene like mdr-1 or mrp aims to protect bone marrow from toxicity of chemotherapeutic agents. The interest in the use of mrp as an alternative to mdr-1 gene transfer for bone marrow protection lies in its different modulation. Indeed, classical P-gp reversal agents, tested in the clinic to decrease mdr-1 tumor resistance, have little or no effect on MRP function. This would allow, in the same patient, the use of reversal agents to decrease P-gp tumor resistance without reversing bone marrow protection of the transduced hematopoietic cells provided by multidrug resistance-associated protein (MRP). As a first step, we have constructed and tested two different mrp-containing vectors with either the Harvey retroviral long terminal repeat (LTR) or PGK as promoters and generated ecotropic producer cells. We have shown by Southern blot analysis that retroviral supernatant from these producer cells can efficiently transmit the mrp gene to target cells. Mrp expression could be detected by fluorescence-activated cell sorting (FACS) analysis in the producer cells. The transduced cells have increased resistance to doxorubicin, vincristine, and etoposide. Furthermore, chemoprotection of the transduced cells was increased after selection with chemotherapeutic agents in the presence of glutathione, a co-factor for MRP function. These data indicate that mrp retroviral vectors may be useful for chemoprotection and selection.

Clinical Gene Therapy Using the Human MDR1 Gene: At the Threshold

Many human tumors such as bladder carcinoma that are initially responsive to chemotherapy eventually fail to respond to treatment. For most drugs, the dose escalation that may be required for a cure cannot be achieved because sensitive tissues such as bone marrow limit cytotoxic therapy. Approaches to prevent or circumvent myelosuppression are therefore a high priority of research on dose intensification protocols. One such strategy is to protect bone marrow cells by virtue of drug resistance gene expression such as the MDR1 gene encoding for P-glycoprotein. In a first set of experiments, bone marrow cells derived from transgenic mice that constitutively express MDR1 were transplanted to lethally irradiated mouse-recipients. In a second set of experiments, a construct consisting of a murine LTR promoter, the delta mouse molony promoter, and the human MDR1 gene was transfected into CD34-positive bone marrow stem cells from rhesus monkeys. The mouse studies demonstrated effective and long-lasting protection against chemotherapy-induced myelosuppression. Furthermore, high and stable expression of the human MDR1 gene was observed in recipient monkey bone marrow cells. On the basis of this preclinical analysis, clinical protocols to test myeloprotection afforded by MDR1 gene transfer so as to intensify chemotherapy in otherwise incurable human cancer were submitted for institutional and state approval. The protocol “phase I study of autologous reinfusion of haematopoietic precursor cells genetically modified by retroviral gene transfer of the multidrug resistance (MDR1) gene in patients with metastatic refractory bladder carcinoma (no. MEC 133.184/1994/54)” has been activated for production of virus supernatant.

Retrovirus‐mediated transfer of the multidrug resistance gene into human haemopoietic progenitor cells

We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34+ cells as a target for human multidrug resistance (MDR1) gene transfer. Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, efficiency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 micrograms/ml taxol. In uninfected control, 1-2% of CFU-GM and CFU-GEMM were found to be drug-resistant, while 14-31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progenitors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction. MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls). (3) Flow cytometric analysis of the expression of CD34 and P-glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P-glycoprotein was found on 17-25% of viable CD34+ cells. (4) Cytochemical localization by APAAP staining of P-glycoprotein. No specific localization was found in untransduced controls, whereas transduced and cultured CB-cells expressed P-glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor cells seems a feasible and attractive approach to generate a drug-resistant haemopoiesis.

Multidrug resistance after retroviral transfer of the human MDR1 gene correlates with P-glycoprotein density in the plasma membrane and is not affected by cytotoxic selection.

Multidrug resistance (MDR) in mammalian cells is associated with the expression of the MDR1 gene encoding P-glycoprotein (P-gp), an and active efflux pump for various lipophilic compounds. MDR transfectants can be isolated after MDR1 gene transfer and selection with cytotoxic drugs; low levels of drug resistance have also been observed in unselected NIH 3T3 mouse cells after retrovirus-mediated transfer of mouse mdr1 cDNA. MDR cell lines possess multiple phenotypic changes, suggesting that P-gp function could be complemented by some additional mechanisms associated with cytotoxic selection. To determine whether cytotoxic selection contributes to the MDR phenotype of MDR1-expressing cells, NIH 3T3 cells infected with a recombinant retrovirus carrying the human MDR1 gene were selected by two different procedures: (i) noncytotoxic selection for increased P-gp expression on the cell surface by multiple rounds of immunofluorescence labeling and flow sorting or (ii) one or more steps of selection with a cytotoxic drug. The levels of MDR in both types of infectants showed an excellent correlation with the P-gp density in the plasma membrane, expressed as immunoreactivity with a P-gp-specific antibody normalized by reactivity with an antibody against an unrelated antigen. Cytotoxic selection conferred no additional increase in resistance relative to P-gp density. These results indicate that P-gp density in the plasma membrane may be sufficient to determine the level of MDR.