In vitro T lymphopoiesis: A model system for stem cell gene therapy for AIDS

Abstract: Stable introduction of therapeutic genes into hematopoietic stem cells has the potential to reconstitute immunity in individuals with HIV infection. However, many important questions regarding the safety and efficacy of this approach remain unanswered and may be addressed in a non-human primate model. To facilitate evaluation of expression of foreign genes in T cells derived from transduced hematopoietic progenitor cells, we have established a culture system that supports the differentiation of rhesus macaque and human CD34⁺ bone marrow derived cells into mature T cells. Thymic stromal monolayers were prepared from the adherent cell fraction of collagenase digested fetal or neonatal thymus. After 10–14 days, purified rhesus CD34⁺ bone marrow-derived cells cultured on thymic stromal monolayers yielded CD3⁺CD4⁺CD8⁺, CD3⁺CD4⁺CD8^?, and CD3⁺CD4^?CD8⁺ cells. Following stimulation with mitogens, these T cells derived from CD34⁺ cells could be expanded over 1,000-fold and maintained in culture for up to 20 weeks. We next evaluated the ability of rhesus CD34⁺ cells transduced with a retroviral vector containing the marker gene neo to undergo in vitro T cell differentiation. CD34⁺ cells transduced in the presence of bone marrow stroma and then cultured on rhesus thymic stroma resulted in T cells containing the retroviral marker gene. These studies should facilitate both in vitro and in vivo studies of hematopoietic stem cell therapeutic strategies for AIDS.     

逆转录病毒载体介导MGMT和MDR1基因增强人脐血CD34＋细胞对联合化疗抗性的研究

To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) and multidrug resistance gene (MDR1) increase resistance to 1,3-Bis(2-Chloroethy1)-1-Nitrosourea (BCNU) and P-glycoprotein effluxed drugs, the present authors obtained a full length cDNA fragment encoding MGMT from liver tissue of a patient with cholelithiasis by RT-PCR. A bicistronic retroviral vector G1Na-MGMT-IRES-MDR1 cDNA was constructed and transfected the packaging cell lines GP + E86 and PA317 by electric performation method, using the medium containing VCR and BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, cord blood CD34+ cells were enriched with a high-gradient magnetic cell sorting system (MACS), and then transfected repeatedly with supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hemapoietic growth factors. PCR, RT-PCR, Southern blot, Northern blot, Western blot, FACS and MTT assay were used to evaluate the transfer and expression of the double genes in cord blood CD34+ cells. The cDNA encoding MGMT was verified by DNA sequencing and the bicistronic retroviral vector was confirmed by restriction endonuclease analysis. The purity of cord blood CD34+ cells was approximately 92% and recover rate was 75%, the highest titer of recombinant amphotropic retrovirus in the supernatant was up to 5.8 x 10(5) cfu/ml. The efficiency of gene transduction was 18% and 20% tested by colony formation and PCR, respectively. No helper virus was found by both nested PCR and rescue assay. The results showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The MTT analysis showed a 4.5 to 7.8-fold increase of resistance of transducted cells to BCNU and P-glycoprotein effluxed drug as compared with the nontransduced cells. This study provided a foundation for ameliorating combination chemotherapy toxicity in tumor clinical trial.     

Retroviral MDR1 gene transfer into marrow-engrafting human peripheral blood progenitor cells results in preferential transgene expression in the immature myeloid compartment rather than in mature myeloid progeny in vivo

BACKGROUND: The objective of multidrug resistance-1 (MDR1) gene therapy is protection of the myeloid cell lineage. It is therefore important to examine the effect of retroviral transduction on myeloid maturation. Transfer of the human MDR1 gene can confer resistance to a variety of cytostatic drugs. For a safe application in humans it is paramount to follow-up the development of transduced cells. METHODS: We transduced human mobilized peripheral blood progenitor cells (PBPC) with a viral vector containing the human MDR1 cDNA and transplanted the transduced cells into non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. The progeny of the transduced cells was analyzed in detail by flow cytometry. RESULTS: A detailed analysis by four-color flow cytometry showed that MDR1 transgene-expressing CD33+ myeloid cells were preferentially negative for the maturation-associated myeloid markers CD11b and CD10, while the untransduced CD33+ myeloid cells expressed significantly higher proportions of these Ag (P<0.01 each). There was no difference in the expression of B- or T-lymphoid Ag among the MDR1-transduced and untransduced lymphoid cells. DISCUSSION: These data indicate that retroviral MDR1 gene transfer results in preferential P-glycoprotein expression in myeloid progenitor cells, which is the target cell population for myelotoxicity of cytostatic drugs.     

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells

Summary Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterialneo ^R gene. The presence of theneo ^R gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of theneo ^R gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4⁺ and CD8⁺ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4⁺ and CD8⁺ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4⁺ or CD8⁺ cells. In other experiments highly purified CD4⁺ and CD8⁺ T cell subpopulations from either TIL or PBMC could be successfully transduced with theneo ^R gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity or vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4⁺ and CD8⁺ gene-modified TIL. Current address: Bone Marrow Transplantation, Hadassah University Hospital, 91120 Jerusalem, Israel     

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34⁺ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34⁺ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34⁺ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold, total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34⁺ cells by 15.7-fold. Maximal expansions of CFC and CD34⁺ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34⁺ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34⁺ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines.     

Circumvention of chemotherapy-induced myelosuppression by transfer of themdr1 gene

Drug-induced myelosuppression is a frequent reason for curtailing chemotherapy in cancer patients. Rescue of myelosuppressed patients with autologous marrow transplants is reasonably advanced and permits an increase in the dose of anticancer drugs. Despite this improvement, patients often relapse with drug resistance disease. The human multidrug resistance (mdr1) gene might make it possible to render hemopoietic stem cells resistant to anticancer drugs after transfer of this gene. By introducing resistant stem cells into patients it might be possible to treat these patients repeatedly with otherwise ablative therapy. This review explores the feasibility ofmdr1 gene therapy.Abbreviations MDR multidrug resistance - ABMT autologous bone marrow transplantation - P-gp P-glycoprotein - RCR replication-competent retrovirus     

High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.

Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore, since it has now become possible to infect CD34+ cells with pseudotyped HIV-1 with a high level of efficiency in vitro, many important questions regarding the effect of HIV-1 on lineage-specific differentiation of hematopoietic progenitors can now be addressed.     

Gene therapy using anticancer drug-resistance genes

Myelosuppression is a major dose-limiting factor in cancer chemotherapy. Introduction of drug-resistance genes into bone marrow cells of cancer patients has been proposed to overcome this limitation. In theory, any gene whose expression protects cells against the toxic effects of chemotherapy should be useful in vivo for this purpose. Among such genes, human multidrug-resistance gene (MDR1) has been studied most extensively for this purpose, and clinical trials of drug-resistance gene therapy have been started in the US for cancer patients who undergo high-dose chemotherapy with autologous hematopoietic stem cell transplantation. In Japan, our clinical protocol of MDR1 gene therapy "A clinical study of drug-resistance gene therapy to improve the efficacy and safety of chemotherapy against breast cancer" has been submitted to the government. To improve the efficacy and safety of this drug-resistance gene therapy, we have constructed a series of MDR1-bicistronic retrovirus vectors using a retrovirus backbone of Harvey murine sarcoma virus and internal ribosome entry site (IRES) from picornavirus to co-express a second gene with the MDR1 gene. MDR1-MGMT bicistronic vectors can be used to protect bone marrow cells of cancer patients from combination chemotherapy with MDR1-related anticancer agents and nitrosoureas. In addition, MDR1-bicistronic retrovirus vectors can be designed to use the MDR1 gene as an in vivo selectable marker to enrich the transduced cells which express therapeutic genes, if disease is curable by the expression of a single-peptide gene in any types of bone marrow cells or peripheral blood cells.     

Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes

Background

The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34⁺/CD38^low and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes.

Methods

CB cells were transduced on Retronectin using an MSCV‐based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13‐MGIN (GALV), 293GPG‐MGIN (VSV‐G) or AM12‐MGIN (amphotropic).

Results

Sorted CD34⁺/CD38^low cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP‐positive cells was 61.8±6.6% (PG13‐MGIN), 26.9±3.5% (293GPG‐MGIN), and 39.3±4.8% (AM12‐MGIN). For transplantation experiments, CD34⁺ cells were pre‐stimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5×10⁵ cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum‐free medium and transduction of CD34⁺ cells using VSV‐G‐pseudotyped vectors under serum‐free conditions was very inefficient. In contrast, transduction with PG13‐MGIN under serum‐free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3±6.6%), and efficient gene transfer to SRCs (46.2±4.8%).

Conclusions

The best conditions for transduction and engraftment of CB SRCs were obtained with GALV‐pseudotyped vectors using serum‐free conditions. Copyright © 2002 John Wiley & Sons, Ltd.

     

Factors affecting retrovirus-mediated gene transfer to human CD34+ cells

BACKGROUND: Retrovirus-mediated gene transfer is a useful technology in studying the biology of hematopoietic stem cells (HSCs) as well as in developing gene therapy products for a variety of human diseases. One of the most important factors determining the success of these studies is the number of HSCs receiving the gene of interest. METHODS: We tested various parameters for their influences on gene transfer efficiency to CD34+ cells derived from bone marrow. Based on a literature survey, three medium formulations of CD34+ cells have been compared for their effects on gene delivery efficiency and differentiation of them. We also tested whether FBS, used in the medium formulation, could be replaced with human serum or synthetic material. RESULTS: Formulation A, consisting of stem cell factor, Flt-3 ligand, thrombopoietin, and IL-3, provided optimum results in that it maintained the highest percentage of CD34+ cells during the culture as well as produced the highest gene delivery efficiency. It was found that the synthetic serum substitute containing bovine serum albumin, insulin and human transferrin could replace the fetal bovine serum present in the original formulation A without compromising gene transfer efficiency. When the transduction procedure was repeated three times, the gene could be delivered in up to 60% of the cell population. Gene delivery efficiency was comparable between CD34+ cells derived from bone marrow and mobilized peripheral blood. CONCLUSIONS: Our data could be useful in designing a procedure for stem cell gene therapy and providing a basis for further improving the conditions for gene transfer to various HSCs.     

Optimization of a retroviral vector for transduction of human CD34 positive cells

Human stem and progenitor cells have recently become objects of intensive studies as an important target for gene therapy and regenerative medicine. Retroviral vectors are among the most effective tools for genetic modification of these cells. However, their transduction efficiency strongly depends on the choice of the ex vivo transduction system. The aim of this study was to elaborate a system for retroviral vector transduction of human CD34 positive cells isolated from cord blood. The retroviral vector pMINV EGFP was chosen for transduction of two human erythroblastoid cell lines: KG-1a (CD34 positive) and K562 (CD34 negative). For vector construction, three promoters and two retroviral vector packaging cell lines were used. To optimize the physicochemical conditions of the transduction process, different temperatures of supernatant harvesting, the influence of centrifugation and the presence of transduction enhancing agents were tested. The conditions elaborated with KG-1a cells were further applied for transduction of CD34 positive cells isolated from cord blood. The optimal efficiency of transduction of CD34 positive cells with pMINV EGFP retroviral vector (26% of EGFP positive cells), was obtained using infective vector with LTR retroviral promoter, produced by TE FLY GA MINV EGFP packaging cell line. The transduction was performed in the presence of serum, at 37 degrees C, with co-centrifugation of cells with viral supernatants and the use of transduction enhancing agents. This study confirmed that for gene transfer into CD34 positive cells, the detailed optimization of each element of the transduction process is of great importance.     

Mobilization of CD34~+CD38~- hematopoietic stem cells after priming in acute myeloid leukemia

AIM: To evaluate quantitatively and qualitatively the different CD34+cell subsets after priming by chemotherapy granulocyte colony-stimulating factor(± G-CSF)in patients with acute myeloid leukemia.METHODS: Peripheral blood and bone marrow sampleswere harvested in 8 acute myeloid leukemia patients during and after induction chemotherapy. The CD34/CD38 cell profile was analyzed by multi-parameter flow cytometry. Adhesion profile was made using CXC chemokine receptor 4(CXCR4)(CD184), VLA-4(CD49d/CD29) and CD47.RESULTS: Chemotherapy ± G-CSF mobilized immature cells(CD34+CD38 population), while the more mature cells(CD34+CD38lowand CD34+CD38+populations) decreased progressively after treatment. Circulating CD34+cells tended to be more sensitive to chemotherapy after priming with G-CSF. CD34+cell mobilization was correlated with a gradual increase in CXCR4 and CD47expression, suggesting a role in cell protection and the capacity of homing back to the marrow.CONCLUSION: Chemotherapy ± G-CSF mobilizes into the circulation CD34+bone marrow cells, of which, the immature CD34+CD38-cell population. Further manipulations of these interactions may be a means with which to control the trafficking of leukemia stem cells to improve patients’ outcomes.     

Long-term expression of gene introduction into normal human T-lymphocytes by retroviral-mediated gene transfer

Human T-lymphocytes are long lived, easily accessible, mature, and capable of proliferation. They are theoretically a suitable target for retroviral mediated gene transfer. To test this hypothesis, normal human T-cells obtained from bone marrow and peripheral blood were stimulated with phytohemagglutinin (PHA) and infected 24 h later with the retroviral vector N2 which carries the bacterial neo gene. T-lymphocytes were propagated in culture for up to 14 weeks with interleukin-2 (IL-2). Analysis by whole cell RNA dot/blot using a single stranded RNA probe demonstrated persistent expression of the neo gene. Preliminary functional studies revealed that both helper and suppressor functions were preserved in the infected cells in culture. These results demonstrate that normal T-cells are capable of long-term expression of genes introduced by retroviral mediated gene transfer and are potential target cells for somatic gene therapy.     

逆病毒载体介导双耐药基因在脐血CD34^＋细胞中的表达和抗性研究

To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and multidrug resistance gene (MDR1) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed. The vector was transduced into the packaging cell lines GP + E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDR1cDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4- to 7.2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontransduced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.     

Establishment of a Humanized APL Model via the Transplantation of PML-RARA-Transduced Human Common Myeloid Progenitors into Immunodeficient Mice

Recent advances in cancer biology have revealed that many malignancies possess a hierarchal system, and leukemic stem cells (LSC) or leukemia-initiating cells (LIC) appear to be obligatory for disease progression. Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia characterized by the formation of a PML-RARα fusion protein, leads to the accumulation of abnormal promyelocytes. In order to understand the precise mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model involving retroviral transduction of PML-RARA into CD34⁺ hematopoietic cells from human cord blood and transplantation of these cells into immunodeficient mice. The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA. As seen in human APL, the induced APL cells showed a low transplantation efficiency in the secondary recipients, which was also exhibited in the transplantations that were carried out using the sorted CD34⁻ fraction. In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA. Common myeloid progenitors (CMP) from CD34⁺/CD38⁺ cells developed APL. These findings demonstrate that CMP are a target fraction for PML-RARA in APL, whereas the resultant CD34⁻ APL cells may share the ability to maintain the tumor.     

Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy.

Recombinant retroviral vectors producing multicistronic mRNAs were constructed. Picornavirus putative internal ribosome entry sites (IRES) were used to confer cap-independent translation of an internal cistron. Internal cistrons were engineered by ligation of various lengths of the IRES of encephalomyocarditis (EMC) virus or polio virus to the E. coli chloramphenicol acetyltransferase (CAT) gene. The IRES/CAT fusions were introduced into retroviral vectors 3' to the translation stop codon of the neomycin phosphotransferase (NEO) gene, and the molecular constructs transfected into retroviral vector packaging lines. Retroviral vector producer cells efficiently express the internal CAT gene product only when the full length IRES is used. Both the EMC/CAT and polio/CAT retroviral vectors produced high titer vector supernatant capable of productive transduction of target cells. To test the generality of this gene transfer system, a retroviral vector containing an IRES fusion to the human adenosine deaminase (ADA) gene was constructed. Producer cell supernatant was used to transduce NIH/3T3 cells, and transduced cells were shown to express NEO, and ADA. Novel three-gene-containing retroviral vectors were constructed by introducing the EMC/ADA fusion into either an existing internal-promoter-containing vector, or a polio/CAT bicistronic vector. Producer cell clones of the three-gene vectors synthesize all three gene products, were of high titer, and could productively transduce NIH/3T3 cells. By utilizing cap-independent translation units, IRES vectors can produce polycistronic mRNAs which enhance the ability of retroviral-mediated gene transfer to engineer cells to produce multiple foreign proteins.     

Prospects for gene therapy

Experiments in animal models and human cells in vitro suggest that gene transfer using retroviral vectors may be useful to treat genetic diseases and to gain information that may improve treatment of other common diseases such as cancer. The approach to treatment of genetic diseases by inserting genes into bone marrow cells and experimental models, and a novel application of gene transfer technology to cancer research are discussed herein.     

Multidrug resistant transgenic mice as a novel pharmacologic tool.

Multidrug resistance resulting from expression of an energy-dependent drug efflux pump encoded by the human MDR1 gene is a major impediment to effective cancer therapy. Pharmacologic intervention aimed at inhibiting this multidrug transporter should improve existing chemotherapy of human cancer, but drug development has been delayed by the difficulty and expense of developing valid animal models. Using recombinant DNA technology, a transgenic mouse has been engineered whose bone marrow is protected from the toxic effects of chemotherapy by expression of the MDR1 gene. This animal system allows the rapid screening of drugs which inhibit the multidrug transporter and heralds a new era of using transgenic animals for pharmacologic screening.     

Increasing drug resistance in human lung cancer cells by mutant-type p53 gene mediated by retrovirus

Human mutant-type (mt) p53 cDNA was synthesized and cloned from human lung cancer cell line GL containing mt-p53 gene by using polymerase chain reaction (PCR). It was confirmed that the mt-p53 cDNA con-tained the complete coding sequence of p53 gene but mutated at codon 245 (G→T) and resulted in glycine to cysteine by sequencing analysis. The retroviral vector pD53M of the mt-p53 was constructed and introduced into the drug-sen-sitive human lung cancer cells GAO in which p53 gene did not mutate. The transfected GAO cells strongly expressed mutant-type p53 protein by immunohistochemistry, showing that pD53M vector could steadily express in GAO cells. The drug resistance to several anticancer agents of GAO cells infected by pD53M increased in varying degrees, with the highest increase of 4-fold, in vitro and in vivo. By quantitative PCR and flow cytometry (FCM) analyses, the expression of MDR1 gene and the activity of P-glycoprotein (Pgp) did not increase, the expression of MRP gene and the activity of m