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W J Zeller

Publications and source records attributed to W J Zeller.

At least 19 recordsLinked to original sources
PubMed2006

Retroviral vector insertions in T-lymphocytes used for suicide gene therapy occur in gene groups with specific molecular functions.

Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.

Animals↗
PubMed2006

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Synergistic activity of imatinib and 17-AAG in imatinib-resistant CML cells overexpressing BCR-ABL--Inhibition of P-glycoprotein function by 17-AAG.

Overexpression of BCR-ABL and P-glycoprotein (Pgp) are two of the known mechanisms of imatinib resistance. As combination therapy may allow to overcome drug resistance, we investigated the effect of combination treatment with imatinib and 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat-shock protein 90 (Hsp90) inhibitor, on different imatinib-sensitive and imatinib-resistant CML cell lines. In imatinib-sensitive cells, combination index (CI) values obtained using the method of Chou and Talalay indicated additive (CI=1) or marginally antagonistic (CI>1) effects following simultaneous treatment with imatinib and 17-AAG. In imatinib-resistant cells both drugs acted synergistically (CI<1). In primary chronic-phase CML cells additive or synergistic effects of the combination of imatinib plus 17-AAG were discernible. Annexin V/propidium iodide staining showed that the activity of imatinib plus 17-AAG is mediated by apoptosis. Combination treatment with imatinib plus 17-AAG was more effective in reducing the BCR-ABL protein level than 17-AAG alone. Monotherapy with 17-AAG decreased P-glycoprotein activity, which may increase intracellular imatinib levels and contribute to the sensitization of CML cells to imatinib. The results suggest that combination of imatinib and 17-AAG may be useful to overcome imatinib resistance in a clinical setting.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Transfer of drug resistance genes in hematopoietic progenitors for chemoprotection: is it still an option?

For numerous malignancies a relationship between the intensity of antineoplastic chemotherapy and tumor response has been demonstrated. Myelotoxicity is the main cause of chemotherapy-associated morbidity and of treatment delays. The concept of myeloprotective cytostatic drug resistance gene transfer to normal hematopoietic stem cells (HSC) therefore sparks great enthusiasm. While initial studies using murine retroviral vectors on murine HSC showed that the concept works, a number of clinical studies in the last decade were not informative because of limitations in transduction efficiency and transgene expression.Furthermore, possible side effects such as unforeseen transgene activity and vector integration-based leukemogenesis have been reported. Among others, these developments raised some scepticism against the feasibility of myeloprotective gene transfer. Recently, considerable improvements have been achieved in vector design, HSC manipulation, selection protocols and risk assessment methods which are discussed in detail here. Based on these experimental studies successful clinical trials can now be anticipated.

Antineoplastic Agents↗
PubMed2003

Rapid detection of retroviral vector integration sites in colony-forming human peripheral blood progenitor cells using PCR with arbitrary primers.

We have developed a highly sensitive polymerase chain reaction (PCR)-based technique termed two-step PCR, which uses arbitrary primers to identify proviral integration sites in retrovirally marked human colony-forming cells. The two-step PCR was established on cell line clones transduced with the SF1m retroviral vector and independently validated by demonstrating identical integration sites with ligation-mediated PCR, a different technique requiring restriction enzyme digestion and adapter ligation for amplifying unknown DNA flanking the provirus. Two-step PCR was performed on peripheral blood progenitor cell (PBPC) colonies that contained as few as 75 cells, which was estimated by quantitative real-time PCR. We were able to amplify and directly sequence proviral integration sites in 35 % of PBPC colonies (25/72, five donors). Identity to the vector long-terminal repeat was confirmed and flanking DNA was found to match with human database sequences, reaffirming specificity. Two-step PCR is a valuable new tool for rapid analysis of genomic target sites for viral vectors, and will aid significantly in understanding clonal development of hematopoiesis and other cell types. Our protocol has the potential for general applicability as the arbitrary primers described here bind to genomic DNA and are thus independent of the vector backbone used.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Imatinib restores expression of CD62L in BCR-ABL-positive cells.

Chronic myelogenous leukemia (CML) is characterized by aberrant trafficking of malignant hematopoietic progenitor cells in the peripheral blood. Expression of the cell adhesion molecule CD62L was reported to be significantly lower in CML patients than in normal controls. We studied whether the transcription of CD62L in CML cells is dependent on the activity of the BCR-ABL tyrosine kinase. Following addition of the Abelson (ABL) tyrosine kinase inhibitor imatinib (formerly STI571) to two BCR-ABL-positive cell lines (BV173, SD-1), we observed a dose-dependent increase in CD62L RNA levels of up to 45-fold by a quantitative, real-time polymerase chain reaction and an increase in the amount of cell surface-bound CD62L of up to 18-fold by quantitative flow cytometry, respectively. These data are validated by an increased CD62L expression in the bone marrow of patients (n=6) with advanced CML who received imatinib. Restoration of defective cell adhesion mediated via the CD62L pathway may be one mechanism of action of imatinib in BCR-ABL-positive leukemias.

Antineoplastic Agents↗
PubMed2003

Prospects and RISC score of viral gene therapy for sarcoma.

Soft tissue sarcomas are a challenge for medical oncology and gene therapy. Protective and sensitising approaches that target normal and malignant tissue, respectively, both have their role for opening the therapeutic window. Recent data show that an intensive maintenance chemotherapy significantly reduces metastatic spread and improves disease-free survival in selected patient groups. However, delays of treatment due to cytopenia are frequent. Cytostatic drug resistance gene transfer to haematopoietic progenitor cells using retroviral vectors may allow further improvement of therapy results. In recent years, retroviral vector design, transduction techniques and engraftment capability of transduced cells have been optimised. Safety considerations of retroviral gene transfer have attracted public attention and can be addressed by analysis of genomic vector integration sites. A data bank project, 'retroviral insertion estimate of chromosomal integration' (RISC), containing > 200 integration sequences, has been set up by the authors' group to recognise critical genomic regions and genes involved with possible transforming capacity. Monitoring these parameters will allow the selection of the most suitable vectors for clinical application. Sarcoma cells seem to be highly susceptible to a variety of vectors, such as recombinant adeno-associated virus-2 (rAAV-2) vectors, adenoviral vectors or oncolytic herpes simplex viruses. Results from the first clinical trials with adenoviral vectors encoding for cytokines are promising. The other systems await further development towards clinical applications. Perspectives for further research are discussed in this review.

Animals↗
PubMed2002

Rationale for combination therapy of chronic myelogenous leukaemia with imatinib and irradiation or alkylating agents: implications for pretransplant conditioning.

The tyrosine kinase activity of the BCR-ABL oncoprotein results in reduced apoptosis and thus prolongs survival of chronic myelogenous leukaemia cells. The tyrosine kinase inhibitor imatinib (formerly STI571) was reported to selectively suppress the proliferation of BCR-ABL-positive cells. Assuming that imatinib could be included in pretransplantation conditioning therapies, we tested whether combinations of imatinib and gamma-irradiation or alkylating agents such as busulfan or treosulfan would display synergistic activity in BCR-ABL-positive chronic myelogenous leukaemia BV173 and EM-3 cell lines. Further, primary cells of untreated chronic myelogenous leukaemia patients were assayed for colony forming ability under combination therapy with imatinib. Additionally, the cytotoxic effect of these combinations on BCR-ABL-negative cells was investigated. In the cell lines a tetrazolium based MTT assay was used to quantify growth inhibition after exposure to cytotoxic drugs alone or to combinations with imatinib. Irradiation was applied prior to exposure to imatinib. Interaction of drugs was analysed using the median-effect method of Chou and Talalay. The combination index was calculated according to the classic isobologram equation. The combination imatinib + gamma-irradiation proved to be significantly synergistic over a broad range of cell growth inhibition levels in both BCR-ABL-positive cell lines and produced the strongest reduction in primary chronic myelogenous leukaemia colony-forming progenitor cells. Combinations of imatinib + busulfan and imatinib + treosulfan showed merely additive to antagonistic effects. Imatinib did not potentiate the effects of irradiation or cytotoxic agents in BCR-ABL-negative cells. Our data provide the basis to further develop imatinib-containing conditioning therapies for stem cell transplantation in chronic myelogenous leukaemia.

Antineoplastic Agents↗
PubMed2002

Gene therapy for sarcoma.

Soft tissue sarcomas are mesenchymal tumors which respond poorly to systemic therapy. Recent studies suggest a higher response rate with an increased doxorubicin dosage. However, this was parallel with a profound hematotoxicity in 75% of patients. Transfer of the human multidrug resistance 1 (MDR1) gene to normal hematopoietic stem cells and transplantation may significantly reduce the hematotoxicity of anthracyclin-based chemotherapy. To test this concept of supportive gene therapy in advance of a clinical study, we transduced mobilized peripheral blood progenitor cells (PBPC) with the retroviral vector SF91m3 containing the human MDR1 gene, transplanted these cells to immune-deficient mice, allowed 6 weeks for engraftment to occur and treated the animals with MDR1-based chemotherapy. In the MDR1-transduced group the human leukocytes were significantly protected from the toxicity of chemotherapy (p < 0.05). While the gene transfer rate was in the range of 10% and thus comparable to recent clinical trials, the gene expression was 59% of transduced cells and thus significantly higher than previously reported for less-advanced vectors. On the other hand, ifosfamide, a drug which has been used successfully for stem cell mobilization, is active in soft tissue sarcoma. Due to these favorable characteristics sarcoma is an attractive target to test the efficacy of MDR1 gene therapy in a clinical setting. Gene therapeutic strategies may also be used to directly target sarcoma cells, e.g. by transfer of suicide genes. We found that adenoassociated virus 2 (AAV-2) vectors efficiently transduce human HS-1 and HT1080 sarcoma cells (>90%) while other tumor cell lines and primary human PBPC were less susceptible. The thymidine kinase (TK) suicide gene was cloned into an AAV-2 vector and a complete kill of TK-transduced HS-1 and HT1080 cells was observed following exposure to aciclovir or ganciclovir (GCV), while >90% of mock-transduced HS-1 cells survived at these dosages. Transplantation of those sarcoma cells to nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice resulted in a survival of >5 months in the AAV-TK-transduced/GCV-treated group, while the mice in the mock-transduced/GCV-treated group had died after 3 weeks. These data show that soft tissue sarcomas are a particularly suitable model system for the development and clinical testing of new gene therapeutic concepts.

Animals↗
PubMed2001

The FBMD-1 stroma cell line secretes a unique moiety which can increase retroviral transduction of lineage-committed and primitive human peripheral blood progenitor cells.

Peripheral blood progenitor cells are a prime target for gene therapy approaches. As recent data point to the relevance of soluble stroma factors for the efficient transduction of progenitor cells, we tested the stroma-conditioned medium (SCM) of the two cell lines FBMD-1 and L88/5 as well as desulfated and O-sulfated heparin (HS dS and HS OS) for their effect on transduction of peripheral blood progenitor cells. We transduced CD34+ cells of nine tumor patients with the retroviral SF-MDR vector containing the human multidrug resistance 1 (MDR1) gene under serum-free conditions on the fibronectin fragment CH-296 with or without SCM. Provirus-specific polymerase chain reaction showed a median 1.6-fold higher integration rate of the transgene into committed progenitor cells for the group with added FBMD-1 SCM (P=.008). This was maintained after 2 (P=.02) and, as a trend, after 5 weeks of stroma-dependent long-term culture. We found a median 1.5-fold increase in rhodamine-123 (Rh-123) exclusion in myeloid lineage-committed progeny cells following transduction in the presence of FBMD-1 SCM (P=.0004). After 2 or 5 weeks of long-term culture, a significantly higher proportion of Rh-123(dull) cells could still be detected in the FBMD-1 SCM transduction group (P=.003 and P=.04, respectively). L88/5 SCM or HS OS or HS dS was not effective as supplement for improving gene transfer. The FBMD-1 stroma cell line appears to secrete a unique moiety, which can increase retroviral transduction of lineage-committed and primitive progenitor cells. The FBMD-1 stroma activity is not attributable to heparan sulfate.

Adolescent↗
PubMed2001

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells.

The ABL-specific tyrosine kinase inhibitor STI571 (formerly CGP57148B) induced cytogenetic remissions in 33% of chronic myelogenous leukemia (CML) patients in a phase I trial (Druker et al 1999). Combination therapy may increase this proportion. We tested whether combinations of STI571 and cytarabine or other chemotherapeutic agents such as hydroxyurea, mafosfamide or etoposide would display synergistic activity in BCR-ABL-positive chronic myelogenous leukemia (CML) cell lines derived from patients in blast crisis. In addition, the toxicity of these combinations on BCR-ABL-negative cells was investigated. A tetrazolium-based MTT assay was used to quantity growth inhibition after 48 h of exposure to cytotoxic agents alone and in simultaneous combination with STI571. The drug interactions were analyzed using the median-effect method of Chou and Talalay. The combination index (CI) was calculated according to the classic isobologram equation. At growth inhibition levels of over 50%, STI571 + cytarabine as well as STI571 + etoposide were significantly synergistic (CI < 1, P < 0.05) in the BCR-ABL-positive cell lines evaluated. At 60% inhibition or higher, a similar synergistic pattern became apparent for STI571 + mafosfamide (P < 0.05), while STI571 + hydroxyurea showed ambiguous, cell line-dependent synergism (BV173), additivity (EM-3) or antagonism (K562) in CML cell lines. Furthermore, the BCR-ABL-negative HL-60, KG1a and normal CD34+ progenitor cells were not affected by 0.8 microM STI571, a concentration which produced more than 50% growth inhibition in all BCR-ABL-positive cells tested, and no potentiation of growth inhibition was observed in these BCR-ABL-negative cells when STI571 was combined with chemotherapeutic agents. Our in vitro data with CML blast crisis cell lines strongly suggest that combinations of STI571 with cytarabine or etoposide be rapidly considered for clinical testing.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Production of reactive oxygen species by phagocytic cells after exposure to glass wool and stone wool fibres - effect of fibre preincubation in aqueous solution.

The potential of four man-made vitreous fibres (MMVFs) (glass wool Code A, stone wool Code G, HT-N and MMVF 21) and of two natural mineral fibres (crocidolite, erionite) to induce production of reactive oxygen species (ROS) by differentiated HL-60 cells (HL-60-M cells) was investigated by determination of luminol-enhanced chemiluminescence (CL). Quartz served as positive control. The same system was used to uncover possible influences of fibre preincubation in aqueous solutions on the ROS-generating potential. Following preincubation in unbuffered saline over about 4 weeks, Code A and G fibres showed decreased ROS-generating potential as compared to freshly suspended fibres. On the other hand, MMVF 21 and HT-N fibres as well as crocidolite and erionite showed no decreased CL after incubation in aqueous solutions. The observed decrease of the ROS-generating potential of Code A and G fibres after preincubation may be an expression of fibre surface alterations (leaching, initiation of dissolution) that influences the response of exposed phagocytic cells. After incubation of both fibres in buffered solutions at different pH values (5.0, 7.4) a reduced ROS-generating potential was still discernible as compared to freshly suspended fibres.

Asbestos, Crocidolite↗
PubMed2000

Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice.

Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% +/- 5% [mean +/- SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P <.01). A population of clearly rhodamine-123(dull) human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% +/- 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol. (Blood. 2000;95:1237-1248)

Adipocytes↗
PubMed2000

MDR1 gene expression in NOD/SCID repopulating cells after retroviral gene transfer under clinically relevant conditions.

We have adapted a recently published protocol for retroviral gene transfer into hematopoietic cells [A. J. Schilz et al. (1998) Blood 92: 3163-3171] with respect to clinical requirements such as large-volume vector stock generation, adequate cell source, high cell numbers, and serum-free conditions. We present data on transduction efficacy and expression of the multidrug resistance 1 (MDR1) gene in human CD34(+) cells from mobilized peripheral blood (PB) mediated by a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector. Using a 1-day cytokine-mediated prestimulation, consisting of human interleukin (IL)-3, IL-6, stem cell factor (SCF), Flt-3 ligand (FL), and thrombopoietin (TPO), followed by a 3-day transduction procedure, we were able to detect up to 51% CD34(+) cells expressing MDR1. Xenotransplantation of transduced cells into NOD/LtSz-scid/scid (NOD/SCID) mice resulted in a mean engraftment level of 23% (0.1 to 87%). As shown by quantitative PCR analysis, a mean of 12.7% (range 0.3 to 55%) of the engrafted human cells in the bone marrow of chimeric mice contained the MDR1 cDNA. Furthermore, enhanced expression of MDR1 above control levels was detected in up to 15% of the engrafted human cell population. Our data suggest that NOD/SCID repopulating cells derived from mobilized PB can be transduced efficiently with existing retroviral vector systems under clinically applicable conditions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Human multidrug resistance-1 gene transfer to long-term repopulating human mobilized peripheral blood progenitor cells.

Mobilized peripheral blood progenitor cells (PBPC) are an attractive target for the retrovirus-mediated transfer of cytostatic drug resistance genes. We analyzed NOD/SCID mouse repopulating CD34+ PBPC from cancer patients following retroviral Transwell transduction in various cytokine combinations with the FMEV-based (Friend-mink cell focus forming/murine embryonic stem cell virus) hybrid vector SF-MDR carrying the human multidrug resistance-1 (MDR1) gene. Five to 10 weeks following transplantation of 2.0 x 10(6) CD34+ PBPC into NOD/SCID mice we observed medium to high levels of human cell engraftment with up to 33%. The extent of vector-marked human cells was assessed by a quantitative real-time polymerase chain reaction (PCR). SF-MDR gene transfer into long-term in vivo repopulating human hematopoietic cells was optimal in the presence of either IL-3/IL-6/SCF/FL or FL/TPO/SCF resulting in three-fold (12.4% +/- 1.7%) or four-fold (16.5% +/- 6.8%) higher average proportions of gene-marked human cells in NOD/SCID mice as compared to IL-3 alone (P < 0.01). In conclusion, we could optimize the engraftment capacity and the retroviral gene transfer to CD34+ PBPC using cocktails of early acting cytokines in combination with the recombinant fibronectin fragment CH-296. Our data suggest that the NOD/SCID model provides a valid assay to estimate the gene transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation settings.

Animals↗
PubMed2000

Differential expression of a recombinant adeno-associated virus 2 vector in human CD34+ cells and breast cancer cells.

The use of autologous hematopoietic stem cell (HSC) grafts after high-dose chemotherapy protocols may be hampered by contamination of the grafts with tumor cells. Because epithelial cells seem to be the natural hosts of adeno-associated virus 2 (AAV-2), we speculated that epithelial tumor cells in HSC grafts might be selective targets for AAV-2-based vectors. To test this hypothesis, the breast cancer cell lines T47D and MCF-7 were infected with a recombinant AAV-2 vector expressing the green fluorescent protein (GFP) gene; in addition, human CD34+ mobilized peripheral progenitor cells were infected with the same vector. At a multiplicity of infection of 100, only 1.39% +/- 0.51% CD34+ cells expressed the GFP gene whereas, 36.06% +/- 6.53% of the infected T47D cells and 41.52% +/- 3.16% of the infected MCF-7 cells expressed the transduced GFP gene. After further optimizing the transduction procedure by using higher multiplicities of infection (100-500) and preincubation of samples with the tyrosine kinase inhibitor genistein, up to 82.52% and 85.35% GFP+ T47D and MCF-7 cells, respectively, were observed. The GFP fluorescence intensity in transduced mammary tumor cells was up to 3 logs higher than that of transduced CD34+ cells. The differential expression of recombinant AAV-2 vectors in hematopoietic and epithelial tumor cells warrants further research with this vector system, including the use of suicide genes for the purging of autologous HSC grafts.

Antigens, CD34↗