ABSTRACT
Nanoparticles consisting of single molecules of DNA condensed with polyethylene glycol-substituted lysine 30-mers efficiently transfect lung epithelium following intrapulmonary administration. Nanoparticles formulated with lysine polymers having different counterions at the time of DNA mixing have distinct geometric shapes: trifluoroacetate or acetate counterions produce ellipsoids or rods, respectively. Based on intracytoplasmic microinjection studies, nanoparticle ellipsoids having a minimum diameter less than the 25 nm nuclear membrane pore efficiently transfect non-dividing cells. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids, whereas the 8-11 nm diameter of rod-like particles is smaller than the nuclear pore diameter. In mice, up to 50% of lung cells are transfected after dosing with a rod-like compacted 6.9 kbp lacZ expression plasmid, and correction of the CFTR chloride channel was observed in humans following intranasal administration of a rod-like compacted 8.3 kbp plasmid. To further investigate the potential size and shape limitations of DNA nanoparticles for in vivo lung delivery, reporter gene activity of ellipsoidal and rod-like compacted luciferase plasmids ranging in size between 5.3 and 20.2 kbp was investigated. Equivalent molar reporter gene activities were observed for each formulation, indicating that microinjection size limitations do not apply to the in vivo gene transfer setting.
Subject(s)
Cystic Fibrosis/therapy , Genetic Therapy/methods , Lung/enzymology , Plasmids/genetics , Transfection/methods , Cell Line , Cystic Fibrosis/metabolism , Epithelial Cells/enzymology , Gene Expression , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Humans , Luciferases/analysis , Luciferases/genetics , Microscopy, Electron, Transmission , Nanostructures , NanotechnologyABSTRACT
We conducted a phase 1 trial of direct injection of an E1, E3-deleted adenovirus encoding interleukin-2 (AdCAIL-2) into subcutaneous deposits of melanoma or breast cancer. Twenty-three patients were injected at seven dose levels (10(7)-10(10) p.f.u). Local inflammation was observed at the site of injection in 60% of patients, but side-effects were otherwise minor. Incomplete local tumor regression occurred at the site of injection in 24% of patients, but no conventional clinical responses were seen. Circulating CD4 and CD8 counts fell significantly 24 h after injection. Post-injection biopsies demonstrated tumor necrosis and lymphocytic infiltration with the predominant tumor-infiltrating cells both CD3- and CD8-positive. Vector-derived sequences were detected in 14 of 18 biopsies examined 7 days after injection and vector-derived hIL-2 mRNA was detected in 80% of 7-day biopsies processed after injection of 10(8) p.f.u. of AdCAIL-2 or higher. While IL-2 was detectable by ELISA in tumor biopsies at 48 h, no protein was detectable in injected tumors after 7 days and no circulating IL-2 was detectable at any time-point. No Ad5E1 sequences were detected either before or after injection indicating absence of replication-competent virus or endogenous E1-like sequence; furthermore, only rare vector shedding was detected. Anti-adenovirus and neutralizing antibody titers were elevated 1 month after injection in all patients. This trial therefore confirms the safety of use of adenoviral vectors for gene delivery in humans and demonstrates successful transgene expression even in the face of pre-existing immunity to adenovirus.
Subject(s)
Adenoviridae/genetics , Breast Neoplasms/secondary , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Interleukin-2/genetics , Melanoma/therapy , Antibodies, Viral/blood , Breast Neoplasms/immunology , Breast Neoplasms/therapy , CD3 Complex/immunology , CD4-Positive T-Lymphocytes/immunology , CD8 Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Dose-Response Relationship, Immunologic , Female , Gene Expression , Humans , Immunohistochemistry , Injections, Intralesional , Lymphocyte Count , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/immunology , Polymerase Chain Reaction , Skin Neoplasms/immunology , Skin Neoplasms/therapyABSTRACT
We investigated whether directly injecting retroviral vectors into preimmune fetuses could result in the transfer and long-term expression of exogenous genes. Twenty-nine preimmune sheep fetuses were injected with helper-free retroviral vector preparations. Twenty-two fetuses survived to term, 4 of which were sacrificed at birth. Of the remaining 18 animals, 3 were controls and 15 had received vector preparations. Twelve of these 15 animals demonstrated transduction of hematopoietic cells when blood and marrow were analyzed by neo(r)-specific PCR. Eight experimental sheep have been followed for 5 years, during which time we have consistently observed proviral DNA and G418-resistant hematopoetic progenitors. The G418-resistant colonies were positive when analyzed by neo(r)-specific PCR. neo(r) gene expression was also demonstrated using several immunological and biochemical methods. The transduction of hematopoietic stem cells was confirmed when lambs transplanted with bone marrow from in utero-transduced sheep exhibited neo(r) activity in marrow and blood. Vector distribution was widespread in primary animals without pathology. PCR analysis indicates that the germ line was not altered. These studies demonstrate that direct injection of an engineered retrovirus is a feasible means of safely delivering a foreign gene to a developing fetus and achieving long-term expression without modifying the germ line of the recipient.
Subject(s)
Fetal Diseases/therapy , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Kanamycin Kinase/genetics , Kanamycin Kinase/metabolism , Retroviridae/genetics , Animals , Blotting, Southern , Enzyme-Linked Immunosorbent Assay , Erythroid Precursor Cells/drug effects , Female , Fetal Diseases/genetics , Fluorescent Antibody Technique , Gene Transfer Techniques , Gentamicins/pharmacology , Hematopoietic Stem Cell Transplantation , In Situ Hybridization, Fluorescence , Injections, Intraperitoneal , Polymerase Chain Reaction , Pregnancy , Sequence Analysis, DNA , SheepSubject(s)
Adenoviridae/genetics , Gene Transfer Techniques , Genetic Therapy , Interleukin-2/genetics , Plasmacytoma/therapy , Female , Genetic Vectors , Humans , Middle AgedABSTRACT
To evaluate whether marrow contributes to relapse after autologous bone marrow transplantation (AuBMT) for acute leukemia, transplanted marrow was marked with the G1N retroviral vector (Genetic Therapy Inc.) containing the neomycin phosphotransferase gene (neo). Between April 1992 and August 1993, 4 patients were transplanted for acute myeloid leukemia (AML) in second complete remission (CR) and 1 patient for acute lymphoid leukemia in first CR. An average of 12.4% (range 5-19%) of transplanted marrow mononuclear cells were exposed to G1N vector for 4 hr. In the vector-treated portion of the marrow, 4.9% of GM-CFU and 3.6% of erythroid burst-forming units (BFU-E) were resistant to G418 in vitro. In the 5 patients, the polymerase chain reaction (PCR) detected the neo sequence on only two occasions after AuBMT. Of 4 patients surviving 1 year after transplantation, only 1 had evidence of gene marked cells by PCR. Two AML patients have relapsed, one of whom had evidence of neo sequences in the bone marrow at day 100 but not at relapse 11 months after AuBMT. The second patient relapsed 18 months after AuBMT but never had PCR evidence of neo sequences before or after relapse. Our results indicate vector-transduced autologous bone marrow from heavily pretreated adults with acute leukemia mark with low efficiency, although vector sequences have been detected in bone marrow and peripheral blood up to 1 year after transplant. Of the 2 relapsed patients, no evidence of vector-marked leukemic blasts have been detected.
Subject(s)
Bone Marrow Transplantation , Gene Transfer Techniques , Genetic Vectors , Leukemia, Lymphoid/therapy , Leukemia, Myeloid/therapy , Retroviridae/genetics , Acute Disease , Adult , Consumer Product Safety , Female , Follow-Up Studies , Hematopoietic Stem Cells , Humans , Male , Middle Aged , Transplantation, AutologousABSTRACT
Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and IL-2 appears to produce dramatic regressions in patients with metastatic melanoma and renal cancer. However, the in vivo mechanism of TIL function is not known. We conducted an UCLA Human Subject Protection Committee, Recombinant DNA Advisory Committee, and FDA-approved clinical trial using genetically-marked TIL to test the hypothesis that these cells have unique, tumor-specific in vivo trafficking patterns. TIL and PBL (as a control effector cell population) were isolated and expanded in parallel in vitro in IL-2-containing medium for 4-6 wk. During the expansion, TIL and PBL were separately transduced with the amphotropic retroviral vectors LNL6 and G1Na. Transduced TIL and PBL were coinfused into patients and their respective numbers measured in tumor, peripheral blood, and normal tissues; integrated provirus could be quantitated and distinguished by DNA PCR. Nine patients were treated (six melanoma, three renal) and received between 4.5 x 10(8) and 1.24 x 10(10) total cells. Both "marked" TIL and PBL could be detected circulating in the peripheral blood, in some patients for up to 99 d after infusion. Marked TIL and/or PBL could be detected in tumor biopsies in six of nine patients as early as day 6 and as late as day 99 after infusion. No convincing pattern of preferential trafficking of TIL vs. PBL to tumor was noted. Moreover, concurrent biopsies of muscle, fat, and skin demonstrated the presence of TIL/PBL in comparable or greater numbers than in tumor in five patients. The results of this double gene marking trial provide interesting insights into the life span and trafficking of adoptively transferred lymphocytes, but do not support the hypothesis that TIL specifically traffic to tumor deposits.
Subject(s)
Lymphocytes, Tumor-Infiltrating/cytology , Adult , Aged , Base Sequence , Carcinoma, Renal Cell/therapy , DNA Primers/chemistry , Evaluation Studies as Topic , Female , Genetic Markers , Genetic Vectors , Humans , Immunization, Passive , Male , Melanoma/therapy , Middle Aged , Molecular Sequence DataABSTRACT
We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady-state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.
Subject(s)
Antigens, CD/analysis , Bone Marrow Transplantation , Breast Neoplasms/therapy , Hematopoietic Stem Cell Transplantation , Multiple Myeloma/therapy , Antigens, CD34 , Breast Neoplasms/pathology , Cyclophosphamide/pharmacology , Female , Filgrastim , Fluorouracil/pharmacology , Follow-Up Studies , Genes, Reporter , Genetic Markers , Genetic Therapy , Graft Survival , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cells/drug effects , Humans , Kanamycin Kinase , Male , Melphalan/pharmacology , Middle Aged , Multiple Myeloma/pathology , Phosphotransferases (Alcohol Group Acceptor)/analysis , Recombinant Proteins/analysis , Recombinant Proteins/pharmacology , Treatment Outcome , Whole-Body IrradiationABSTRACT
Patients with solid tumors are increasingly being treated by autologous bone marrow transplantation (BMT). Although response rates appear to be increased, disease recurrence is the commonest cause of treatment failure. Whether relapse is entirely due to residual disease in the patient or arises also from infiltrating malignant cells contained in the autologous marrow transplant has not been resolved. If the latter explanation is correct, then purging would be required as part of the transplantation procedure. We used retrovirally mediated transfer of the neomycin-resistance gene to mark BM harvested from eight patients with neuroblastoma in clinical remission. The marked marrow cells were subsequently reinfused as part of an autologous BMT. At relapse, we sought the marker gene in malignant cell populations. Three patients have relapsed, and in each the marker gene was detected by phenotypic and genetic analyses of resurgent malignant cells at medullary and extramedullary sites. Analysis of neuroblast DNA for discrete marker gene integration sites suggested that at least 200 malignant cells, each capable of tumor formation, were introduced with the autologous marrow transplant and contributed to relapse. Thus, autologous BMTs administered to patients with this solid tumor may contain a multiplicity of malignant cells that subsequently contribute to relapse. The marker-gene technique we describe should permit evaluation of the mechanisms of relapse and the efficacy of purging in patients receiving autologous marrow transplantation for other solid tumors that infiltrate the marrow.
Subject(s)
Bone Marrow Transplantation , Neuroblastoma/therapy , Base Sequence , Child , Child, Preschool , Drug Resistance, Microbial/genetics , Female , Gene Transfer Techniques , Humans , Male , Molecular Sequence Data , Neomycin/pharmacology , Neuroblastoma/pathology , Polymerase Chain Reaction , Transplantation, AutologousABSTRACT
Gene therapy for chronic myelogenous leukaemia (CML) may provide a therapeutic option for patients who are ineligible for bone marrow transplantation. To determine the feasibility of such an approach we evaluated the transduction efficiency of CML progenitor colonies from seven patients in chronic phase. Vector transduction was optimized using the CML-derived K562 cell line and applied to CML mononuclear cells. After vector exposure, optimal gene transfer was noted when CML mononuclear cell cultures contained stem cell factor, IL-3, GM-CSF and erythropoietin. The addition of IL-6 to this combination decreased transduction efficiency. Using these conditions, 20.4% +/- 2.4 (SE) of erythroid colonies (CFU-GEMM and BFU-E) and 20.2% +/- 4.7 of CFU-GM colonies were G418 resistant. This compares with a transduction efficiency of 5.9% +/- 1.1 and 6.4% +/- 1.5, respectively, for erythroid and CFU-GM colonies using marrow obtained from normal donors. Only a modest increase in gene transfer was noted when CML cells were stimulated with cytokines for the 24 h preceding vector exposure. Vector DNA in colonies expressing the BCR/ABL transcript was documented by performing PCR analysis on individual colonies. The relatively high gene transfer rate in CML suggests that this disease might be very suitable for gene therapy.
Subject(s)
Gene Transfer Techniques , Genetic Vectors , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Retroviridae/genetics , Base Sequence , Cytokines/pharmacology , Drug Resistance/genetics , Gentamicins/pharmacology , Humans , Molecular Sequence Data , Neoplastic Stem Cells/drug effects , Polymerase Chain Reaction , Time Factors , Tumor Cells, CulturedABSTRACT
If residual cancer cells in harvested bone marrow could be marked and subsequently detected in patients at relapse, valuable information would be obtained about the source of recurrent disease after autologous marrow transplantation. If normal progenitor cells were also marked, the study would provide useful data on the susceptibility of these human cells to gene transfer and their capacity to express newly introduced genes. We transferred the neomycin-resistance gene (NeoR) into bone marrow cells harvested from 20 children with acute myeloid leukemia (n = 12) or neuroblastoma (n = 8) in clinical and cytological remission using a retrovirus vector. The cells were then returned to the patients as part of an autologous bone marrow transplantation protocol. Two AML and three neuroblastoma patients have relapsed. In all, the resurgent cells contained the NeoR marker by analysis with PCR. These results prove that so-called remission marrow can contribute to relapse in patients who receive autologous transplants. The gene marking technique is now being used to evaluate techniques of pretransplant purging.
Subject(s)
Bone Marrow Transplantation/pathology , Clone Cells , Gene Transfer Techniques , Hematopoietic Stem Cells/cytology , Humans , Leukemia, Myeloid/diagnosis , Leukemia, Myeloid/pathology , Lymphocyte Subsets/pathology , Neuroblastoma/diagnosis , Neuroblastoma/pathology , Recurrence , Transplantation, AutologousSubject(s)
Biomarkers/analysis , Bone Marrow Purging , Bone Marrow Transplantation , Busulfan , Cyclophosphamide , Genetic Markers , Interleukin-2 , Leukemia, Myeloid/therapy , Mesna , Phosphotransferases (Alcohol Group Acceptor)/genetics , Acute Disease , Adolescent , Base Sequence , Cell Separation , Child , Child, Preschool , Clinical Protocols , Combined Modality Therapy , Drug Resistance , Evaluation Studies as Topic , Genetic Vectors , Hematopoietic Stem Cells , Humans , Immunologic Factors , Infant , Informed Consent , Kanamycin Kinase , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/genetics , Leukemia, Myeloid/pathology , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/analysis , Recombinant Fusion Proteins , Remission Induction , Research Design , Retroviridae/genetics , Safety , Transplantation, AutologousABSTRACT
Replication-defective retroviral vectors were used for ex vivo gene transfer into rat liver grafts under conditions mimicking clinical liver transplantation. Supernatant containing single- and double-gene vectors encoding for either the human IL-7 and/or neomycin phosphotransferase genes were used to perfuse the liver grafts during cold ischemia before transplantation. Whole liver grafts were perfused with vector supernatant or medium only. Reduced-size liver grafts (50% hepatectomy) were similarly perfused either immediately after reduction or 24 hr later after induction of active hepatocyte division. After transplantation of these grafts in orthotopic position, the liver tissue was removed at specified intervals, and genomic DNA and mRNA were examined for proviral sequences and expression. Stable integration of the proviral sequences was detected only in reduced-size grafts transplanted 24 hr after hepatectomy. Proviral message of both neomycin phosphotransferase and human IL-7 were present up to 21 days after transduction. This study demonstrates efficient ex vivo gene transfer to donor liver grafts. Gene transfer to livers before transplantation carries the potential to modulate immunogenicity and alter the antigraft immune response.
Subject(s)
Gene Transfer Techniques , Liver Transplantation/methods , Animals , Gene Expression , Genes , Genetic Vectors , Interleukin-7/genetics , Male , RNA, Messenger/genetics , Rats , Rats, Inbred Lew , RetroviridaeABSTRACT
Two human melanoma cell lines were transduced with the human interleukin (IL)-7 and IL-2 genes using retroviral-mediated gene transfer. Stable, high-level cytokine expression was achieved. The in vitro growth of transduced tumors was unaltered. Neither of the IL-2-transduced melanoma cell lines grew in athymic mice, whereas one IL-7-transduced melanoma line showed retarded in vivo growth. This is consistent with animal studies suggesting a predominantly T-cell response to IL-7-transduced tumors and a more nonspecific response to IL-2-transduced tumors. Both IL-7- and IL-2-transduced melanoma cell lines could induce cytotoxic lymphocytes in mixed lymphocyte-tumor cultures. The expression of putative melanoma antigens (MAGE)-1 and MAGE-3 was unaltered by cytokine transduction. In one cell line, IL-7 transduction resulted in a marked inhibition of the immunosuppressive peptide transforming growth factor (TGF)beta 1. The results allow a comparison of immunobiologic properties of IL-7- and IL-2-transduced human melanoma cell lines in consideration of their use in genetically engineered tumor vaccines. IL-7 transduction results in stable cytokine expression and phenotypic alterations that appear to be favorable for enhanced immunogenicity and it deserves clinical testing.
Subject(s)
Cytotoxicity, Immunologic , Interleukin-2/metabolism , Interleukin-7/metabolism , Melanoma/immunology , Melanoma/pathology , Animals , Antigens, Neoplasm , Base Sequence , Cell Division , Cell Line , DNA Primers , DNA, Viral/biosynthesis , Gene Transfer Techniques , Humans , Interleukin-2/biosynthesis , Interleukin-7/biosynthesis , Melanoma/metabolism , Melanoma-Specific Antigens , Mice , Mice, Nude , Molecular Sequence Data , Neoplasm Proteins/analysis , Neoplasm Proteins/biosynthesis , Neoplasm Transplantation , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Retroviridae/genetics , Transforming Growth Factor beta/analysis , Transforming Growth Factor beta/biosynthesis , Transplantation, Heterologous , Tumor Cells, Cultured , Virus IntegrationABSTRACT
The contribution of infused bone marrow cells to long-term haemopoietic recovery in patients undergoing autologous bone marrow transplantation is unknown. Such information would help to clarify the role of this procedure in cancer therapy and would aid in the development of strategies to reduce the risk of subsequent aplasia. By transferring a neomycin resistance marker gene into the marrow cells of 20 patients before transplantation, we were able to trace the pattern of haemopoietic reconstitution postinfusion. The marker gene was present and expressed in all haemopoietic lineages in vivo in 15 of 18 evaluable patients at 1 month post-transplantation, in 8 of 9 patients at 6 months, and in 5 of 5 at 1 year. The marker has remained detectable for up to 18 months--the duration of our study. Our findings indicate that harvested bone marrow consistently contributes to long-term multilineage recovery of haemopoiesis after autologous marrow transplantation in cancer patients. These results provide a rationale for the continued exploration of more ablative preparative regimens with single or sequential autologous marrow transplants.
Subject(s)
Bone Marrow Transplantation , Leukemia, Myeloid/therapy , Neuroblastoma/therapy , Adolescent , Adult , Child , Child, Preschool , Gene Amplification , Genetic Markers , Humans , Leukemia, Myeloid/genetics , Neuroblastoma/genetics , Polymerase Chain ReactionSubject(s)
Genetic Therapy , Genetic Vectors , Retroviridae/isolation & purification , Virus Replication , 3T3 Cells , Animals , Genes, Viral , Haplorhini , Humans , Leukemia, Experimental/microbiology , Lymph Nodes/microbiology , Lymphoma/microbiology , Mice , Moloney murine leukemia virus/genetics , Retroviridae/genetics , Retroviridae/pathogenicity , Retroviridae/physiologyABSTRACT
Bone marrow harvested for autologous bone-marrow transplantation may contain residual malignant cells even when it is judged to be in remission. Genetic marking and subsequent detection of these cells in recipients would give useful information about the origin of relapse after transplantation. We transferred the neomycin-resistance gene into bone-marrow cells harvested from children with acute myeloid leukaemia in remission. Two patients have relapsed since reinfusion of the marked cells. In both, the resurgent blast cells contained the neomycin-resistance gene marker; thus, remission marrow can contribute to disease recurrence. This method of tracking malignant cells should enable the development of better marrow purging strategies.
Subject(s)
Bone Marrow Transplantation , Genetic Markers , Leukemia, Myeloid, Acute/therapy , Bone Marrow Cells , Child , Drug Resistance, Microbial/genetics , Female , Humans , Immunophenotyping , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/pathology , Neomycin , Receptors, Immunologic , Recurrence , Transplantation, AutologousABSTRACT
Autologous bone marrow transplantation (ABMT) is widely used as treatment for malignant disease. Although the major cause of treatment failure is relapse, it is unknown if this arises entirely because of residual disease in the patient or whether contaminating cells in the rescuing marrow contribute. Attempts to purge marrow of its putative residual malignant cells may delay hematopoietic reconstitution and are of uncertain efficacy. We now describe how retrovirus-mediated gene transfer may be used to elucidate the source of relapse after ABMT for acute myeloid leukemia and to evaluate the efficacy of purging. Clonogenic myeloid leukemic blast cells in patient marrow can be transduced with the NeoR gene-containing helper-free retrovirus, LNL6, with an efficacy of 0% to 23.5% (mean, 10.5%). Transduced colonies grow in selective media and the presence of the marker gene can be confirmed in individual malignant colonies by polymerase chain reaction. If such malignant cells remain in harvested "remission" marrow, they will therefore be marked after exposure to LNL6. Detection of the marker gene in the malignant cells present at any later relapse would be firm evidence that residual disease contributed to disease recurrence, and would permit rapid subsequent evaluation of purging techniques. The technique also marks normal marrow progenitors from patients with acute myeloblastic leukemia. These colony-forming cells can be detected in long-term marrow cultures at a frequency of 1% to 18% for up to 10 weeks after exposure to the vector. Animal models and analysis of probability tables both suggest that these levels of marking in vitro are sufficient to provide information about the mechanisms of relapse and the biology of marrow regeneration in vivo. These preclinical data form part of the basis for current clinical studies of gene transfer into marrow before ABMT.
Subject(s)
Bone Marrow Transplantation , Bone Marrow/pathology , Gentamicins/pharmacology , Hematopoietic Stem Cells/pathology , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/surgery , Transfection , Cell Division/drug effects , Cells, Cultured , Colony-Forming Units Assay , Genetic Markers , Hematopoietic Stem Cells/drug effects , Humans , Kanamycin Kinase , Phosphotransferases/genetics , Probability , Recurrence , Retroviridae/genetics , Transplantation, AutologousABSTRACT
Disseminated neuroblastoma is a malignancy of children often treated by intensive chemotherapy/radiotherapy followed by autologous bone marrow transplantation (ABMT). A high proportion of those treated subsequently relapse. It is unknown if relapse is a consequence of residual disease in the patient or of contaminating malignant cells remaining in the infused marrow, which, of necessity, is harvested and stored prior to ablative chemotherapy/radiotherapy. The assumption that residual cells in the infused marrow contribute to relapse has lead to the adoption of marrow purging prior to reinfusion. However, neither the necessity nor the efficacy of the procedure have been established. We now show how retroviral-mediated gene transfer using the LNL6 vector may resolve this issue. Clonogenic neuroblastoma cells in patient marrow can be transduced and the NEOR gene detected by observing individual neuroblastoma cell colony growth in G418, and by polymerase chain reaction (PCR) of individual colonies. Efficiency of transduction is between 0 and 13.5%. If marrow is exposed to LNL6 prior to infusion and marked cells are detected at the time of relapse, this would demonstrate that infused marrow contributed to disease recurrence. The technique could then be used to analyze the efficacy of marrow purging techniques. Since normal progenitor cells from these patients are also marked, the technique can be used to study factors that modify reconstitution and transducibility of infused marrow. Clinical studies using this approach have now begun.
Subject(s)
Bone Marrow Transplantation , Neoplasm Recurrence, Local , Neuroblastoma/pathology , Retroviridae/genetics , Transfection , Base Sequence , DNA/analysis , Drug Resistance/genetics , Genetic Markers , Gentamicins/pharmacology , Humans , Molecular Sequence Data , Neuroblastoma/genetics , Neuroblastoma/surgery , Phenotype , Polymerase Chain Reaction , Transduction, Genetic , Transplantation, AutologousABSTRACT
Retroviral mediated gene transfer has proven to be an effective method of transferring genetic material into many different mammalian cells. Presented in this short review is a history of retroviral-mediated gene transfer as it relates to hematopoietic- and blood-derived cells. Gene transfer into mouse, primate, and fetal sheep and primate hematopoietic stem cells is discussed. Gene transfer into lymphocytes, in particular tumor-infiltrating lymphocytes, is also addressed. The use of such lymphocytes in ongoing and submitted clinical protocols utilizing retroviral-mediated gene transfer are presented.