Robert C. Moen

Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.

BACKGROUND AND METHODS Treatment with tumor-infiltrating lymphocytes (TIL) plus interleukin-2 can mediate the regression of metastatic melanoma in approximately half of patients. To optimize this treatment approach and define the in vivo distribution and survival of TIL, we used retroviral-mediated gene transduction to introduce the gene coding for resistance to neomycin into human TIL before their infusion into patients--thus using the new gene as a marker for the infused cells. RESULTS Five patients received the gene-modified TIL. All the patients tolerated the treatment well, and no side effects due to the gene transduction were noted. The presence and expression of the neomycin-resistance gene were demonstrated in TIL from all the patients with Southern blot analysis and enzymatic assay for the neomycin phosphotransferase coded by the bacterial gene. Cells from four of the five patients grew successfully in high concentrations of G418, a neomycin analogue otherwise toxic to eukaryotic cells. With polymerase-chain-reaction analysis, gene-modified cells were consistently found in the circulation of all five patients for three weeks and for as long as two months in two patients. Cells were recovered from tumor deposits as much as 64 days after cell administration. The procedure was safe according to all criteria, including the absence of infections virus in TIL and in the patients. CONCLUSIONS These studies demonstrate the feasibility and safety of using retroviral gene transduction for human gene therapy and have implications for the design of TIL with improved antitumor potency, as well as for the possible use of lymphocytes for the gene therapy of other diseases.

Gene-marking to trace origin of relapse after autologous bone-marrow transplantation

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.

Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients

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.

Gene marking and autologous bone marrow transplantation.

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.

Development of a clinical protocol for hepatic gene transfer: Lessons learned in preclinical studies

ABSTRACT: Strategies for hepatic gene therapy have been proposed that involve isolation of primary hepatocytes and introduction of recombinant genes into these cells in culture, followed by autologous hepatocellular transplantation (HCT). Consideration of clinical applications requires data suggesting that HCT can be performed safely in human subjects in addition to data indicating that recombinant gene expression can reverse a disease process. This report describes preclinical studies that underlie a clinical trial of HCT in which hepatocytes would be labeled with a marker gene to facilitate assessment of engraftment in the recipient. Human hepatocytes were harvested from liver segments preserved in Belzars solution and transduced with an amphotropic retroviral vector carrying a recombinant marker gene (neomycin phosphotransferase II). Human hepatocytes were recovered from monolayer culture, stained with the fluorescent dye 1,1′-dioctadecyl-3,3,3,3′-tetra-methylindo-carbocyanine perchlorate (DiI) and transplanted into severe combined immunodeficient mice by splenic injection. Engrafted hepatocytes were identified in the liver and spleen of severe combined immunodeficient mice but not immunocompetent controls. Two large animal models of HCT are described. In a dog model, neomycin phosphotransferase II-containing hepatocytes were identified in the liver 7 wk after transplantation. In a baboon model, autologous HCT with DiI-stained cells demonstrated that transplanted cells assume a normal morphology and constitute up to 5% of hepatocytes. These data demonstrate transduction and transplantation of human hepatocytes and the feasibility of HCT in large animals. On the basis of these studies, the proposed clinical trial for gene transfer and transplantation in human subjects has been approved by the National Institutes of Health and the Food and Drug Administration. These studies illustrate the nature and extent of preclinical data required to gain approval for clinical trials involving gene transfer into human subjects. These data also illustrate the limitations of existing methods that may be used for hepatic gene therapy in the future.

EVALUATION OF RETROVIRAL MEDIATED GENE TRANSFER BY IN SITU HYBRIDIZATION

Genetically engineered retroviral vectors can be used to transfer genes into eucaryotic cells. The application of these vectors for human gene therapy is being evaluated in vitro and in animal models. The time course of vector mediated gene transfer into the human erythroleukemia cell line, K562, was followed by in situhybridization (ISH) to determine if this technique could be used to follow retroviral mediated gene transfer. The initial binding of vector to K562 cells was readily detected. Little vector RNA was detected during particle uptake, conversion of the vector RNA to dsDNA, and integration of the dsDNA into the host genome. However, by 18h after vector binding cytoplasmic RNA from the transferred gene was readily detected with steady state levels reached by 48h, demonstrating the ability of ISH to follow vector mediated gene transfer. In an autologous monkey bone marrow transplant model attempts to transfer a human adenosine deaminase (hADA) gene has resulted in hADA activity 0.5% that of endogenous monkey ADA. To determine if this represents a low level of activity in a large number of cells or the converse, ISH was used to show that 6 of 673 peripheral blood mononuclear cells contained retroviral RNA, implying an appropriate level of hADA gene expression in those cells infected by the vector. Similar percentages of gene transfer are seen in in vitro studies with primate and human bone marrow colony forming assays. The ability of ISH to detect expression of transferred genes in single cells provides a powerful technique for the refinement of methods for human gene therapy.

1008 THEORETICAL AND BIOLOGICAL CONSIDERATIONS IN SUCCESSFUL MISMATCHED BONE MARROW TRANSPLANTATION

Six children with severe combined immunodeficiency disease have received bone marrow transplants from a haplotype mismatched parent. The marrow was treated by monoclonal antibody and complement prior to infusion. Complicating factors involved the presence of prior maternal graft versus host in 1, prior thymus transplantation in 1 and presence of sufficient native immunity to require ablation in one. Two children died without engraftment. The survivors have B and T cell engraftment as demonstrated by normal in vitro T cell proliferative responses, normal immunoglobul in and/or functional antibody levels except one of the patients appears to have IgA deficiency. The longest period of follow up is 20 months and the shortest is three months. In 1 patient, thymus biopsy after transplant confirmed a normal distribution of cells and the presence of a marker for dendritic cells. This marker was not present in a patient who was not successfully engrafted.These data show: 1. Successful engraftment can be accomplished with haplotype mismatched marrow even in the face of maternal graft versus host disease. 2. Haplotype mismatched marrow can home to the host thymus and be appropriately differentiated. 3. Criteria for prior ablative therapy need to be established; thymus immunohistochemistry may be helpful in this regard. 4. Mechanisms of the immune response can be inferred from the nature of the reconstitution.