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21.
Sangiolo D Lesnikova M Nash RA Jensen MC Nikitine A Kiem HP Georges GE 《Gene therapy》2007,14(21):1549-1554
Several clinical studies of gene-modified T cells have shown limited in vivo function of the cells, immunogenicity of the transgene, and lack of a selective advantage for gene-modified T cells. To address these problems, we developed a lentiviral vector (LV) that provides a selectable, proliferative advantage and potentially decreases immunogenicity for transduced T cells. The bicistronic vector expressed two genes linked with an internal ribosomal entry site. One gene is a variant of the inosine monophosphate dehydrogenase 2, inosine monophosphate dehydrogenase (IMPDH(IY)), conferring resistance to the immunosuppressive drug mycophenolate mofetil (MMF). The other is a suicide gene, herpes simplex virus thymidine kinase (HSV-TK), rendering proliferating cells sensitive to ablation with ganciclovir, fused to the selectable transmembrane marker DeltaCD34 (DeltaCD34/TK). Cells transduced with LV-DeltaCD34/TK.IMPDH(IY) were efficiently enriched by immunomagnetic selection for CD34, proliferated in 0.5-5 microM MMF, and were killed by 0.5-25 microg ml(-1) ganciclovir. We demonstrate efficient selection and killing of gene-modified cells and suggest LV-DeltaCD34/TK.IMPDH(IY)-transduced T cells could be used to facilitate allogeneic hematopoietic cell engraftment. The expression of IMPDH(IY) would allow in vivo selection with MMF, and DeltaCD34/TK expression would allow rapid and safe elimination of transduced T cells if graft-versus-host disease developed. 相似文献
22.
Most of the current hematopoietic stem cell (HSC) -directed gene therapy applications have focused on the replacement of defective or deficient genes in an autologous setting. More recently HSC gene therapy applications have also included the enhancement or improvement of HSC features. Allogeneic HSCs have been used to facilitate and improve allogeneic transplantation and to achieve tolerance to transplanted cells, tissues or organs. Different gene transfer approaches addressing a variety of immunomodulatory mediators contributing to graft tolerance or immunological ignorance may have a critical role in improving long-term graft survival. Allogeneic tissues are frequently recognized by allospecific T cells as foreign and are rapidly rejected in the absence of immunosuppression. The higher susceptibility to cancer and infectious diseases of immunosuppressed patients led to investigation of new therapies to induce graft-specific tolerance. Peripheral tolerance to allogeneic grafts can be achieved by a variety of mechanisms including clonal deletion, suppression caused by regulatory T cells and anergy induction associated with microchimerism effect. In the last decades, potential candidates to confer allograft protection were identified. In this review, we summarize ongoing strategies and developments in genetic manipulation of cells, tissues and organs for allogeneic transplantation including modulating the effector arm of the immune response. 相似文献
23.
Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity 总被引:55,自引:0,他引:55
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I G Schmidt-Wolf R S Negrin H P Kiem K G Blume I L Weissman 《The Journal of experimental medicine》1991,174(1):139-149
C.B-17 severe combined immune deficient (SCID) mice, which lack functional B and T lymphocytes, allow xenografts and, therefore, can be used to study the biology of human malignancies. Two different human B cell lymphoma cell lines, SU-DHL-4 and OCI-Ly8, which both harbor the t(14;18) chromosomal translocation, were injected into C.B-17 SCID mice. Mice injected intravenously or intraperitoneally developed tumors and died in a dose-dependent manner. The presence of tumor cells in various murine tissues could be demonstrated by a clonogenic tumor assay, staining of frozen sections with a monoclonal antibody (mAb) against a human B cell antigen (CD19), and with the polymerase chain reaction technique. A protocol using cytotoxic effector cells was developed and used to selectively deplete the tumor cells from bone marrow. These cells were developed by growing peripheral blood mononuclear cells in the presence of interferon gamma (IFN-gamma), anti-CD3 mAb, and interleukin 2 (IL-2). The timing of IFN-gamma treatment was critical and optimal if IFN-gamma was added before IL-2 treatment. The cells that were stimulated by IFN-gamma, followed by IL-2, could be expanded by treatment with a mAb directed against CD3. These cells could be further activated by IL-1, but not by tumor necrosis factor alpha. With this protocol, a tumor cell kill of 3 logs was obtained as measured by a clonogenic assay. Interestingly, despite their high cytotoxic activity against lymphoma cells, these cells had little toxicity against a subset of normal human hematopoietic precursor cells (granulocyte/macrophage colony-forming units). These cells were further tested by treating murine bone marrow contaminated with the human lymphoma cell line SU-DHL-4, and injecting these cells into SCID mice to assay for tumor growth in vivo. The animals injected with bone marrow contaminated with SU-DHL-4 cells had enhanced survival if the bone marrow was treated with the cytokine-induced killer cells before infusion. The SCID mouse provides a useful in vivo model for evaluation of new therapeutic approaches for lymphoma treatment. The cytokine-induced killer cells generated as described here could have an important impact on bone marrow purging for autologous bone marrow transplantation as well as for adoptive immunotherapy. 相似文献
24.
The optimal stem cell source for stem cell gene therapy has yet to be determined. Most large-animal studies have utilized peripheral blood or marrow-derived cells collected after administration of granulocyte colony-stimulating factor (G-SCF) and stem cell factor (SCF); however, SCF is unavailable for clinical use in the United States and the European Union. A recent study in a competitive repopulation assay in the rhesus macaque showed very inefficient marking of G-CSF-mobilized (G/only) peripheral blood (G-PBSC) CD34(+) cells relative to G-CSF and SCF-mobilized cells using vectors with an amphotropic pseudotype. Because G-PBSC would be the preferred target cell population for most clinical stem cell gene therapy applications, we asked whether we could achieve efficient transduction and engraftment of G-PBSC using Phoenix-GALV-pseudotyped vectors. We transplanted three baboons with G/only mobilized CD34(+) cells transduced with GALV-pseudotyped retroviral vectors. We observed high-level, persistent engraftment of gene-modified G-PBSC in all animals with gene marking levels in granulocytes up to 60%. We analyzed amphotropic (PIT2) and GALV (PIT1) receptor expression in G/only cells and found preferential expression of PIT1 after G/only, which may explain the inferior results with amphotropic pseudotypes. These findings demonstrate that high stem cell gene transfer levels can be achieved using G-CSF-mobilized PBSC with Phoenix-GALV-pseudotyped vectors. 相似文献
25.
Seal S Hockenbery DM Spaulding EY Kiem HP Abbassi N Deeg HJ 《Experimental hematology》2008,36(12):1660-1672
OBJECTIVE: Clonal marrow cells from patients with early myelodysplastic syndrome (MDS) undergo apoptosis in response to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Cells from advanced MDS are resistant to TRAIL. Two isoforms of the Flice inhibitory protein (FLIP) short (FLIPS) and FLIP long (FLIPL), which modulate TRAIL signals, showed disease-stage-dependent differential regulation. Therefore, we aimed at characterizing potential differential effects of FLIPL and FLIPS, on TRAIL and TNF-alpha-induced apoptosis in model leukemic cell lines. MATERIALS AND METHODS: Using lentiviral constructs, FLIPL and FLIPS, as well as a green fluorescent protein control were overexpressed in ML-1 cells, which constitutively express very low levels of FLIP and are highly sensitive to apoptosis induction. Cells were then exposed to TRAIL or TNF-alpha, and effects on the extrinsic and intrinsic pathways of apoptosis induction were assessed. RESULTS: Overexpression of FLIP reduced TRAIL and TNF-alpha-induced apoptosis in ML-1 cells. However, while FLIPL completely abrogated apoptosis, FLIPS allowed for BID cleavage and caspase-3 activation. Concurrently, there was a decline of Bcl-xL and X-linked inhibitor of apoptosis protein (XIAP) in FLIPS cells followed by apoptosis. Further, inhibition of nuclear factor-kappaB (NF-kappaB) activation in TNF-alpha-treated cells resulted in profound apoptosis in FLIPS, but not in FLIPL-overexpressing cells, consistent with the observations in patients with early stage MDS. Inhibition of NF-kappaB had only minimal effects on TRAIL signaling. CONCLUSION: Thus, FLIPL and FLIPS exerted differential effects in myeloid leukemic cell lines in response to TRAIL and TNF-alpha. It might be possible to therapeutically exploit those differences with effector molecules specific for the FLIP isoforms. 相似文献
26.
Foamy virus (FV) vectors have shown great promise for hematopoietic stem cell (HSC) gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit. 相似文献
27.
The cure of a human immunodeficiency virus (HIV)-1–infected patient following allogeneic transplantation from a CCR5-null donor and potential cure of two patients transplanted with CCR5 wild-type hematopoietic stem cells (HSC) have provided renewed optimism that a potential alternative to conventional antiretroviral therapy (ART) is forthcoming. While allogeneic grafts have thus far suggested complete eradication of viral reservoirs, it has yet to be observed following autologous HSC transplantation. Development of curative autologous transplantation strategies would significantly increase the number of treatable patients, eliminating the need for matched donors and reducing the risks of adverse events. Recent studies suggest gene therapy may provide a mechanism for developing curative therapies. Expression of cellular/artificial restriction factors or disruption of CCR5 has been shown to limit viral replication and provide protection of genetically modified cells. However, significant obstacles remain with regards to the depletion of established viral reservoirs in an autologous transplantation setting devoid of the “allo-effect”. Here, we discuss results from early-stage clinical trials and recent findings in animal models of gene modified HSC transplantation. Finally, we propose innovative combination therapies that may aid in the reduction and/or elimination of viral reservoirs in HIV-1–infected patients and promote the artificial development of a natural controller phenotype.Gene therapy approaches have recently come to the forefront of alternative methods in the quest for the development of curative strategies for the treatment of human immunodeficiency virus (HIV)-1–infected patients. Nearly all of these studies have been aimed at developing infection-resistant immune cell populations highlighting the findings published by Hütter et al., in which a single HIV-1–infected patient, known as the “Berlin patient”, was effectively cured of HIV following allogeneic hematopoietic stem cell transplantation (HSCT) from a CCR5-null donor.1 While several questions remain concerning what aspects of treatment or if multiple interventions led to the cure of this patient, a new role for gene therapy in the pursuit of a cure for HIV/acquired immunodeficiency syndrome has emerged.Although current antiretroviral therapy (ART) is perhaps one of the most vital breakthroughs in the modern era of medicine, this therapy does not equate a cure. Strategies to genetically modify target cells have evolved over the last two decades; however, for the most part, the underlying theme of preventing the expression of cellular factors needed for viral infection and/or the expression of restrictive factors has remained relatively unchanged (see ref. 2 for review of previously characterized methods for developing infection-resistant cells). Few approaches aimed at targeting proviral DNA have been developed; however, recent breakthroughs in genetic engineering of sequence specific nucleases may pave the way for future targeted therapies aimed at reducing viral reservoirs (reviewed in ref. 3). This review discusses the early-stage clinical findings, the current progress made in preclinical animal models, the ambitious goals needed to achieve a functional cure with relation to findings in elite controllers, and the innovative therapies that may be needed to eliminate or reduce viral reservoirs in combination with autologous, gene modified HSCT. Here, a “functional cure” is defined as conditions that lead to the complete and sustainable control of plasma viremia at levels that are currently achievable by conventional highly active ART (HAART). We refer to “eradication” as a sterilizing cure in which no replication competent forms of HIV exist. 相似文献
28.
Robert D. Okada David K. Glover Kiem N. Nguyen Gerald Johnson III 《European journal of nuclear medicine and molecular imaging》1995,22(7):600-607
The purpose of the current study was to clarify the myocardial kinetics of technetium-99m sestamibi when the latter is administered during reperfusion. Sestamibi has in the past been given to patients following thrombolytic therapy to document reperfusion and assess salvage. However, the factors which affect sestamibi kinetics during reperfusion are not clearly defined. In this study the left circumflex coronary artery was occluded for 2 h in six dogs (group 1) and for 3 h in six dogs (group 2), followed by reperfusion. Five additional dogs were not reperfused (group 3). Sestamibi was administered during reperfusion in groups 1 and 2, and during ongoing occlusion in group 3. Regional myocardial sestamibi activity was monitored for 3 h using miniature implanted radiation detectors and gamma camera imaging. Group 1 dogs had no infarcts, group 2 had moderate infarcts (mean: 13.9%), and group 3 had large infarcts (mean: 25.2%). Three-hour fractional myocardial clearances were significantly greater for reperfused infarcted (group 2) (0.23±0.02 SEM) and for nonreperfused infarcted myocardium (group 3) (0.24±0.02) compared to control (0.10±0.01) and reperfused non-infarcted myocardium (group 1) (0.07±0.02;P<0.01). Quantitative image analysis demonstrated a significant reduction in the left circumflex/left anterior descending count ratios from initial to final scans for group 2 (0.74±0.03 to 0.65±0.03,P<0.05), and a trend towards a reduction in the count ratios from initial to final scans for group 3 (0.38±0.04 to 0.30 ±0.04;P = 0.06). Thus, probe-derived myocardial sestamibi kinetics following reperfusion do differentiate non-infarcted from infarcted myocardium. Although the detection of accelerated clearance can be demonstrated by quantitative analysis of gamma camera images in dogs, this may be technically difficult in clinical situations. 相似文献
29.
30.
MS Thakar C Bonfim BM Sandmaier P O'Donnell L Ribeiro T Gooley HJ Deeg ME Flowers R Pasquini R Storb AE Woolfrey HP Kiem 《Pediatric hematology and oncology》2012,29(6):568-578
Allogeneic hematopoietic cell transplantation (HCT) is the only known cure for patients with Fanconi anemia (FA) who develop aplasia or leukemia. However, transplant regimens typically contain high-dose alkylators, which are poorly tolerated in FA patients. Furthermore, as many patients lack human leukocyte antigen (HLA)-matched family donors, alternative donors are used, which can increase the risk of both graft rejection and graft-versus-host disease (GVHD). To improve on these three concerns, we developed a multi-institutional clinical trial using a fludarabine (FLU)-based conditioning regimen with limited alkylators/low-dose radiation, HLA-haploidentical marrow, followed by reduced-dose cyclophosphamide (CY) to treat three FA patients with aplasia. All three patients engrafted with 100% donor CD3 chimerism at 1 month. One patient died early from disseminated toxoplasmosis infection. Of the two survivors, one had significant pretransplant co-morbidities and inadequate immunosuppression, and developed severe acute GVHD. The other patient had only mild acute and no chronic GVHD. With a follow-up of 2 and 3?years, respectively, both patients are doing well, are transfusion-independent, and maintain full donor chimerism. The patient with severe GVHD has resolving oral GVHD and good quality of life. We conclude that using low-intensity conditioning, HLA-haploidentical marrow, and reduced-dose CY for in vivo T-cell depletion can correct life-threatening aplasia in FA patients. 相似文献