ABSTRACT
Sphingolipids (SLs) are a class of essential, bioactive lipids. The SL family includes over 4000 distinct molecules, characterized by their sphingoid base (long-chain aliphatic amine) backbone. SLs are key components of cell membranes, yet their roles go well beyond structure. SLs are involved in many cellular processes including cell differentiation, apoptosis, growth arrest and senescence. As cancer cells routinely display increased growth properties and escape from cell death, it has been suggested that enzymes involved in SL synthesis or catabolism may be altered in cancer cells. In this review, we discuss the role of SL pathway enzymes in cancer, and in acquired resistance to therapy. The use of inhibitors and gene silencing approaches targeting these SL pathways is also explored. Finally, we elaborate on the role of SL pathway enzymes in the tumor microenvironment and their effect on immune cell function.
Subject(s)
Immunity , Neoplasms/immunology , Sphingolipids/metabolism , Animals , Apoptosis , Cell Cycle , Cell Differentiation , Drug Resistance, Neoplasm , Humans , Lipid Metabolism , Molecular Targeted Therapy , Signal TransductionABSTRACT
The purpose of the study was to examine the effect of lentivirus-mediated IL-10 gene therapy to target lung allograft rejection in a mouse orthotopic left lung transplantation model. IL-10 may regulate posttransplant immunity mediated by IL-17. Lentivirus-mediated trans-airway luciferase gene transfer to the donor lung resulted in persistent luciferase activity up to 6 months posttransplant in the isograft (B6 to B6); luciferase activity decreased in minor-mismatched allograft lungs (B10 to B6) in association with moderate rejection. Fully MHC-mismatched allograft transplantation (BALB/c to B6) resulted in severe rejection and complete loss of luciferase activity. In minor-mismatched allografts, IL-10-encoding lentivirus gene therapy reduced the acute rejection score compared with the lentivirus-luciferase control at posttransplant day 28 (3.0 ± 0.6 vs. 2.0 ± 0.6 (mean ± SD); p = 0.025; n = 6/group). IL-10 gene therapy also significantly reduced gene expression of IL-17, IL-23, and retinoic acid-related orphan receptor (ROR)-γt without affecting levels of IL-12 and interferon-γ (IFN-γ). Cells expressing IL-17 were dramatically reduced in the allograft lung. In conclusion, lentivirus-mediated IL-10 gene therapy significantly reduced expression of IL-17 and other associated genes in the transplanted allograft lung and attenuated posttransplant immune responses after orthotopic lung transplantation.
Subject(s)
Down-Regulation , Genetic Therapy/methods , Graft Rejection/prevention & control , Interleukin-10/therapeutic use , Interleukin-17/genetics , Lentivirus/genetics , Lung Transplantation , Animals , Disease Models, Animal , Female , Graft Rejection/genetics , Graft Rejection/metabolism , Graft Survival/genetics , Interleukin-10/genetics , Interleukin-17/biosynthesis , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , RNA/genetics , Real-Time Polymerase Chain Reaction , Transplantation, HomologousABSTRACT
Cell-fate control gene therapy (CFCGT)-based strategies can augment existing gene therapy and cell transplantation approaches by providing a safety element in the event of deleterious outcomes. Previously, we described a novel enzyme/prodrug combination for CFCGT. Here, we present results employing novel lentiviral constructs harboring sequences for truncated surface molecules (CD19 or low-affinity nerve growth factor receptor) directly fused to that CFCGT cDNA (TmpkF105Y). This confers an enforced one-to-one correlation between cell marking and eradication functions. In-vitro analysis demonstrated the full functionality of the fusion product. Next, low-dose 3'-azido-3'-deoxythymidine (AZT) administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced clonal K562 cells suppressed tumor growth; furthermore, one integrated vector on average was sufficient to mediate cytotoxicity. Further, in a murine xenogeneic leukemia-lymphoma model we also demonstrated in-vivo control over transduced Raji cells. Finally, in a proof-of-principle study to examine the utility of this cassette in combination with a therapeutic cDNA, we integrated this novel CFCGT fusion construct into a lentivector designed for treatment of Fabry disease. Transduction with this vector restored enzyme activity in Fabry cells and retained AZT sensitivity. In addition, human Fabry patient CD34(+) cells showed high transduction efficiencies and retained normal colony-generating capacity when compared with the non-transduced controls. These collective results demonstrated that this novel and broadly applicable fusion system may enhance general safety in gene- and cell-based therapies.
Subject(s)
Antigens, CD19/genetics , Nucleoside-Phosphate Kinase/genetics , Receptor, Nerve Growth Factor/genetics , Animals , Cell Death/drug effects , Cell Death/genetics , Cell Line, Tumor , Fabry Disease/genetics , Genetic Vectors , HEK293 Cells , Humans , Lentivirus/genetics , Leukemia-Lymphoma, Adult T-Cell/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Protein Engineering , Recombinant Fusion Proteins/genetics , Transformation, Genetic , Zidovudine/toxicityABSTRACT
Activity and specificity of chemotherapeutic agents against solid tumors can be augmented via the targeted or localized delivery of 'suicide' genes. Selective activation of specific prodrugs in cells expressing the 'suicide' gene drives their elimination by apoptosis, while also enabling the killing of adjacent bystander cells. Strong bystander effects can compensate for poor 'suicide' gene delivery, and depend on the prodrugs used and mechanisms for the acquisition of activated drug by the bystander population, such as the presence of gap junctional intercellular communications. Although a number of 'suicide' gene therapies for cancer have been developed and characterized, such as herpes simplex virus-derived thymidine kinase (HSV-tk)-based activation of ganciclovir, their limited success highlights the need for the development of more robust approaches. Limiting activation kinetics and evolution of chemoresistance are major obstacles. Here we describe 'suicide' gene therapy of cancer based on the lentivirus-mediated delivery of a thymidine-active human deoxycytidine kinase variant. This enzyme possesses substrate plasticity that enables it to activate a multitude of prodrugs, some with distinct mechanisms of action. We evaluated the magnitude and mechanisms of bystander effects induced by different prodrugs, and show that when used in combination, they can synergistically enhance the bystander effect while avoiding off-target toxicity.
Subject(s)
Astrocytoma/therapy , Deoxycytidine Kinase/biosynthesis , Genes, Transgenic, Suicide , Genetic Therapy/methods , Glioblastoma/therapy , Thymidine/pharmacology , Astrocytoma/enzymology , Astrocytoma/genetics , Cell Line, Tumor , Deoxycytidine Kinase/genetics , Gene Transfer Techniques , Genetic Engineering , Glioblastoma/enzymology , Glioblastoma/genetics , Humans , Thymidine Kinase/biosynthesis , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , TransfectionABSTRACT
Recent investigations have demonstrated that adenoviral and lentiviral vectors encoding HER-2 can be utilized in cancer immunotherapy. However, it is not known whether both viral systems elicit a similar immune response. Here, we compare the immune response in mice induced by dendritic cells (DCs) infected with either recombinant adenovirus or lentivirus encoding rat HER-2 (rHER-2). Both vaccine types yielded similar control of tumor growth, but we found clear differences in their immune responses 10 days after DC immunization. Adenovirus rHER-2-transduced DCs elicited locally and systemically high frequencies of CD4+ and CD8+ T cells, while lentivirus rHER-2-transduced DCs predominantly led to CD4+ T-cell infiltration at the tumor site. Splenocytes from mice immunized with lentivirus rHER-2-transduced DCs secreted higher levels of interferon (IFN)-γ, mainly by CD4+ T cells, following stimulation by RM-1-mHER-2 tumors. In contrast, the adenovirus vaccinated group exhibited CD4+ and CD8+ T cells that both contributed to IFN-γ production. Besides an established cellular immune response, the rHER-2/DC vaccine elicited a significant humoral response that was highest in the adenovirus group. DC subsets and regulatory T cells in the spleen were also differentially modulated in the two vaccine systems. Finally, adoptive transfer of splenocytes from both groups of immunized mice strongly inhibited in vivo tumor growth. Our results suggest that not only the target antigen but also the virus system may determine the nature and magnitude of antitumor immunity by DC vaccination.
Subject(s)
Adenoviridae , Dendritic Cells/transplantation , Genes, erbB-2/genetics , Genetic Vectors/administration & dosage , Immunotherapy/methods , Lentivirus , Neoplasms/therapy , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Interferon-gamma/metabolism , Mice , Neoplasms/immunology , RatsABSTRACT
Allogeneic transplantation of hematopoietic stem cells and lymphocytes is a curative treatment for malignant and non-malignant disease. However, the primary complication limiting the safety of transplantation is graft-versus-host disease (GvHD), which is mediated by donor T cells. Strategies for pre- and post-transplant manipulation of graft cells are not yet optimal for balancing GvHD severity with beneficial Graft-versus-Leukemia (GvL) effects. Emerging cell fate control gene therapy (CFCGT)-based strategies, such as 'suicide' gene therapy for donor T cell regulation, can supplement existing transplantation approaches by providing a safety element to reduce GvHD. Past uses of CFCGT in the clinic have provided proof-of-principle that GvHD can be controlled by such a strategy. However, there exists a need for improved transgene delivery and suicide control systems. Recently, lentiviral vectors (LVs) have emerged as effective gene delivery vehicles for the clinic. Combining lentiviral gene delivery with newer generations of 'suicide' systems that possess improved enzyme/prodrug specificities, activities, and reduced immunogenicity, could provide the necessary degree of control required to more successfully manage GvHD. Improving the safety of transplantation through successful CFCGT will serve to expand the potential donor pool and the spectrum of disorders that can be treated by this therapeutic schema.
Subject(s)
Genetic Vectors , Graft vs Host Disease/therapy , Lentivirus/genetics , Cell Lineage , Genetic Therapy , Graft vs Host Disease/prevention & control , Hematopoietic Stem Cell Transplantation , Humans , Transplantation, HomologousABSTRACT
Spermatogonial transplantation will provide a new way to study spermatogenesis in domestic animals, disseminate male genetics and produce transgenic animals, if efficiency can be improved. We evaluated a 'surgical' method for transplanting donor cells into testes of ram lambs, where the head of the epididymis is reflected, and a catheter introduced into the extra-testicular rete testis. We also tested transduction of ram spermatogonia with a lentiviral (LV) vector as a means to identify permanent colonization, and introduce genes into donor cells. Eight ram lambs, 11- to 13-week olds, were the recipients: in five, spermatogonia were injected into one testis, and the contralateral testis was an un-manipulated control: in two, spermatogonia were injected into one testis and the contralateral was sham-injected: in one, both testes were injected. Six lambs received spermatogonia labelled with a cell-tracking dye and these were collected 1 or 2 weeks after transplantation; three lambs received spermatogonia transduced with a LV vector driving the expression of enhanced Green Fluorescence Protein and these were collected after 2 months. Donor cells were detected by immunohistochemistry in tubules of seven of nine recipient testes. Approximately 22% of tubule cross-sections contained donor cells immediately after transplantation, and 0.2% contained virally transduced cells 2 months after transplantation. The onset of spermatogenesis was delayed, and there were lesions in both injected and sham-injected testes. Despite the effects of the surgery, elongated spermatids were present in one recipient testis 2 months after surgery. The results suggest that, after modifying the surgical and transduction techniques, this approach will be a means to produce good colonization by donor spermatogonia in sheep testes.
Subject(s)
Rete Testis , Sheep , Spermatogonia/metabolism , Spermatogonia/transplantation , Transduction, Genetic/veterinary , Animals , Cell Survival , Gene Expression , Genetic Vectors , Green Fluorescent Proteins/genetics , Immunohistochemistry/veterinary , Lentivirus/genetics , Lentivirus/growth & development , Male , Seminiferous Tubules/cytology , SpermatogenesisABSTRACT
Adoptively transferred T cells possess anticancer activities partially mediated by T-cell FasL engagement of Fas tumor targets. However, antigen-induced T-cell activation and clonal expansion, which stimulates FasL activity, is often inefficient in tumors. As a gene therapy approach to overcome this obstacle, we have created oncoretroviral vectors to overexpress FasL or non-cleavable FasL (ncFasL) on murine T cells of a diverse T-cell receptor repertoire. Expression of c-FLIP was also engineered to prevent apoptosis of transduced cells. Retroviral transduction of murine T lymphocytes has historically been problematic, and we describe optimized T-cell transduction protocols involving CD3/CD28 co-stimulation of T cells, transduction on ice using concentrated oncoretrovirus, and culture with IL-15. Genetically modified T cells home to established prostate cancer tumors in vivo. Co-stimulated T cells expressing FasL, ncFasL and ncFasL/c-FLIP each mediated cytotoxicity in vitro against RM-1 and LNCaP prostate cancer cells. To evaluate the compatibility of this approach with current prostate cancer therapies, we exposed RM-1, LNCaP, and TRAMP-C1 cells to radiation, mitoxantrone, or docetaxel. Fas and H-2(b) expression were upregulated by these methods. We have developed a novel FasL-based immuno-gene therapy for prostate cancer that warrants further investigation given the apparent constitutive and inducible Fas pathway expression in this malignancy.
Subject(s)
Fas Ligand Protein/metabolism , Immunotherapy/methods , Prostatic Neoplasms/therapy , Retroviridae/genetics , Transduction, Genetic , Animals , Antineoplastic Agents/pharmacology , Cell Line , Cell Line, Tumor , Cells, Cultured , Docetaxel , Fas Ligand Protein/immunology , Gene Expression Regulation/drug effects , Humans , Male , Mice , Mice, Inbred C57BL , Mitoxantrone/pharmacology , Phenotype , Prostatic Neoplasms/genetics , Prostatic Neoplasms/immunology , Retroviridae/immunology , T-Lymphocytes/immunology , Taxoids/pharmacologyABSTRACT
A deficiency in alpha-galactosidase A (alpha-gal A) activity causes Fabry disease. Virus-based delivery of genes can correct cells and establish a sustained supply of therapeutic proteins. Recombinant lentiviral vectors (LVs) show promise in this context. We first demonstrate LV-mediated marking of peripheral blood (PB) cells by transduction/transplantation of hematopoietic stem/progenitor cells. Stable enGFP expression was observed in PB for 37 weeks. Next, we transplanted Fabry mice with bone marrow mononuclear cells (BMMNCs) transduced a single time with a LV encoding the human alpha-gal A cDNA. Sustained expression of functional alpha-gal A in Fabry mice was observed over 24 weeks. Plasma alpha-gal A activity from treated Fabry mice was two-fold higher than wild-type controls. Increased alpha-gal A activity, often to supra-normal levels, and reduction of globotriaosylceramide, a glycolipid that accumulates in Fabry disease, was observed in all organs assessed. In secondary bone marrow transplantations, Fabry mice showed multilineage marking of PB, splenocytes and BMMNCs, along with therapeutic levels of alpha-gal A activity in plasma and organs over 20 weeks. Lastly, we transduced mobilized PB CD34(+) cells from a Fabry patient and observed corresponding enzymatic increases. Thus a single LV-mediated transduction of primitive hematopoietic cells can result in sustained correction for Fabry disease.
Subject(s)
Fabry Disease/therapy , Genetic Therapy/methods , Hematopoietic Stem Cells/enzymology , alpha-Galactosidase/genetics , Animals , B-Lymphocytes/enzymology , Fabry Disease/enzymology , Flow Cytometry , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Hematopoietic Stem Cell Transplantation , Humans , Lentivirus/genetics , Mice , Mice, Knockout , Models, Animal , Transduction, Genetic/methods , alpha-Galactosidase/metabolismABSTRACT
Sphingomyelinases hydrolyse sphingomyelin to ceramide, a process involved in signal-transduction routes leading to apoptosis and various other cellular responses. In the present study, we investigated the sphingomyelinase content of caveolae, invaginated plasma-membrane microdomains that contain a variety of signalling molecules. These structures are highly enriched in sphingomyelin as well as in ceramide, which suggests that metabolism of these lipids might, to some extent, occur locally. By cell fractionation, we demonstrate that, in addition to a previously reported minute amount of acidic sphingomyelinase activity, a substantial amount of neutral sphingomyelinase activity resides in caveolae of human skin fibroblasts. This caveolar neutral sphingomyelinase activity was also detected in Niemann-Pick disease type A fibroblasts, which are completely devoid of functional acidic sphingomyelinase. Neutral (but not acidic) sphingomyelinase activity was specifically inhibited by a peptide that corresponds to the scaffolding domain of caveolin, which suggests a direct molecular interaction between the two proteins. In addition, this finding implies a cytosolic orientation of the caveolar neutral sphingomyelinase. Interestingly, stimulation of fibroblasts with tumour necrosis factor alpha (TNFalpha) resulted in a partial shift of its p55 receptor to caveolin-enriched membrane fractions and the appearance of caveolin-sensitive neutral sphingomyelinase activity in the non-caveolar fractions. These results suggest that (part of) the presently identified caveolar neutral sphingomyelinase activity is involved in TNFalpha signalling.
Subject(s)
Caveolins/pharmacology , Signal Transduction/physiology , Sphingomyelin Phosphodiesterase/metabolism , Tumor Necrosis Factor-alpha/metabolism , Caveolae/metabolism , Caveolins/chemistry , Cells, Cultured , Cholesterol/deficiency , Cholesterol/metabolism , Enzyme Activation/drug effects , Humans , Niemann-Pick Diseases/pathology , Protein Structure, Tertiary , Sphingolipids/metabolism , Sphingomyelin Phosphodiesterase/antagonists & inhibitorsABSTRACT
Fabry disease is a lipid storage disorder resulting from mutations in the gene encoding the enzyme alpha-galactosidase A (alpha-gal A; EC ). We previously have demonstrated long-term alpha-gal A enzyme correction and lipid reduction mediated by therapeutic ex vivo transduction and transplantation of hematopoietic cells in a mouse model of Fabry disease. We now report marked improvement in the efficiency of this gene-therapy approach. For this study we used a novel bicistronic retroviral vector that engineers expression of both the therapeutic alpha-gal A gene and the human IL-2Ralpha chain (huCD25) gene as a selectable marker. Coexpression of huCD25 allowed selective immunoenrichment (preselection) of a variety of transduced human and murine cells, resulting in enhanced intracellular and secreted alpha-gal A enzyme activities. Of particular significance for clinical applicability, mobilized CD34(+) peripheral blood hematopoietic stem/progenitor cells from Fabry patients have low-background huCD25 expression and could be enriched effectively after ex vivo transduction, resulting in increased alpha-gal A activity. We evaluated effects of preselection in the mouse model of Fabry disease. Preselection of transduced Fabry mouse bone marrow cells elevated the level of multilineage gene-corrected hematopoietic cells in the circulation of transplanted animals and improved in vivo enzymatic activity levels in plasma and organs for more than 6 months after both primary and secondary transplantation. These studies demonstrate the potential of using a huCD25-based preselection strategy to enhance the clinical utility of ex vivo hematopoietic stem/progenitor cell gene therapy of Fabry disease and other disorders.
Subject(s)
Fabry Disease/therapy , Genetic Therapy/methods , Receptors, Interleukin-2/genetics , alpha-Galactosidase/genetics , 3T3 Cells , Animals , Bone Marrow Transplantation , Disease Models, Animal , Fabry Disease/metabolism , Fabry Disease/pathology , Gene Expression , Genetic Vectors , HeLa Cells , Humans , Mice , Mice, Inbred C57BL , Receptors, Interleukin-2/metabolism , Retroviridae/genetics , alpha-Galactosidase/metabolismABSTRACT
Fabry disease is an X-linked metabolic disorder caused by a deficiency of alpha-galactosidase A (alpha-Gal A). Lack of this lysosomal hydrolase results in the accumulation of galactose-terminal glycosphingolipids in a number of tissues, including vascular endothelial cells. Premature death is predominantly associated with vascular conditions of the heart, kidneys and brain. Historically, treatment has largely been palliative. Alternative treatments for many lysosomal storage diseases have been developed, including allogeneic organ and bone marrow transplantation, enzyme replacement therapy, and gene therapy. Significant clinical risks still exist with allogeneic transplantations. Alpha-Gal A enzyme replacement therapy has been implemented in clinical trials. This approach has been effective but may have limitations for long-term systemic or cost-effective correction. As an alternative, gene therapy approaches, involving a variety of gene delivery systems, have been pursued for the amelioration of Fabry disease. Fabry disease is a compelling disorder for gene therapy, as target cells are readily accessible and relatively low levels of enzyme correction may suffice to reduce storage. Importantly, metabolic cooperativity effects are also manifested in Fabry disease, wherein corrected cells secrete alpha-Gal A that can correct bystander cells. In addition, a broad therapeutic window probably exists, and mouse models of Fabry disease have been generated to assist studies. As an example, in vitro and in vivo studies using alpha-Gal A-transduced haematopoietic cells from Fabry mice have demonstrated enzymatic correction of recipient cells and dissemination of alpha-Gal A upon transplantation, leading to reduced lipid storage in a number of clinically relevant organs. This corrective enzymatic effect has recently been shown to be even further enhanced upon pre-selection of therapeutically transduced cells prior to transplantation. This review will briefly detail current gene delivery methods and summarize results to date in the context of gene therapy for Fabry disease.
Subject(s)
Fabry Disease/therapy , Genetic Therapy , Animals , Fabry Disease/genetics , Gene Transfer Techniques , Genetic Vectors , Hematopoietic Stem Cell Transplantation , Humans , Mice , Transduction, GeneticABSTRACT
Fabry disease is a compelling target for gene therapy as a treatment strategy. A deficiency in the lysosomal hydrolase alpha-galactosidase A (alpha-gal A; EC ) leads to impaired catabolism of alpha-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop vascular occlusions that cause cardiovascular, cerebrovascular, and renal disease. Unlike for some lysosomal storage disorders, there is limited primary nervous system involvement in Fabry disease. The enzyme defect can be corrected by gene transfer. Overexpression of alpha-gal A by transduced cells results in secretion of this enzyme. Secreted enzyme is available for uptake by nontransduced cells presumably by receptor-mediated endocytosis. Correction of bystander cells may occur locally or systemically after circulation of the enzyme in the blood. In this paper we report studies on long-term genetic correction in an alpha-gal A-deficient mouse model of Fabry disease. alpha-gal A-deficient bone marrow mononuclear cells (BMMCs) were transduced with a retrovirus encoding alpha-gal A and transplanted into sublethally and lethally irradiated alpha-gal A-deficient mice. alpha-gal A activity and Gb3 levels were analyzed in plasma, peripheral blood mononuclear cells, BMMCs, liver, spleen, heart, lung, kidney, and brain. Primary recipient animals were followed for up to 26 weeks. BMMCs were then transplanted into secondary recipients. Increased alpha-gal A activity and decreased Gb3 storage were observed in all recipient groups in all organs and tissues except the brain. These effects occurred even with a low percentage of transduced cells. The findings indicate that genetic correction of bone marrow cells derived from patients with Fabry disease may have utility for phenotypic correction of patients with this disorder.
Subject(s)
Bone Marrow Transplantation , Fabry Disease , Fabry Disease/therapy , Gene Transfer Techniques , Lipid Metabolism , alpha-Galactosidase/genetics , Animals , Bone Marrow Cells/metabolism , Fabry Disease/metabolism , Gene Expression , Mice , Mice, Knockout , Organ Specificity , Time FactorsABSTRACT
A major lipid-signaling pathway in mammalian cells implicates the generation of ceramide from the ubiquitous sphingolipid sphingomyelin (SM). Hydrolysis of SM by a sphingomyelinase present in acidic compartments has been reported to mediate, via the production of ceramide, the apoptotic cell death triggered by stress-inducing agents. In the present study, we investigated whether the ceramide formed within or accumulated in lysosomes indeed triggers apoptosis. A series of observations strongly suggests that ceramide involved in stress-induced apoptosis is not endolysosomal: 1) Although short-chain ceramides induced apoptosis, loading cells with natural ceramide through receptor-mediated endocytosis did not result in cell death. 2) Neither TNF-alpha nor anti-CD95 induced the degradation to ceramide of a natural SM that had been first introduced selectively into acidic compartments. 3) Stimulation of SV40-transformed fibroblasts by TNF-alpha or CD40 ligand resulted in apoptosis equally well in cells derived from control individuals and from patients affected with Farber disease, having a genetic defect of acid ceramidase activity leading to lysosomal accumulation of ceramide. Also, induction of apoptosis using anti-CD95 (Fas) or anti-CD40 antibodies, TNF-alpha, daunorubicin, and ionizing radiation was similar in control and Farber disease lymphoid cells. In all cases, apoptosis was preceded by a comparable increase of intracellular ceramide levels. 4) Retroviral-mediated gene transfer and overexpression of acid ceramidase in Farber fibroblasts, which led to complete metabolic correction of the ceramide catabolic defect, did not affect the cell response to TNF-alpha and CD40 ligand.
Subject(s)
Apoptosis/physiology , Ceramides/physiology , Lysosomes/metabolism , Stress, Physiological/pathology , Apoptosis/drug effects , Apoptosis/radiation effects , CD40 Antigens/pharmacology , Cell Line, Transformed , Cell Survival , Daunorubicin/pharmacology , Humans , Hydrogen-Ion Concentration , Hydrolysis , Lysosomal Storage Diseases/metabolism , Lysosomal Storage Diseases/pathology , Signal Transduction , Sphingomyelins/metabolism , Tumor Necrosis Factor-alpha/pharmacology , fas Receptor/pharmacologyABSTRACT
It has been slightly more than 10 years since the first proof-of-concept studies were performed, which demonstrated the feasibility of gene transfer into the heart and vasculature of experimental animals. Since that time there has been a dramatic increase in the nature and sophistication of gene transfer techniques and also in the number of cardiovascular diseases that are potential targets for gene-based therapies. In this article, the authors review the current strategies for gene delivery, including viral and nonviral approaches. The authors also highlight several biologic processes within the cardiovascular system, including restenosis, experimental angiogenesis, heart failure, and atherosclerosis-conditions for which gene therapy shows promise. It is hoped that this will provide an update of this therapeutic strategy for the year 2000.
Subject(s)
Cardiovascular Diseases/therapy , Gene Transfer Techniques , Genetic Therapy , Cardiovascular Diseases/genetics , Humans , In Vitro TechniquesABSTRACT
Fabry disease is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). We previously engineered a retrovirus encoding human alpha-gal A and demonstrated enzymatic correction of patient cells. Further, we demonstrated metabolic cooperativity, in that corrected cells secrete alpha-gal A that can be taken up and utilized by bystander cells in vitro. In the present study, we created a system to examine and quantitate this phenomenon in vivo. To differentiate from endogenous alpha-gal A, we constructed a retroviral vector (pUMFG/alpha-gal A/FLAG) containing a fusion form of alpha-gal A with a specific tag sequence at the carboxy terminus. The catalytic activity of the fusion protein was identical to wild-type alpha-gal A. The fusion protein was overexpressed in and secreted by transduced patient cells. In uptake studies, the fusion protein was detected in the lysosome-enriched fraction of recipient cells. We then examined the effectiveness of the pUMFG/alpha-g A/FLAG retroviral vector in vivo. Murine bone marrow (BM) cells were transduced and transplanted into irradiated hosts. After 9 weeks, proviral DNA was detected by PCR in peripheral blood and BM mononuclear cells. More importantly, specific fusion protein enzymatic activity could be demonstrated in those cells and in plasma. Thus, we have demonstrated that overexpressed alpha-gal A enters the circulation from transduced BM cells and is stable over a significant period of time.
Subject(s)
Bone Marrow Cells/enzymology , Fabry Disease/therapy , Genetic Therapy , Leukocytes, Mononuclear/enzymology , Transduction, Genetic , alpha-Galactosidase/genetics , Animals , Bone Marrow Transplantation , Cells, Cultured , DNA, Viral/analysis , Fibroblasts/metabolism , Genetic Vectors , Humans , Leukocytes, Mononuclear/virology , Male , Mice , Polymerase Chain Reaction , Recombinant Fusion Proteins/metabolism , Retroviridae/genetics , Skin/cytology , alpha-Galactosidase/metabolismABSTRACT
Fabry disease is a lysosomal storage disorder that is due to a deficiency in alpha-galactosidase A (alpha-gal A). Previously we have shown that a recombinant retrovirus synthesized for the transfer of the human alpha-gal A coding sequence was able to engineer enzymatic correction of the hydrolase deficiency in fibroblasts and lymphoblasts from Fabry patients. The corrected cells secreted alpha-gal A that was taken up and utilized by uncorrected bystander cells, thus demonstrating metabolic cooperativity. In separate experiments we used transduced murine bone marrow cells and successfully tested and quantitated this phenomenon in vivo. In the present studies, which were designed to bring this therapeutic approach closer to clinical utility, we establish that cells originating from the bone marrow of numerous Fabry patients and normal volunteers can be effectively transduced and that these target cells demonstrate metabolic cooperativity. Both isolated CD34+-enriched cells and long-term bone marrow culture cells, including nonadherent hematopoietic cells and adherent stromal cells, were transduced. The transferred gene generates increased intracellular alpha-gal A enzyme activity in these cells. Further, it causes functional correction of lipid accumulation and provides for long-term alpha-gal A secretion. Collectively, these results indicate that a multifaceted gene transfer approach to bone marrow cells may be of therapeutic benefit for patients with Fabry disease.
Subject(s)
Bone Marrow Cells/enzymology , Fabry Disease/pathology , Hematopoietic Stem Cells/enzymology , alpha-Galactosidase/metabolism , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Cell Adhesion , Cells, Cultured , DNA, Viral/analysis , Fabry Disease/enzymology , Fabry Disease/genetics , Fabry Disease/therapy , Genetic Therapy , Genetic Vectors/genetics , Glycosphingolipids/metabolism , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Humans , Lysosomes/enzymology , Mice , Proviruses/isolation & purification , Retroviridae/genetics , Retroviridae/isolation & purification , Stromal Cells/enzymology , Stromal Cells/metabolism , Stromal Cells/pathology , Transfection , alpha-Galactosidase/geneticsABSTRACT
The achievement of positive outcomes in many clinical protocols involving hematopoietic stem cells (HSCs) has been handicapped by the limited numbers of marrow repopulating cells available to actually bring about therapy. This insufficiency has been especially problematic in stem cell transplantation and gene therapy. A number of studies have been initiated to attempt expansion of HSCs, mainly by manipulation of key cytokines in cell suspension cultures. Unfortunately, these expansion methods usually lead to altered properties in the amplified cells, mainly by reducing their self-renewal and multi-lineage differentiative potentials. Here we discuss our ongoing work, utilizing a unique endothelial cell line that supports primitive hematopoiesis, to attempt to generate expansion of primate HSCs that retain their elementary properties. Genetic marking of early hematopoietic cells to facilitate tracking will be mentioned as will the development and employment of assay systems designed to evaluate the long-term functional attributes of the expanded cells.
Subject(s)
Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Genetic Therapy , Hematopoiesis , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Animals , Cells, Cultured , Coculture Techniques , Genetic Markers , Genetic Vectors , Hematopoietic Stem Cells/physiology , Humans , Papio , Retroviridae/geneticsABSTRACT
Farber disease is a rare severe lysosomal storage disorder due to a deficient activity of the enzyme acid ceramidase (AC). Patients have granulomas along with lipid-laden macrophages that accumulate in a number of tissues, leading to multiple diverse clinical symptoms. There is no therapy for the disorder and most patients succumb to the disease in early childhood. The severity of the disease progression seems to correlate with the amount of the accumulated ceramide substrate. Since the cDNA for human AC has been elucidated we sought to establish if genetic transfer of this sequence would lead to enzymatic and, especially, functional correction of the catabolic defect in Farber patient cells. To do this, a novel amphotropic recombinant retrovirus was constructed that engineers transfer of the human AC cDNA. On infection of patient fibroblasts, AC enzyme activity in cell extracts was completely restored. Further, substrate-loading assays of intact living cells showed a fully normalized catabolism of lysosomal ceramide. Lastly, as reported for some other corrected enzymatic defects of lysosomes, metabolic cooperativity was seen, in that functionally corrected patient fibroblasts secreted AC that was taken up through the mannose 6-phosphate receptor and used by uncorrected fibroblasts as well as recipient Farber lymphoblastoid cells. This overall transduction and uptake scenario for Farber disease allows future treatment of this severe disorder to be envisioned using gene transfer approaches.
Subject(s)
Amidohydrolases/genetics , Gene Transfer Techniques , Genetic Vectors , Lysosomal Storage Diseases , Retroviridae , Acid Ceramidase , Amidohydrolases/metabolism , Cell Line, Transformed , Cells, Cultured , Ceramidases , DNA, Complementary , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , Genetic Engineering , Humans , Lysosomal Storage Diseases/therapy , Recombination, GeneticABSTRACT
The chimeric receptor, RARalpha/VDR, contains the DNA-binding domain of the retinoic acid receptor (RARalpha) and the ligand-binding domain of the vitamin D receptor (VDR). The ligand-binding properties of RARalpha/VDR are equivalent to that of VDR, with an observed Kd for 1alpha,25 dihydroxy-vitamin D3 (D3) of 0.5 nM. In CV-1 cells, both RARalpha and RARalpha/VDR induce comparable levels of ligand-mediated transcriptional activity from the retinoic acid responsive reporter gene, beta(RARE)3-TK-luciferase, in the presence of the ligand predicted from the receptor ligand-binding domain. Two chimeric RAR receptors were constructed which contained the ligand-binding domain of the estrogen receptor (ER): RARalpha/ER and ER/RARalpha/ER. Both RARalpha/ER and ER/RARalpha/ER bind beta-estradiol with high affinity, and are transcriptionally active only from palindromic RAREs (TREpal and/or (TRE3)3). Only RARalpha/VDR matched in kind and degree the functional characteristics of RARalpha: (1) maximally active from the beta(RARE); (2) moderately active from the TREs; (3) inactive from the retinoic X receptor response elements (RXREs) ApoA1 and CRBP II; (4) forms heterodimers with RXRalpha; and (5) binds to the betaRARE. F9 embryonal carcinoma cell lines were generated which express RARalpha/VDR mRNA (F9RARalpha/VDR cells) and compared with F9 wild-type (F9-Wt) cells, which do not express VDR mRNA. Treatment with all-trans retinoic acid (tRA) inhibits cell growth and induces the differentiation morphology in both F9-Wt and F9-RARalpha/VDR cells; whereas, treatment with D3 is similarly effective only for F9-RARalpha/VDR cells. It is concluded RARalpha/VDR is an useful 'tool' to pinpoint, or to augment transcription from RAREs in gene pathways controlled by RAR without inhibiting the retinoid responsiveness of endogenous RARs.