Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells

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.     

Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.

Fabry disease is an X-linked metabolic disorder due to a deficiency of alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). Patients accumulate glycosphingolipids with terminal alpha-galactosyl residues that come from intracellular synthesis, circulating metabolites, or from the biodegradation Of senescent cells. Patients eventually succumb to renal, cardio-, or cerebrovascular disease. No specific therapy exists. One possible approach to ameliorating this disorder is to target corrective gene transfer therapy to circulating hematopoietic cells. Toward this end, an amphotropic virus-producer cell line has been developed that produces a high titer (>10(6) i.p. per ml) recombinant retrovirus constructed to transduce and correct target cells. Virus-producer cells also demonstrate expression of large amounts of both intracellular and secreted alpha-gal A. To examine the utility of this therapeutic vector, skin fibroblasts from Fabry patients were corrected for the metabolic defect by infection with this recombinant virus and secreted enzyme was observed. Furthermore, the secreted enzyme was found to be taken up by uncorrected cells in a mannose-6-phosphate receptor-dependent manner. In related experiments, immortalized B cell lines from Fabry patients, created as a hematologic delivery test system, were transduced. As with the fibroblasts, transduced patient B cell lines demonstrated both endogenous enzyme correction and a small amount of secretion together with uptake by uncorrected cells. These studies demonstrate that endogenous metabolic correction in transduced cells, combined with secretion, may provide a continuous source of corrective material in trans to unmodified patient bystander cells (metabolic cooperativity).     

Preselective gene therapy for Fabry disease

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.     

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzyme deficiency leads to impaired catabolism of alpha-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal alpha-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human alpha-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, alpha-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20-35% of that of the normal mice. The transduced animals continued to show higher alpha-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or alpha-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.     

Gene therapy for Fabry disease

Fabry disease is an X-linked metabolic disorder caused by a deficiency ofα-galactosidase A (α-Gal A). Lack of this lysosomal hydrolase results in theaccumulation of galactose-terminal glycosphingolipids in a number of tissues,including vascular endothelial cells. Premature death is predominantly associated withvascular conditions of the heart, kidneys and brain. Historically, treatment has largelybeen palliative. Alternative treatments for many lysosomal storage diseases have beendeveloped, including allogeneic organ and bone marrow transplantation, enzymereplacement therapy, and gene therapy. Significant clinical risks still exist withallogeneic transplantations. α-Gal A enzyme replacement therapy has beenimplemented in clinical trials. This approach has been effective but may havelimitations for long-term systemic or cost-effective correction. As an alternative, genetherapy approaches, involving a variety of gene delivery systems, have been pursuedfor the amelioration of Fabry disease. Fabry disease is a compelling disorder for genetherapy, as target cells are readily accessible and relatively low levels of enzymecorrection may suffice to reduce storage. Importantly, metabolic cooperativity effectsare also manifested in Fabry disease, wherein corrected cells secrete α-Gal A that cancorrect bystander cells. In addition, a broad therapeutic window probably exists, andmouse models of Fabry disease have been generated to assist studies. As an example,in vitro and in vivo studies using α-Gal A-transduced haematopoietic cells from Fabrymice have demonstrated enzymatic correction of recipient cells and dissemination ofα-Gal A upon transplantation, leading to reduced lipid storage in a number ofclinically relevant organs. This corrective enzymatic effect has recently been shown tobe even further enhanced upon pre-selection of therapeutically transduced cells priorto transplantation. This review will briefly detail current gene delivery methods andsummarize results to date in the context of gene therapy for Fabry disease.     

Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer

Fabry disease is a systemic disease caused by genetic deficiency of a lysosomal enzyme, alpha-galactosidase A (alpha-gal A), and is thought to be an important target for enzyme replacement therapy. We studied the feasibility of gene-mediated enzyme replacement for Fabry disease. The adeno-associated virus (AAV) vector containing the alpha-gal A gene was injected into the right quadriceps muscles of Fabry knockout mice. A time course study showed that alpha-gal A activity in plasma was increased to approximately 25% of normal mice and that this elevated activity persisted for up to at least 30 weeks without development of anti-alpha-gal A antibodies. The alpha-gal A activity in various organs of treated Fabry mice remained 5-20% of those observed in normal mice. Accumulated globotriaosylceramide in these organs was completely cleared by 25 weeks after vector injection. Reduction of globotriaosylceramide levels was also confirmed by immunohistochemical and electronmicroscopic analyses. Echocardiographic examination of treated mice demonstrated structural improvement of cardiac hypertrophy 25 weeks after the treatment. AAV vector-mediated muscle-directed gene transfer provides an efficient and practical therapeutic approach for Fabry disease.     

Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb(3); also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified alpha-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered alpha-gal A, (ii) to assess the pharmacokinetics of i.v.-administered alpha-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb(3). alpha-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified alpha-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of alpha-gal A, nine of the 10 patients had significantly reduced Gb(3) levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of alpha-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb(3) burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.     

Long-term correction of globotriaosylceramide storage in Fabry mice by recombinant adeno-associated virus-mediated gene transfer

Fabry disease is an X-linked recessive inborn metabolic disorder characterized by systemic and vascular accumulation of globotriaosylceramide (Gb(3)) caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). The condition is associated with an increased morbidity and mortality due to renal failure, cardiac disease, and early onset of stroke. Hemizygous males are primarily affected clinically with variable expression in heterozygous females. Gene-therapy trials have been initiated recently in alpha-gal A knockout mouse models of Fabry disease by using a variety of viral vectors. In the present investigation we administered single i.v. injections of 1 x 10(10) genomes of recombinant adeno-associated virus (rAAV) encoding the human alpha-gal A gene driven by a modified chicken beta-actin (CAG) promoter to alpha-gal A knockout (Fabry) mice. Transgenic mice were analyzed for expression of alpha-gal A activity and Gb(3) levels in liver, kidney, heart, spleen, small intestine, lung, and brain. Administration of the rAAV-CAG-hAGA vector resulted in stable expression of alpha-gal A in organs of the Fabry mice for >6 months. alpha-Gal A activity in the organs became equal to or higher than that of wild-type mice. Accumulated Gb(3) in the liver, heart, and spleen was reduced to that of wild-type mice with lesser but significant reductions in kidney, lung, and small intestine. Injection of the rAAV-CAG-hAGA construct into skeletal muscle did not result in expression of alpha-gal A in it or in other tissues. This study provides a basis for a simple and efficient gene-therapy approach for patients with Fabry disease and is indicative of its potential for the treatment of other lysosomal storage disorders.     

Restoration of normal lysosomal function in mucopolysaccharidosis type VII cells by retroviral vector-mediated gene transfer.

Retroviral vectors were constructed containing a rat beta-glucuronidase cDNA driven by heterologous promoters. Vector-mediated gene transfer into human and canine beta-glucuronidase-deficient mucopolysaccharidosis type VII fibroblasts completely corrected the deficiency in beta-glucuronidase enzymatic activity. In primary cultures of canine mucopolysaccharidosis type VII retinal pigment epithelial cells, which contain large amounts of undegraded glycosaminoglycan substrates, vector correction restored normal processing of specific glycosaminoglycans in the lysosomal compartment. In canine mucopolysaccharidosis type VII bone marrow cells, beta-glucuronidase was expressed at high levels in transduced cells. Thus, the vector-encoded beta-glucuronidase was expressed at therapeutic levels in the appropriate organelle and corrected the metabolic defect in cells exhibiting the characteristic pathology of this lysosomal storage disorder.     

Reconstitution of lymphoid development and function in ZAP-70-deficient mice following gene transfer into bone marrow cells

Mutations in the ZAP-70 protein tyrosine kinase gene result in a severe combined immunodeficiency (SCID) characterized by a selective inability to produce CD8(+) T cells and a signal transduction defect in peripheral CD4(+) cells. Transplantation of genetically modified hematopoietic progenitor cells that express the wild-type ZAP-70 gene may provide significant benefit to some of these infants. The feasibility of stem cell gene correction for human ZAP-70 deficiency was assessed using a ZAP-70 knock-out model. ZAP-70-deficient murine bone marrow progenitor cells were transduced with a retroviral vector expressing the human ZAP-70 gene. Engraftment of these cells in irradiated ZAP-70-deficient animals resulted in the development of mature CD4(+) and CD8(+) T cells. In marked contrast, both populations were absent in ZAP-70(-/-) mice undergoing transplantation with bone marrow progenitor cells transduced with a control vector. Importantly, ZAP-70-reconstituted T cells proliferated in response to T-cell receptor stimulation. Moreover, these ZAP-70-expressing T cells demonstrated a diverse T-cell receptor repertoire as monitored by the relative usage of each T-cell receptor beta chain hypervariable region subfamily. The presence of ZAP-70 in B cells did not affect either lipopolysaccharide- or lipopolysaccharide/interleukin-4-mediated immunoglobulin isotype switching. Altogether, these data indicate that retroviral-mediated gene transfer of the ZAP-70 gene may prove to have a therapeutic benefit for patients with ZAP-70-SCID.     

Correction of cardiac abnormalities in fabry mice by direct intraventricular injection of a recombinant lentiviral vector that engineers expression of alpha-galactosidase A.

BACKGROUND: Recombinant lentiviral vectors (LVs) offer the possibility of stable, long-term expression of transgenes even in non-dividing cells. In the present study this vector system was applied to a clinically relevant cardiovascular problem. METHODS AND RESULTS: Fabry disease results from deficient activity of alpha-galactosidase A (alpha-gal A) and cardiac abnormalities are a common and an important cause of death in patients with the disease. A therapeutic LV that delivers the alpha-gal A cDNA has been synthesized. In vitro studies established efficient transduction of the H9c2 rat cardiomyocytes and showed overexpression of enGFP (control) and alpha-gal A. In in vivo studies, the enGFP cDNA was transferred into C57BL/6 mouse hearts by direct intraventricular injection. Next, in a mouse model of Fabry disease, the recombinant therapeutic construct was delivered analogously. In cardiac tissue, alpha-gal A activity rose to 23% of normal levels at day 7 after LV injection, which is encouraging because levels of correction approximating 5% of normal may be curative for this disorder. There was also a corresponding reduction in globotriaosylceramide accumulation. Other organs assayed showed no detectable changes in alpha-gal A activity levels in injected animals. CONCLUSION: A localized benefit of directly injecting a therapeutic LV into the heart has been shown, confirming the utility of this delivery system for research and therapy for a variety of cardiovascular disorders.     

Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)-deficient mice and corrects their immune and metabolic defects

Adenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA(-/-) mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach.     

A bicistronic therapeutic retroviral vector enables sorting of transduced CD34+ cells and corrects the enzyme deficiency in cells from Gaucher patients

Corrective gene transfer for therapeutic intervention in metabolic and hematopoietic disorders has been hampered by the relatively inefficient transduction of human hematopoietic stem cells. To overcome this, a bicistronic recombinant retrovirus has been generated that delivers both a therapeutic glucocerebrosidase (GC) cDNA for the treatment of Gaucher disease, and a small murine cell surface antigen (heat-stable antigen [HSA]) as a selectable marker. An amphotropic retroviral- producing cell clone was created, and filtered supernatant was used to transduce NIH 3T3 cells. Sorting of transduced cells by flow cytometry enabled separation into populations based on cell surface fluorescence intensity derived from the expressed HSA. Significant increases in GC enzyme activity were seen for the transduced and especially the transduced and sorted cells. Similarly, increases in GC specific activity were seen in transduced and sorted skin fibroblasts from a patient with Gaucher disease. To streamline future transfer and sorting protocols for hematopoietic cells, transformed B-cell lines from Gaucher patients were created. Type I B cells were transduced and sorted, and large increases in GC specific activity occurred with concomitant increases in integrated retroviral copy numbers. In addition, toward the goal of using this selectable approach for corrective gene transfer to bone marrow stem cells, CD34+ cells were isolated from normal BM donors, transduced, and sorted based on cell surface expression of HSA. Proviral DNA was detected in approximately 40% of clonogenic progenitor colonies derived from unsorted, transduced CD34+ cells, demonstrating the high titer of the vector. However, after sorting, 100% of the colonies had the corrective GC cDNA, demonstrating the efficiency of this selective system for human hematopoietic progenitors. It is expected that strategies based on this approach will allow sorting of transduced cells of many types before implantation of transduced cells to animals or patients. This vector system may also be used to simplify manipulations and studies on retroviral-mediated gene delivery in vitro and for in vivo models.     

Phenotypic correction of Fanconi anemia group C knockout mice

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc -/- nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity of fancc -/- hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc -/- bone marrow cells were transduced with the use of retrovirus carrying the human fancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment of fancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells. (Blood. 2000;95:700-704)     

Growth factors and stromal support generate very efficient retroviral transduction of peripheral blood CD34+ cells from Gaucher patients

We have achieved high-efficiency gene transfer into nonmobilized peripheral blood (PB) CD34+ cells from patients with Gaucher's disease using a clinically acceptable retroviral supernatant transduction protocol. In our studies, bone marrow (BM) and PB CD34+ cells were transduced using a high titer (10(8) particles/mL) retroviral supernatant once a day for 4 consecutive days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), with or without an irradiated allogeneic BM stromal layer. The growth factors alone resulted in 29% +/- 10% gene transfer of PB CD34+ clonogenic cells in contrast with 71% +/- 17% gene transfer efficiency using stroma with the growth factors; a 2.5-fold increase. The increase in gene transfer efficiency was less prominent when BM CD34+ cells were used (40% +/- 16% without and 57% +/- 8% with stroma, a 1.5-fold increase). The overall transduction efficiency of both PB and BM CD34+ cells was lower when the cells were transduced over a stromal cell layer without added growth factors. The combination of IL-3, IL-6, and SCF with stroma transduced 75% of primitive long-term culture initiating cells (PB LTC- ICs) in comparison with 34% of LTC-ICs when IL-3, IL-6, and SCF were used without stromal support. Using this clinically acceptable supernatant/cytokines/stroma transduction protocol, correction of the glucocerebrosidase (GC) deficiency in the progeny cells of PBLTC-ICs from Gaucher's-disease patients has been accomplished. Efficient transduction of the PB CD34+ cells using this transduction protocol may allow repeated delivery of "GC-corrected" hematopoietic stem and progenitor cells to Gaucher's-disease patients.     

Gene transfer of the uroporphyrinogen III synthase cDNA into haematopoietic progenitor cells in view of a future gene therapy in congenital erythropoietic porphyria

Congenital erythropoietic porphyria (CEP) is an inherited metabolic disorder characterized by an overproduction and accumulation of porphyrins in bone marrow. This autosomal recessive disease results from a deficiency of uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the haem biosynthetic pathway. It is phenotypically heterogeneous: patients with mild disease have cutaneous involvement, while more severely affected patients are transfusion dependent. The cloning of UROIIIS cDNA and genomic DNA has allowed the molecular characterization of the genetic defect in a number of families. To date, 22 different mutations have been characterized. Allogeneic bone marrow transplantation is the only curative treatment available for the severe, transfusion-dependent, cases. When bone marrow transplantation cannot be performed owing to the absence of a suitable donor, the autografting of genetically modified cells is an appealing alternative. The best approach to somatic gene therapy in this disease involves the use of recombinant retroviral vectors to transduce cells ex vivo, followed by autologous transplantation of the genetically modified cells. We investigated retroviral transfer in deficient human fibroblasts, immortalized lymphoblasts as well as bone marrow cells, and obtained a complete restoration of the enzymatic activity and full metabolic correction.Using K562 cells, an erythroleukaemic cell line, the expression of the transgene remained stable during 3 months and during erythroid differentiation of the cells. Finally, a 1.6- to 1.9-fold increase in enzyme activity compared to the endogenous level was found in normal CD34+ cells, a population of heterogeneous cells known to contain the progenitor/stem cells for long-term expression. The future availability of a mouse model of the disease will permit ex vivo gene therapy experiments on the entire animal.     

Efficient retroviral transduction of human bone marrow progenitor and long-term culture-initiating cells: partial reconstitution of cells from patients with X-linked chronic granulomatous disease by gp91-phox expression

The primary immunodeficiencies are attractive candidates for the development of gene therapy approaches based on the transduction of hematopoietic cells. We have constructed a high-titer recombinant retrovirus for expression of gp91-phox, deficiencies of which cause the X-linked form of chronic granulomatous disease (X-CGD). We have used this vector to transduce human bone marrow, using either unfractionated mononuclear cells or purified CD34+ cells as targets and evaluated several infection protocols. Efficient gene transfer to progenitors and long-term culture-initiating cells (LTC-IC) was obtained for each target population. Importantly for potential clinical application, this could be achieved without the use of exogenous cytokines or polybrene. Progenitors representing each of the lineages detectable in vitro were transduced at equal efficiencies. The vector was shown partially to restore gp91-phox deficiency and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in transduced cells derived from X- CGD patients. These data demonstrate that it is possible to transduce primitive human hematopoietic cells efficiently and reconstitute NADPH oxidase.     

Gene therapy of RAG-2-/- mice: sustained correction of the immunodeficiency

Patients with mutations of either RAG-1 or RAG-2 genes suffer from severe combined immunodeficiency (SCID) characterized by the lack of T and B lymphocytes. The only curative treatment today consists of hematopoietic stem cell (HSC) transplantation, which is only partially successful in the absence of an HLA genoidentical donor, thus justifying research to find an alternative therapeutic approach. To this end, RAG-2-deficient mice were used to test whether retrovirally mediated ex vivo gene transfer into HSCs could provide long-term correction of the immunologic deficiency. Murine RAG-2-/-Sca-1(+) selected bone marrow cells were transduced with a modified Moloney leukemia virus (MLV)-based MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) retroviral vector containing the RAG-2 cDNA and transplanted into RAG-2-/- sublethally irradiated mice (3Gy). Two months later, T- and B-cell development was achieved in all mice. Diverse repertoire of T cells as well as proliferative capacity in the presence of mitogens, allogeneic cells, and keyhole limpet hemocyanin (KLH) were shown. B-cell function as shown by serum Ig levels and antibody response to a challenge by KLH also developed. Lymphoid subsets and function were shown to be stable over a one-year period without evidence of any detectable toxicity. Noteworthy, a selective advantage for transduced lymphoid cells was evidenced by comparative provirus quantification in lymphoid and myeloid lineages. Altogether, this study demonstrates the efficiency of ex vivo RAG-2 gene transfer in HSCs to correct the immune deficiency of RAG-2-/- mice, constituting a significant step toward clinical application.     

Aging accentuates and bone marrow transplantation ameliorates metabolic defects in Fabry disease mice

Fabry disease is an X-linked metabolic disorder caused by a deficiency of alpha-galactosidase A (alpha-Gal A). The enzyme defect leads to the systemic accumulation of glycosphingolipids with alpha-galactosyl moieties consisting predominantly of globotriaosylceramide (Gb3). In patients with this disorder, glycolipid deposition in endothelial cells leads to renal failure and cardiac and cerebrovascular disease. Recently, we generated alpha-Gal A gene knockout mouse lines and described the phenotype of 10-week-old mice. In the present study, we characterize the progression of the disease with aging and explore the effects of bone marrow transplantation (BMT) on the phenotype. Histopathological analysis of alpha-Gal A -/0 mice revealed subclinical lesions in the Kupffer cells in the liver and macrophages in the skin with no gross lesions in the endothelial cells. Gb3 accumulation and pathological lesions in the affected organs increased with age. Treatment with BMT from the wild-type mice resulted in the clearance of accumulated Gb3 in the liver, spleen, and heart with concomitant elevation of alpha-Gal A activity. These findings suggest that BMT may have a potential role in the management of patients with Fabry disease.     

Survival and retrovirus infection of murine hematopoietic stem cells in vitro: effects of 5-FU and method of infection.

Gene replacement therapy for diseases of the hematopoietic system requires efficient gene transfer to pluripotent hematopoietic stem cells. We have systematically compared a number of protocols for retrovirus-mediated gene transfer into murine repopulating hematopoietic stem cells. Recipients of infected bone marrow cells were analyzed for the presence of the transduced provirus 4 months after transplantation. Our results show that 5-fluorouracil (5-FU) pretreatment of donor animals was required for efficient gene transfer and that 5-FU-treated bone marrow retained more repopulating activity in culture than untreated bone marrow. A comparison of retrovirus-mediated gene transfer by co-cultivation of bone marrow cells with retrovirus producer cells as opposed to gene transfer by culturing bone marrow cells in retrovirus-containing supernatant revealed that gene transfer by cocultivation was more efficient than supernatant infection. However, the repopulating ability of bone marrow cells cocultured with retrovirus producer cells was reduced compared to bone marrow cells cultured in virus-containing medium.