scholarly journals Functional reconstitution of the NADPH-oxidase by adeno-associated virus gene transfer

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 761-765 ◽  
Author(s):  
AJ Thrasher ◽  
M de Alwis ◽  
CM Casimir ◽  
C Kinnon ◽  
K Page ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Nadph Oxidase ◽  
Fungal Infections ◽  
Retroviral Vectors ◽  
Phagocytic Cells ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem ◽  
Quiescent Cells ◽  
Somatic Gene

Chronic granulomatous disease (CGD) comprises a heterogeneous group of inherited conditions characterized biochemically by disordered function of a unique multicomponent enzyme system present in phagocytic cells, the NADPH-oxidase. Clinically, it is characterized by recurrent bacterial and fungal infections that are relatively resistant to treatment by conventional means. Curative bone marrow transplantation has been successfully achieved in a small number of cases, but the wider application of this procedure is limited by availability of suitable donor material. Somatic gene therapy would overcome this problem, and several groups have now shown correction of the biochemical defect in hematopoietic cells by retrovirus-mediated gene transfer. However, the failure of the current generation of retroviral vectors to efficiently transduce quiescent cells greatly restricts their potential for gene transfer to pluripotent hematopoietic stem cells. Given these limitations, we have constructed vectors based on adeno-associated virus and used these to transfer a functional copy of the p47phox gene to immortalized B cells derived from patients with p47phox-deficient autosomal recessive CGD. We show stable expression of protein and restoration of NADPH-oxidase function in these cells in the absence of selection. Adeno-associated virus vectors may overcome some of the limitations of retroviral gene delivery systems and may therefore be a useful vehicle for curative gene therapy of CGD and other primary immunodeficiencies.

Genetic Therapy for Beta-Thalassemia: From the Bench to the Bedside

Hematology ◽  
2010 ◽  
Vol 2010 (1) ◽  
pp. 445-450 ◽  
Author(s):  
Paritha Arumugam ◽  
Punam Malik
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Bone Marrow Cells ◽  
Genetic Disorder ◽  
Globin Gene ◽  
Retroviral Vectors ◽  
Beta Thalassemia ◽  
Great Success ◽  
Hematopoietic Stem

AbstractBeta-thalassemia is a genetic disorder with mutations in the β-globin gene that reduce or abolish β-globin protein production. Patients with β-thalassemia major (Cooley's anemia) become severely anemic by 6 to 18 months of age, and are transfusion dependent for life, while those with thalassemia intermedia, a less-severe form of thalassemia, are intermittently or rarely transfused. An allogeneically matched bone marrow transplant is curative, although it is restricted to those with matched donors. Gene therapy holds the promise of “fixing” one's own bone marrow cells by transferring the normal β-globin or γ-globin gene into hematopoietic stem cells (HSCs) to permanently produce normal red blood cells. Requirements for effective gene transfer for the treatment of β-thalassemia are regulated, erythroid-specific, consistent, and high-level β-globin or γ-globin expression. Gamma retroviral vectors have had great success with immune-deficiency disorders, but due to vector-associated limitations, they have limited utility in hemoglobinopathies. Lentivirus vectors, on the other hand, have now been shown in several studies to correct mouse and animal models of thalassemia. The immediate challenges of the field as it moves toward clinical trials are to optimize gene transfer and engraftment of a high proportion of genetically modified HSCs and to minimize the adverse consequences that can result from random integration of vectors into the genome by improving current vector design or developing novel vectors. This article discusses the current state of the art in gene therapy for β-thalassemia and some of the challenges it faces in human trials.

scholarly journals Canine model for gene therapy: inefficient gene expression in dogs reconstituted with autologous marrow infected with retroviral vectors

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 742-747 ◽  
Author(s):  
RB Stead ◽  
WW Kwok ◽  
R Storb ◽  
AD Miller
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Transient Gene Expression ◽  
Retroviral Vectors ◽  
Canine Model ◽  
Preclinical Model ◽  
Hematopoietic Stem ◽  
In Vivo Selection

Successful retroviral gene transfer into murine hematopoietic stem cells indicates the potential for somatic gene therapy in the treatment of certain human hereditary diseases. We developed a canine model to test the applicability of these techniques to a preclinical model of human marrow transplantation. Previously we reported that canine CFU-GM could be infected with retroviral vectors carrying either the gene for a mutant dihydrofolate reductase (DHFR) or neomycin phosphotransferase (NEO). This study reports six lethally irradiated dogs transplanted with autologous marrow cocultivated with retroviral vector-producing cells. This procedure conferred drug resistance to 3% to 13% of the CFU- GM. Three dogs infected with either the NEO or DHFR virus engrafted, but we detected no drug-resistant CFU-GM. Three dogs were given marrow infected with a DHFR virus and received methotrexate (MTX) as in vivo selection; all three had evidence of engraftment. In the surviving dog, we detected 0.03% to 0.1% MTX-resistant CFU-GM at 3 to 5 weeks posttransplant during in vivo selection. These results indicate that we can reconstitute lethally irradiated dogs with autologous marrow exposed to retroviral vectors and suggest that gene transfer into hematopoietic cells is feasible on a large scale. However, the low- level transient gene expression indicates that considerable obstacles remain before human gene therapy can be considered.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy

Blood ◽  
2009 ◽  
Vol 114 (17) ◽  
pp. 3546-3556 ◽  
Author(s):  
Barbara Cassani ◽  
Eugenio Montini ◽  
Giulietta Maruggi ◽  
Alessandro Ambrosi ◽  
Massimiliano Mirolo ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cell ◽  
Gene Transfer ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
Cell Clones ◽  
Integration Sites

Abstract Gene transfer into hematopoietic stem cells by γ-retroviral vectors (RVs) is an effective treatment for inherited blood disorders, although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T lymphocytes from adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients 10 to 30 months after infusion of autologous, genetically corrected CD34+ cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single-cell level, primary T-cell clones were isolated from 2 patients. T-cell clones harbored either 1 (89.8%) or 2 (10.2%) vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism, and T-cell receptor-driven functions. Analysis of RV integration sites indicated a high diversity in T-cell origin, consistently with the polyclonal T-cell receptor-Vβ repertoire. Quantitative transcript analysis of 120 genes within a 200-kb window around RV integration sites showed modest (2.8- to 5.2-fold) dysregulation of 5.8% genes in 18.6% of the T-cell clones compared with controls. Nonetheless, affected clones maintained a stable phenotype and normal in vitro functions. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols. The trials described herein have been registered at http://www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

Gene transfer therapy for heritable disease: cell and expression targeting

1993 ◽  
Vol 339 (1288) ◽  
pp. 217-224 ◽  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Target Cells ◽  
Somatic Gene Therapy ◽  
Hematopoietic Stem ◽  
Large Size ◽  
Efficient Gene ◽  
Somatic Gene ◽  
Transfer Strategies ◽  
Therapy Model

Gene therapy is defined as the delivery of a functional gene for expression in somatic tissues with the intent to cure a disease. Different gene transfer strategies may be required to target different tissues. Adenosine deaminase (ADA) deficiency is a good gene therapy model for targeting a rare population of pluripotent hematopoietic stem cells capable of self-renewal. We present evidence for the highly efficient gene transfer and sustained expression of hum an ADA in human primitive hematopoietic progenitors using retroviral supernatant with a supportive stromal layer. A stem cell-enriched (CD34 + ) fraction was also successfully transduced. Duchenne muscular dystrophy (DMD) is also a good model for somatic gene therapy. Two of the challenges presented by this model are the large size of the gene and the large number of target cells. Germline gene transfer and correction of the phenotype has been demonstrated in transgenic mdx mice using both a full-length and a truncated form of the dystrophin cDNA. We present here a deletion mutagenesis strategy to truncate the dystrophin cDNA such that it can be accommodated by retroviral and adenoviral vectors useful for somatic gene therapy.

scholarly journals Canine model for gene therapy: inefficient gene expression in dogs reconstituted with autologous marrow infected with retroviral vectors

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 742-747 ◽  
Author(s):  
RB Stead ◽  
WW Kwok ◽  
R Storb ◽  
AD Miller
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Transient Gene Expression ◽  
Retroviral Vectors ◽  
Canine Model ◽  
Preclinical Model ◽  
Hematopoietic Stem ◽  
In Vivo Selection

Abstract Successful retroviral gene transfer into murine hematopoietic stem cells indicates the potential for somatic gene therapy in the treatment of certain human hereditary diseases. We developed a canine model to test the applicability of these techniques to a preclinical model of human marrow transplantation. Previously we reported that canine CFU-GM could be infected with retroviral vectors carrying either the gene for a mutant dihydrofolate reductase (DHFR) or neomycin phosphotransferase (NEO). This study reports six lethally irradiated dogs transplanted with autologous marrow cocultivated with retroviral vector-producing cells. This procedure conferred drug resistance to 3% to 13% of the CFU- GM. Three dogs infected with either the NEO or DHFR virus engrafted, but we detected no drug-resistant CFU-GM. Three dogs were given marrow infected with a DHFR virus and received methotrexate (MTX) as in vivo selection; all three had evidence of engraftment. In the surviving dog, we detected 0.03% to 0.1% MTX-resistant CFU-GM at 3 to 5 weeks posttransplant during in vivo selection. These results indicate that we can reconstitute lethally irradiated dogs with autologous marrow exposed to retroviral vectors and suggest that gene transfer into hematopoietic cells is feasible on a large scale. However, the low- level transient gene expression indicates that considerable obstacles remain before human gene therapy can be considered.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Abstract Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

scholarly journals Gene Therapy for β-Thalassemia

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 45-50 ◽  
Author(s):  
Punam Malik ◽  
Paritha I. Arumugam
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Retroviral Vectors ◽  
Thalassemia Intermedia ◽  
Hematopoietic Stem ◽  
Variable Expression

AbstractGene transfer for β-thalassemia requires gene transfer into hematopoietic stem cells using integrating vectors that direct regulated expression of β globin at therapeutic levels. Among integrating vectors, oncoretroviral vectors carrying the human β-globin gene and portions of the locus control region (LCR) have suffered from problems of vector instability, low titers and variable expression. In recent studies, human immunodeficiency virus–based lentiviral (LV) vectors were shown to stably transmit the human β-globin gene and a large LCR element, resulting in correction of β-thalassemia intermedia in mice. Several groups have since demonstrated correction of the mouse thalassemia intermedia phenotype, with variable levels of β-globin expression. These levels of expression were insufficient to fully correct the anemia in thalassemia major mouse model. Insertion of a chicken hypersensitive site-4 chicken insulator element (cHS4) in self-inactivating (SIN) LV vectors resulted in higher and less variable expression of human β-globin, similar to the observations with cHS4-containing retroviral vectors carrying the human γ-globin gene. The levels of β-globin expression achieved from insulated SIN-LV vectors were sufficient to phenotypically correct the thalassemia phenotype from 4 patients with human thalassemia major in vitro, and this correction persisted long term for up to 4 months, in xeno-transplanted mice in vivo. In summary, LV vectors have paved the way for clinical gene therapy trials for Cooley’s anemia and other β-globin disorders. SIN-LV vectors address several safety concerns of randomly integrating viral vectors by removing viral transcriptional elements and providing lineage-restricted expression. Flanking the proviral cassette with chromatin insulator elements, which additionally have enhancer-blocking properties, may further improve SIN-LV vector safety.

Expression vectors for human adenosine deaminase gene therapy

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 832-839 ◽  
Author(s):  
Kateri A. Moore ◽  
Frederick A. Fletcher ◽  
Raye Lynn Alford ◽  
Deborah K. Villalon ◽  
Dianne H. Hawkins ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Adenosine Deaminase ◽  
Retroviral Vectors ◽  
Expression Vectors ◽  
Mouse Bone Marrow ◽  
Human Genetic Disease ◽  
Hematopoietic Stem

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one of these vectors (pΔNN2ADA), human ADA was detected in 60–85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.Key words: gene therapy, adenosine deaminase, retrovirus vectors, immunodeficiency.

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