Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 16 de 16
Filter
1.
Blood ; 121(14): e98-107, 2013 Apr 04.
Article in English | MEDLINE | ID: mdl-23386128

ABSTRACT

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34(+) hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34(+) HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60(+), yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60(+)), with variable yield (6 to >500 TRA-1-60(+) iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP-reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34(+) HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14(+) monocytes do not yield iPSC colonies under these reprogramming conditions.


Subject(s)
Cell Lineage/genetics , Cellular Reprogramming/genetics , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Transgenes/genetics , Antigens, CD34/metabolism , Base Sequence , Cell Culture Techniques/methods , Cell Line , Cell Separation/methods , DNA Fingerprinting , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Gene Rearrangement, B-Lymphocyte/genetics , Gene Rearrangement, T-Lymphocyte/genetics , Genome-Wide Association Study , Humans , Immunologic Deficiency Syndromes/pathology , Integrases/genetics , Kruppel-Like Factor 4 , Lentivirus/genetics , Lymphocytes/cytology , Lymphocytes/physiology , Molecular Sequence Data , Monocytes/cytology , Monocytes/physiology , Sendai virus/genetics , Teratoma/pathology , Transduction, Genetic/methods
2.
Blood ; 115(4): 783-91, 2010 Jan 28.
Article in English | MEDLINE | ID: mdl-19965657

ABSTRACT

Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial resulted in only short-term marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector, achieved more than 20% marking in 2 patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated 3 adult patients using busulfan conditioning and an MFGS retroviral vector encoding gp91(phox), achieving early marking of 26%, 5%, and 4% of neutrophils, respectively, with sustained long-term marking of 1.1% and 0.03% of neutrophils in 2 of the patients. Gene-marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those 2 patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection. This study was registered at www.clinicaltrials.gov as #NCT00394316.


Subject(s)
Genetic Therapy/methods , Granulomatous Disease, Chronic/therapy , Membrane Glycoproteins/genetics , Moloney murine leukemia virus/genetics , NADPH Oxidases/genetics , Neutrophils/enzymology , Adult , Aspergillosis/therapy , Busulfan/therapeutic use , Chromosomes, Human, X/genetics , Combined Modality Therapy , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/metabolism , Hematopoietic Stem Cell Transplantation , Humans , Male , Membrane Glycoproteins/metabolism , Monocytes/enzymology , Myeloablative Agonists/therapeutic use , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Neutropenia/therapy , Oxidants/metabolism , Respiratory Burst/physiology , Staphylococcal Infections/therapy , Superoxides/metabolism , Thrombocytopenia/therapy , Transduction, Genetic , Transplantation Conditioning/methods , Transplantation, Autologous , Young Adult
3.
Stem Cells Dev ; 16(3): 361-70, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17610366

ABSTRACT

Hematopoietic stem cell (HSC) graft cell dose impacts significantly on allogeneic transplant. Similarly, HSC gene therapy outcome is affected by loss of repopulating cells during culture required for ex vivo retrovirus transduction. Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a central role in marrow trafficking of HSCs, and maneuvers that enhance CXCR4 activation might positively impact outcome in settings of limiting graft dose. CD26/dipeptidyl peptidase IV (DPP-IV) is an ectoenzyme protease that cleaves SDF-1, thus reducing CXCR4 activation. We show that injection of irradiated nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with >or=2 micromol Diprotin A (a tripeptide specific inhibitor of CD26 protease activity) at the time of transplant of human granulocyte colony-stimulating factor (G-CSF) mobilized CD34(+) peripheral blood cells (CD34(+) PBCs) results in a >3.4-fold enhancement of engraftment of human cells. We also show that CD26 on residual stromal cells in the irradiated recipient marrow milieu, and not any CD26 activity in the human CD34(+) PBC graft itself, plays the critical role in regulating receptivity of this environment for the incoming graft. Human marrow stromal cells also express CD26, raising the possibility that Diprotin A treatment could significantly enhance engraftment of HSCs in humans in settings of limiting graft dose just as we observed in the NOD/SCID mouse human xenograft model.


Subject(s)
Antigens, CD34/metabolism , Blood Cells/metabolism , Bone Marrow Transplantation , Oligopeptides/metabolism , Animals , Blood Cells/cytology , Blood Cells/radiation effects , Cell Movement , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Oligopeptides/administration & dosage , Transplantation, Heterologous
4.
Methods Mol Biol ; 412: 365-83, 2007.
Article in English | MEDLINE | ID: mdl-18453124

ABSTRACT

Polymorphonuclear neutrophils (PMN) have a remarkable capacity for generation of large amounts of reactive oxygen species in response to a variety of infectious or inflammatory stimuli, a process known as the respiratory burst that involves activation of a multicomponent NADPH oxidase. Given their short life span, PMN are not amenable to most molecular biology methods for studying activation of this oxidant-generating system. We have explored a variety of methods for introduction of components of the phagocytic oxidase (phox system) into the promyelocytic erythroleukemia cell line, K-562. Here, we describe a series of cloned K-562 cell lines that were retrovirally transduced for stable production of one or more essential components of the phagocytic oxidase (phox) complex. We outline methods for the use of these transfectable cells for investigating structure, function, and signaling requirements for assembly and activation of the phox system. These versatile lines can be used to examine effects of genetic polymorphisms or mutations in phox components associated with chronic granulomatous disease, to serve as a system for testing gene therapy vectors designed to correct the defective oxidase, to study cross-functioning with recently described phox component homologs, or to explore signaling components involved in regulation of the respiratory burst.


Subject(s)
K562 Cells , Models, Biological , NADPH Oxidases/physiology , Neutrophils/enzymology , Blotting, Western , Cell Fractionation , Cell Membrane/metabolism , Humans , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Plasmids , Protein Transport , Transfection/methods
5.
Exp Hematol ; 33(4): 460-8, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15781337

ABSTRACT

OBJECTIVE: WHIM (warts, hypogammaglobulinemia, recurrent bacterial infection, myelokathexis) syndrome is an autosomal dominant immune deficiency with severe chronic neutropenia and marrow neutrophil apoptosis. Carboxy-termini truncating mutations in the chemokine receptor CXCR4 have been identified in WHIM patients. We created a retrovirus encoding mutated CXCR4 (truncating point mutation 1000C-->T [R334X] inherited heterozygously in several WHIM patients) in order to transducer healthy human CD34 stem cells and K562 to overexpress mutated CXCR4 and determined its effect on receptor responses to stromal-derived factor-1 (SDF1). METHODS: Retrovirus vector was engineered to coexpress WHIM-associated R334X mutated CXCR4 together with green fluorescent protein (GFP). Control vectors included similar constructs with wild-type CXCR4 (WT-CXCR4) or only GFP. CD34+ cells and K562 were transduced with these vectors. Populations of 100% transduced K562 were established by sorting GFP+ cells by flow cytometry. We performed migration and calcium flux assays of transduced CD34+ cells and transduced/sorted K562. We also examined receptor recycling in response to SDF1. RESULTS: Healthy human CD34+ cells and/or human erythroleukemia K562 cells transduced to express mutated CXCR4, WT-CXCR4, or GFP alone demonstrated that mutated CXCR4 was associated with enhanced calcium flux and enhanced migration. There was also decreased receptor internalization and enhanced recovery of surface mutated CXCR4 in response to SDF1 compared with WT-CXCR4. CONCLUSION: We propose that decreased internalization of WHIM-associated mutated CXCR4 leads to prolongation/enhancement of signaling in response to SDF1 and that this may provide the biochemical basis for the autosomal dominant abnormalities of cell trafficking and function associated with WHIM syndrome.


Subject(s)
Endocytosis , Genetic Diseases, Inborn/etiology , Receptors, CXCR4/genetics , Receptors, CXCR4/metabolism , Agammaglobulinemia , Bacterial Infections , Cell Line , Chemokine CXCL12 , Chemokines, CXC/pharmacology , Dose-Response Relationship, Drug , Humans , Kinetics , Neutropenia , Signal Transduction/drug effects , Syndrome , Transduction, Genetic , Warts
6.
Exp Hematol ; 32(8): 709-19, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15308322

ABSTRACT

OBJECTIVE: We characterized a novel in vivo selectable fusion protein, green fluorescence protein-O6-benzylguanine (BG)-resistant O6-methylguanine-methyltransferase (GFP-MGMT* [*refers to mutant MGMT]) used to delineate optimum selection regimens for transduced hematopoietic stem cells (HSC) ex vivo and in vivo. MATERIALS AND METHODS: We transduced human or mouse HSC with retrovirus vector encoding GFP-MGMT* where BG-resistant forms of human P140K-hMGMT* and mouse P144K-mMGMT* were studied. We evaluated selection of transduced HSC ex vivo and in vivo using either BG/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or BG/temozolomide (TMZ) combinations, evaluating transduction marking by flow cytometry and real-time TaqMan PCR. RESULTS: GFP-MGMT* transduction confers nuclear-localized GFP fluorescence and BG resistance. Optimum selection ex vivo of GFP-MGMT*-transduced HSC occurred with BG (2.5-10 microM)/BCNU (5-10 microM) or TMZ (100-200 microM), which increases marking while preserving maximum viable transduced cells. Starting at low levels (0.1%) or high levels (>30%) of in vivo bone marrow gene making in mice, in vivo selection with BG/BCNU (20/6 mg/kg) (weeks 4 and 5) or BG/TMZ (20/60 mg/kg) (daily x 5 at week 4) increased bone marrow marking to 8.58% +/- 3.52% or 82.0% +/- 3.4% GFP+ cells, respectively, in the low- or high-level initial marking mice. CONCLUSIONS: GFP-MGMT* is an informative tool to explore optimization of in vivo selection regimens using BG/BCNU or BG/TMZ to increase gene marking of HSC. Both timing and dosing of selection regimens and the starting level of marking may all be important to the level of selective increase of in vivo marking achieved.


Subject(s)
Cell Nucleus/metabolism , Guanine/analogs & derivatives , Guanine/pharmacology , Hematopoietic Stem Cells/metabolism , Luminescent Proteins/metabolism , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Cells, Cultured , Genetic Vectors , Green Fluorescent Proteins , Mice , Mice, Inbred C57BL , Transduction, Genetic
7.
Blood ; 111(1): 209-18, 2008 Jan 01.
Article in English | MEDLINE | ID: mdl-17875809

ABSTRACT

Leukocyte adhesion deficiency type-1 (LAD-1) is an autosomal recessive immunodeficiency caused by mutations in the beta2 integrin, CD18, that impair CD11/CD18 heterodimer surface expression and/or function. Absence of functional CD11/CD18 integrins on leukocytes, particularly neutrophils, leads to their incapacity to adhere to the endothelium and migrate to sites of infection. We studied 3 LAD-1 patients with markedly diminished neutrophil CD18 expression, each of whom had a small population of lymphocytes with normal CD18 expression (CD18(+)). These CD18(+) lymphocytes were predominantly cytotoxic T cells, with a memory/effector phenotype. Microsatellite analyses proved patient origin of these cells. Sequencing of T-cell subsets showed that in each patient one CD18 allele had undergone further mutation. Interestingly, all 3 patients were young adults with inflammatory bowel disease. Somatic reversions of inherited mutations in primary T-cell immunodeficiencies are typically associated with milder clinical phenotypes. We hypothesize that these somatic revertant CD18(+) cytotoxic T lymphocytes (CTLs) may have altered immune regulation. The discovery of 3 cases of reversion mutations in LAD-1 at one center suggests that this may be a relatively common event in this rare disease.


Subject(s)
CD18 Antigens/genetics , Leukocyte-Adhesion Deficiency Syndrome/genetics , Leukocyte-Adhesion Deficiency Syndrome/immunology , Mosaicism , T-Lymphocytes, Cytotoxic/physiology , Adult , CD8 Antigens/genetics , Female , Genes, Recessive , Humans , Immunologic Memory/genetics , Immunophenotyping , Integrin alpha Chains/genetics , Male , Mutation , Neutropenia/genetics , Superantigens/genetics
8.
Stem Cells ; 25(7): 1807-13, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17464090

ABSTRACT

We used the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model to assess the repopulation potential of subpopulations of mobilized human CD34+ peripheral blood progenitor cells (PBPC). First, PBPC were transduced with gamma-retrovirus vector RD114-MFGS-CFP, which requires cell division for successful transduction, at 24 hours, 48 hours, and 72 hours to achieve 96% cyan fluorescent protein (CFP)-positive cells. Cells were sorted 12 hours after the last transduction into CFP-positive (divided cells) and CFP-negative populations. CFP-positive cells were transplanted postsort, whereas the CFP-negative cells were retransduced and injected at 120 hours. The CFP-negative sorted and retransduced cells contained markedly fewer vector copies and resulted in a 32-fold higher overall engraftment and in a 13-fold higher number of engrafted transgene positive cells. To assess cell proliferation as an underlying cause for the different engraftment levels, carboxyfluorescein succinimidyl ester-labeling of untransduced PBPC was performed to track the number of cell divisions. At 72 hours after initiation of culture, when 95% of all cells have divided, PBPC were sorted into nondivided and divided fractions and transplanted into NOD/SCID mice. Nondivided cells demonstrated 45-fold higher engraftment than divided cells. Late dividing PBPC in ex vivo culture retain high expression of the stem cell marker CD133, whereas rapidly proliferating cells lose CD133 in correlation to the number of cell divisions. Our studies demonstrate that late dividing progenitors transduced with gamma-retroviral vectors contribute most to NOD/SCID engraftment and transgene marking. Confining the gamma-retroviral transduction to CD133-positive cells on days 3 and 4 could greatly reduce the number of transplanted vector copies, limiting the risk of leukemia from insertional mutagenesis. Disclosure of potential conflicts of interest is found at the end of this article.


Subject(s)
Diabetes Mellitus/metabolism , Gammaretrovirus/genetics , Genetic Vectors , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Transduction, Genetic , AC133 Antigen , Adult , Animals , Antigens, CD/metabolism , Cell Division , Cell Movement , Cells, Cultured , Flow Cytometry , Gammaretrovirus/physiology , Glycoproteins/metabolism , Green Fluorescent Proteins/metabolism , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/virology , Humans , Mice , Mice, SCID , Peptides/metabolism , Phenotype , Time Factors
9.
Blood ; 109(1): 78-84, 2007 Jan 01.
Article in English | MEDLINE | ID: mdl-16946301

ABSTRACT

WHIM(warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis) syndrome is a rare immunodeficiency caused in many cases by autosomal dominant C-terminal truncation mutations in the chemokine receptor CXCR4. A prominent and unexplained feature of WHIM is myelokathexis (hypercellularity with apoptosis of mature myeloid cells in bone marrow and neutropenia). We transduced healthy human CD34(+) peripheral blood-mobilized stem cells (PBSCs) with retrovirus vector encoding wild-type (wt) CXCR4 or WHIM-type mutated CXCR4 and studied these cells ex vivo in culture and after engraftment in a nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenograft model. Neither wt CXCR4 nor mutated CXCR4 transgene expression itself enhanced apoptosis of neutrophils arising in transduced PBSC cultures even with stimulation by a CXCR4 agonist, stromal cell-derived factor-1 (SDF-1 [CXCL12]). Excess wt CXCR4 expression by transduced human PBSCs enhanced marrow engraftment, but did not affect bone marrow (BM) apoptosis or the release of transduced leukocytes into PB. However, mutated CXCR4 transgene expression further enhanced BM engraftment, but was associated with a significant increase in apoptosis of transduced cells in BM and reduced release of transduced leukocytes into PB. We conclude that increased apoptosis of mature myeloid cells in WHIM is secondary to a failure of marrow release and progression to normal myeloid cell senescence, and not a direct effect of activation of mutated CXCR4.


Subject(s)
Agammaglobulinemia/genetics , Bacterial Infections/etiology , Bone Marrow Cells/pathology , Immunologic Deficiency Syndromes/genetics , Neutropenia/genetics , Receptors, CXCR4/physiology , Warts/genetics , Amino Acid Substitution , Animals , Apoptosis , Bone Marrow Cells/metabolism , Calcium Signaling , Cell Movement , Chemokine CXCL12 , Chemokines, CXC/physiology , Colony-Forming Units Assay , Disease Susceptibility , Gene Dosage , Graft Survival , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Mutation, Missense , Peripheral Blood Stem Cell Transplantation , Point Mutation , Radiation Chimera , Receptors, CXCR4/genetics , Recombinant Fusion Proteins/physiology , Recurrence , Transgenes , Transplantation, Heterologous
10.
Blood ; 110(1): 67-73, 2007 Jul 01.
Article in English | MEDLINE | ID: mdl-17369490

ABSTRACT

Retroviral gene therapy can restore immunity to infants with X-linked severe combined immunodeficiency (XSCID) caused by mutations in the IL2RG gene encoding the common gamma chain (gammac) of receptors for interleukins 2 (IL-2), -4, -7, -9, -15, and -21. We investigated the safety and efficacy of gene therapy as salvage treatment for older XSCID children with inadequate immune reconstitution despite prior bone marrow transplant from a parent. Subjects received retrovirus-transduced autologous peripherally mobilized CD34(+) hematopoietic cells. T-cell function significantly improved in the youngest subject (age 10 years), and multilineage retroviral marking occurred in all 3 children.


Subject(s)
Genetic Therapy/methods , Immunity/drug effects , Receptors, Interleukin-2/administration & dosage , X-Linked Combined Immunodeficiency Diseases/immunology , X-Linked Combined Immunodeficiency Diseases/therapy , Hematopoietic Stem Cell Transplantation/methods , Humans , Infant , Mutation , Receptors, Interleukin-2/genetics , Retroviridae/genetics , T-Lymphocytes/immunology , Transduction, Genetic , Transplantation, Autologous , X-Linked Combined Immunodeficiency Diseases/genetics
11.
Blood ; 107(8): 3091-7, 2006 Apr 15.
Article in English | MEDLINE | ID: mdl-16384923

ABSTRACT

X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency and early mortality, the only potential cure being hematopoietic stem cell (HSC) transplantation or gene therapy. Current clinical gene therapy protocols targeting HSCs are based upon ex vivo gene transfer, potentially limited by the adequacy of HSC harvest, transduction efficiencies of repopulating HSCs, and the potential loss of their engraftment potential during ex vivo culture. We demonstrate an important proof of principle by showing achievement of durable immune reconstitution in XSCID dogs following intravenous injection of concentrated RD114-pseudotyped retrovirus vector encoding the corrective gene, the interleukin-2 receptor gamma chain (gamma c). In 3 of 4 dogs treated, normalization of numbers and function of T cells were observed. Two long-term-surviving animals (16 and 18 months) showed significant marking of B lymphocytes and myeloid cells, normalization of IgG levels, and protective humoral immune response to immunization. There were no adverse effects from in vivo gene therapy, and in one dog that reached sexual maturity, sparing of gonadal tissue from gene transfer was demonstrated. This is the first demonstration that in vivo gene therapy targeting HSCs can restore both cellular and humoral immunity in a large-animal model of a fatal immunodeficiency.


Subject(s)
Genetic Therapy , Genetic Vectors/administration & dosage , Receptors, Interleukin-2/genetics , Recovery of Function/genetics , Severe Combined Immunodeficiency/therapy , Transduction, Genetic , Animals , Antibody Formation/genetics , Antibody Formation/immunology , B-Lymphocytes/immunology , Dogs , Genetic Therapy/methods , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/immunology , Immunization , Receptors, Interleukin-2/immunology , Recovery of Function/immunology , Retroviridae , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/immunology , T-Lymphocytes/immunology , Transduction, Genetic/methods , Transplantation, Autologous
12.
J Biol Chem ; 281(48): 37045-56, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17015440

ABSTRACT

The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91(phox) subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22(phox) subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91(phox) subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91(phox) subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production.


Subject(s)
Cytochrome b Group/chemistry , Cytochrome b Group/physiology , NADPH Oxidases/chemistry , NADPH Oxidases/physiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acid Sequence , Chryseobacterium/metabolism , Glycosylation , Humans , Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase/chemistry , Membrane Glycoproteins/metabolism , Molecular Sequence Data , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Neutrophils/metabolism , Phagocytosis , Recombinant Proteins/chemistry , Superoxides/metabolism , Trypsin/chemistry
13.
Mol Ther ; 14(2): 202-11, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16600688

ABSTRACT

We have recently reported that the RD114-pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the NADPH-oxidase gp91phox (approved gene symbol CYBB) defect in CD34(+) cells from patients with X-linked chronic granulomatous disease in the NOD/SCID mouse model. Considering clinical use of this vector, we transplanted autologous mobilized peripheral blood CD34(+) progenitor cells, transduced with the RD114-MFGS-gp91phox vector, into two healthy rhesus macaques following nonmyeloablative conditioning. The moderately high levels of in vivo marking seen in the first months following transduction decreased and stabilized at about 8 months posttransplant. Marking for both healthy animals after 15 months was 0.3 to 1.3 vector copies per 100 cells in lymphocytes, neutrophils, and monocytes. Vector insertion analyses performed by linear amplification-mediated PCR and sequencing identified 32 and 45 separate insertion sites in the animals. Identical insertion sites were found in myeloid cells and lymphocytes, demonstrating the successful transduction of lymphomyeloid progenitors. Some inserts landed in the vicinity of genes controlling cell cycle and proliferation. Statistical analyses of insertion sites 1 year posttransplant suggest a high diversity of insertion sites despite low marking.


Subject(s)
Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells , Membrane Glycoproteins/genetics , NADPH Oxidases/genetics , Transduction, Genetic , Animals , Antigens, CD34/analysis , Gammaretrovirus/genetics , Gene Dosage , Genetic Vectors , Macaca mulatta , Male , NADPH Oxidase 2 , Transgenes , Transplantation Conditioning , Transplantation, Autologous , Virus Integration , Whole-Body Irradiation
14.
Blood ; 102(7): 2692-4, 2003 Oct 01.
Article in English | MEDLINE | ID: mdl-12805054

ABSTRACT

Interferon-gamma receptor-1 (IFNgammaR1) deficiency is a rare inherited immunodeficiency. We performed a nonmyeloablative allogeneic stem cell transplantation on a boy with complete IFNgammaR1 deficiency and refractory disseminated Myco- bacterium avium infection. Despite the patient's profound immune defect, early donor stem cell engraftment was low. Full donor engraftment was accomplished only following multiple donor lymphocyte infusions. Detection of IFNgammaR1 expression on peripheral blood monocytes and neutrophils corresponded with establishment of stable, complete donor hematopoietic chimerism. However, expression of, and signaling through IFNgammaR1 disappeared shortly thereafter. Disseminated Mycobacterium avium infection persisted and the patient died. Coculture of Myco- bacterium avium with normal myeloid cells resulted in an IFNgamma signaling defect similar to that observed in vivo. Active disseminated Mycobacterium avium infection may significantly compromise normal immune reconstitution following allogeneic stem cell transplantation. Patients with IFNgammaR1 deficiency should receive transplants before developing refractory mycobacterial infections.


Subject(s)
Mycobacterium avium Complex , Mycobacterium avium-intracellulare Infection/complications , Mycobacterium avium-intracellulare Infection/immunology , Receptors, Interferon/deficiency , Stem Cell Transplantation , Child, Preschool , Humans , Immunocompromised Host , Male , Monocytes/immunology , Receptors, Interferon/genetics , Signal Transduction , Interferon gamma Receptor
15.
Stem Cells ; 22(7): 1128-33, 2004.
Article in English | MEDLINE | ID: mdl-15579633

ABSTRACT

Hematopoietic stem cells (HSCs) lose marrow reconstitution potential during ex vivo culture. HSC migration to stromal cell-derived factor (SDF)-1 (CXCL12) correlates with CXC chemokine receptor 4 (CXCR4) expression and marrow engraftment. We demonstrate that mobilized human CD34+ peripheral blood stem cells (CD34+ PBSCs) lose CXCR4 expression during prolonged culture. We transduced CD34+ PBSCs with retrovirus vector encoding human CXCR4 and achieved 18-fold more CXCR4 expression in over 87% of CD34+ cells. CXCR4-transduced cells yielded increased calcium flux and up to a 10-fold increase in migration to SDF-1. Six-day cultured CXCR4-transduced cells demonstrated significant engraftment in nonobese diabetic/severe combined immunodeficient mice under conditions in which control transduced cells resulted in low or no engraftment. We conclude that transduction-mediated overexpression of CXCR4 significantly improves marrow engraftment of cultured PBSCs.


Subject(s)
Bone Marrow Cells/cytology , Gene Transfer Techniques , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/cytology , Receptors, CXCR4/biosynthesis , Receptors, CXCR4/genetics , Transgenes , Animals , Antigens, CD34/biosynthesis , Calcium/metabolism , Cell Movement , Flow Cytometry , Graft Survival , Humans , Membrane Glycoproteins/metabolism , Mice , Mice, SCID , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Signal Transduction , Time Factors
16.
Blood ; 102(8): 2789-97, 2003 Oct 15.
Article in English | MEDLINE | ID: mdl-12829597

ABSTRACT

In previous studies amphotropic MFGS-gp91phox (murine onco-retrovirus vector) was used in a clinical trial of X-linked chronic granulomatous disease (X-CGD) gene therapy to achieve transient correction of oxidase activity in 0.1% of neutrophils. We later showed that transduced CD34+ peripheral blood stem cells (CD34+ PBSCs) from this trial transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in correction of only 2.5% of human neutrophils. However, higher rates of transduction into stem cells are required. In the current study we demonstrate that the same vector (MFGS-gp91phox) pseudo-typed with RD114 envelope in a 4-day culture/transduction regimen results in a 7-fold increase in correction of NOD/SCID mouse repopulating X-CGD CD34+ PBSCs (14%-22% corrected human neutrophils; human cell engraftment 13%-67%). This increase may result from high expression of receptor for RD114 that we demonstrate on CD34+CD38- stem cells. Using RD114-MFGS encoding cyan fluorescent protein to allow similar studies of normal CD34+ PBSCs, we show that progressively higher levels of gene marking of human neutrophils (67%-77%) can be achieved by prolongation of culture/transduction to 6 days, but with lower rates of human cell engraftment. Our data demonstrate the highest reported level of functional correction of any inherited metabolic disorder in human cells in vivo with the NOD/SCID mouse system using onco-retrovirus vector.


Subject(s)
Granulomatous Disease, Chronic/genetics , Membrane Glycoproteins/genetics , NADPH Oxidases , Oxidoreductases/genetics , Animals , Antigens, CD34/biosynthesis , Flow Cytometry , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Granulomatous Disease, Chronic/therapy , Green Fluorescent Proteins , Hematopoietic Stem Cells , Humans , Luminescent Proteins/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , NADP/metabolism , NADPH Oxidase 2 , Neutrophils/metabolism , RNA, Messenger/metabolism , Retroviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/metabolism , Time Factors , Transgenes , Ultracentrifugation , beta 2-Microglobulin/genetics
SELECTION OF CITATIONS
SEARCH DETAIL