Retroviral-Mediated Gene Transfer of gp91phox Into Bone Marrow Cells Rescues Defect in Host Defense Against Aspergillus fumigatus in Murine X-Linked Chronic Granulomatous Disease

Abstract The X-linked form of chronic granulomatous disease (X-CGD), an inherited deficiency of the respiratory burst oxidase, results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase cytochrome b. The goal of this study was to evaluate the impact of retroviral-mediated gene transfer of gp91phox on host defense against Aspergillus fumigatus in a murine model of X-CGD. Retrovirus vectors constructed using the murine stem cell virus (MSCV) backbone were used for gene transfer of the gp91phox cDNA into murine X-CGD bone marrow cells. Transduced cells were transplanted into lethally irradiated syngeneic X-CGD mice. After hematologic recovery, superoxide production, as monitored by the nitroblue tetrazolium (NBT) test, was detected in up to ≈80% of peripheral blood neutrophils for at least 28 to 35 weeks after transplantation. Neutrophil expression of recombinant gp91phox and superoxide production were significantly less than wild-type neutrophils. However, 9 of 9 mice with ≈50% to 80% NBT+ neutrophils after gene transfer did not develop lung disease after respiratory challenge with 150 to 500 A fumigatus spores, doses that produced disease in 16 of 16 control X-CGD mice. In X-CGD mice transplanted with mixtures of wild-type and X-CGD bone marrow, ≥5% wild-type neutrophils were required for protection against A fumigatus challenge. These data suggest that expression of even low levels of recombinant gp91phox can substantially improve phagocyte function in X-CGD, although correction of very small percentage of phagocytes may not be sufficient for protection against A fumigatus.

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Retroviral-Mediated Gene Transfer of gp91phox Into Bone Marrow Cells Rescues Defect in Host Defense Against Aspergillus fumigatus in Murine X-Linked Chronic Granulomatous Disease

Blood ◽

10.1182/blood.v89.1.41.41_41_48 ◽

1997 ◽

Vol 89 (1) ◽

pp. 41-48 ◽

Cited By ~ 3

Author(s):

Helga Björgvinsdóttir ◽

Chunjin Ding ◽

Nancy Pech ◽

Mary A. Gifford ◽

Ling Lin Li ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Aspergillus Fumigatus ◽

Chronic Granulomatous Disease ◽

Host Defense ◽

Bone Marrow Cells ◽

Superoxide Production ◽

Granulomatous Disease ◽

Wild Type ◽

Marrow Cells

The X-linked form of chronic granulomatous disease (X-CGD), an inherited deficiency of the respiratory burst oxidase, results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase cytochrome b. The goal of this study was to evaluate the impact of retroviral-mediated gene transfer of gp91phox on host defense against Aspergillus fumigatus in a murine model of X-CGD. Retrovirus vectors constructed using the murine stem cell virus (MSCV) backbone were used for gene transfer of the gp91phox cDNA into murine X-CGD bone marrow cells. Transduced cells were transplanted into lethally irradiated syngeneic X-CGD mice. After hematologic recovery, superoxide production, as monitored by the nitroblue tetrazolium (NBT) test, was detected in up to ≈80% of peripheral blood neutrophils for at least 28 to 35 weeks after transplantation. Neutrophil expression of recombinant gp91phox and superoxide production were significantly less than wild-type neutrophils. However, 9 of 9 mice with ≈50% to 80% NBT+ neutrophils after gene transfer did not develop lung disease after respiratory challenge with 150 to 500 A fumigatus spores, doses that produced disease in 16 of 16 control X-CGD mice. In X-CGD mice transplanted with mixtures of wild-type and X-CGD bone marrow, ≥5% wild-type neutrophils were required for protection against A fumigatus challenge. These data suggest that expression of even low levels of recombinant gp91phox can substantially improve phagocyte function in X-CGD, although correction of very small percentage of phagocytes may not be sufficient for protection against A fumigatus.

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Variable correction of host defense following gene transfer and bone marrow transplantation in murine X-linked chronic granulomatous disease

Blood ◽

10.1182/blood.v97.12.3738 ◽

2001 ◽

Vol 97 (12) ◽

pp. 3738-3745 ◽

Cited By ~ 56

Author(s):

Mary C. Dinauer ◽

Mary A. Gifford ◽

Nancy Pech ◽

Ling Lin Li ◽

Patricia Emshwiller

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Gene Transfer ◽

Nadph Oxidase ◽

Host Defense ◽

Marrow Transplantation ◽

Oxidase Activity ◽

Granulomatous Disease ◽

Wild Type ◽

Nadph Oxidase Activity

Chronic granulomatous disease (CGD) is an inherited immunodeficiency in which the absence of the phagocyte superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase results in recurrent bacterial and fungal infections. A murine model of X-linked CGD (X-CGD) was used to explore variables influencing reconstitution of host defense following bone marrow transplantation and retroviral-mediated gene transfer. The outcomes of experimental infection with Aspergillus fumigatus, Staphylococcus aureus, orBurkholderia cepacia were compared in wild-type, X-CGD mice, and transplanted X-CGD mice that were chimeric for either wild-type neutrophils or neutrophils with partial correction of NADPH oxidase activity after retroviral-mediated gene transfer. Host defense to these pathogens was improved in X-CGD mice even with correction of a limited number of neutrophils. However, intact protection against bacterial pathogens required relatively greater numbers of oxidant-generating phagocytes compared to protection against A fumigatus. The host response also appeared to be influenced by the relative level of cellular NADPH oxidase activity, particularly forA fumigatus. These results may have implications for developing effective approaches for gene therapy of CGD.

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High-level reconstitution of respiratory burst activity in a human X- linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox

Blood ◽

10.1182/blood.v88.5.1834.bloodjournal8851834 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1834-1840 ◽

Cited By ~ 1

Author(s):

C Ding ◽

A Kume ◽

H Bjorgvinsdottir ◽

RG Hawley ◽

N Pech ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Respiratory Burst ◽

Oxidase Activity ◽

Bone Marrow Cells ◽

Granulomatous Disease ◽

Respiratory Burst Activity ◽

High Level ◽

Respiratory Burst Oxidase ◽

Marrow Cells

The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91phox, MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91phox in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91phox transcripts was also observed in primary and secondary murine colony-forming unit- spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high- level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function.

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High-level reconstitution of respiratory burst activity in a human X- linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox

Blood ◽

10.1182/blood.v88.5.1834.1834 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1834-1840 ◽

Cited By ~ 42

Author(s):

C Ding ◽

A Kume ◽

H Bjorgvinsdottir ◽

RG Hawley ◽

N Pech ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Respiratory Burst ◽

Oxidase Activity ◽

Bone Marrow Cells ◽

Granulomatous Disease ◽

Respiratory Burst Activity ◽

High Level ◽

Respiratory Burst Oxidase ◽

Marrow Cells

Abstract The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91phox, MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91phox in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91phox transcripts was also observed in primary and secondary murine colony-forming unit- spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high- level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function.

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Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

Biochemical Journal ◽

10.1042/bj3310733 ◽

1998 ◽

Vol 331 (3) ◽

pp. 733-742 ◽

Cited By ~ 8

Author(s):

Masafumi YOSHIMURA ◽

Yoshito IHARA ◽

Tetsuo NISHIURA ◽

Yu OKAJIMA ◽

Megumu OGAWA ◽

...

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

Stromal Cells ◽

Bone Marrow Cells ◽

Adherent Cell ◽

Wild Type ◽

Spleen Cells ◽

Bisecting Glcnac ◽

Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

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Enhancement of Adenovirus-Mediated Gene Transfer to Human Bone Marrow Cells

Leukemia & Lymphoma ◽

10.3109/10428199809050904 ◽

1998 ◽

Vol 29 (5-6) ◽

pp. 439-451 ◽

Cited By ~ 7

Author(s):

Tsutomu Watanabe ◽

Linda Kelsey ◽

Ana Ageitos ◽

Charles Kuszynski ◽

Kazuhiko Ino ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Bone Marrow Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Marrow Cells

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Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Blood ◽

10.1182/blood.v90.6.2148 ◽

1997 ◽

Vol 90 (6) ◽

pp. 2148-2159 ◽

Cited By ~ 118

Author(s):

Harshal H. Nandurkar ◽

Lorraine Robb ◽

David Tarlinton ◽

Louise Barnett ◽

Frank Köntgen ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Bone Marrow Cells ◽

White Blood Cells ◽

Null Mutation ◽

Wild Type ◽

Normal Numbers ◽

Interleukin 11 ◽

Marrow Cells

Abstract Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

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Retroviral gene transfer of a donor class I MHC gene to recipient bone marrow cells induces tolerance to alloantigens in vivo

Transplantation Proceedings ◽

10.1016/s0041-1345(96)00464-2 ◽

1997 ◽

Vol 29 (1-2) ◽

pp. 1130 ◽

Cited By ~ 18

Author(s):

W. Wong ◽

S.A. Stranford ◽

P.J. Morris ◽

K.J. Wood

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Bone Marrow Cells ◽

Class I ◽

Class I Mhc ◽

Retroviral Gene Transfer ◽

Marrow Cells

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Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells

Blood ◽

10.1182/blood.v90.9.3304 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3304-3315 ◽

Cited By ~ 40

Author(s):

Marti F.A. Bierhuizen ◽

Yvonne Westerman ◽

Trudi P. Visser ◽

Wati Dimjati ◽

Albertus W. Wognum ◽

...

Keyword(s):

Bone Marrow ◽

Green Fluorescent Protein ◽

Gene Transfer ◽

Fluorescent Protein ◽

Bone Marrow Cells ◽

Retroviral Vector ◽

Egfp Expression ◽

Green Fluorescent ◽

Marrow Cells

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

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