scholarly journals Enhanced Host Defense After Gene Transfer in the Murine p47phox-Deficient Model of Chronic Granulomatous Disease

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2268-2275 ◽  
Author(s):  
Michael Mardiney ◽  
Sharon H. Jackson ◽  
S. Kaye Spratt ◽  
Fei Li ◽  
Steven M. Holland ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Chronic Granulomatous Disease ◽  
Host Defense ◽  
Ex Vivo ◽  
Pseudomonas Cepacia ◽  
Granulomatous Disease ◽  
Human Immune System ◽  
Human Form

Abstract The p47phox−/− mouse exhibits a phenotype similar to that of human chronic granulomatous disease (CGD) and, thus, is an excellent model for the study of gene transfer technology. Using the Moloney murine leukemia virus–based retroviral vector MFG-S encoding the human form of p47phox, we performed ex vivo gene transfer into Sca-1+ p47phox−/− marrow progenitor cells without conditioning of donors with 5-fluorouracil. Transduced progenitors were transplanted into moderately irradiated (500 cGy), G-CSF preconditioned sibling p47phox−/− mice. Using the fluorescent probe dihydrorhodamine 123 (DHR), in vivo biochemical correction of the superoxide-generating NADPH oxidase system was detected by flow cytometry in 12.3% ± 0.9% of phorbol myristate acetate–stimulated peripheral blood neutrophils at 4 weeks and 2.6% ± 1.0% at 14 weeks after transplantation. Following gene therapy, mice were challenged with the CGD pathogen Burkholderia (formerly Pseudomonas) cepacia and bacteremia levels were assessed at 24 hours and 7 days after inoculation. At both time points, bacteremia levels in gene corrected p47phox−/− mice were significantly lower than untreated p47phox−/− mice (0.89 ± 0.30 colonies v 237.7 ± 83.6 colonies at 24 hours, P < .02; 4.0 ± 2.0 colonies v 110.2 ± 26.5 colonies at 7 days, P < .0014). More importantly, Kaplan-Meier survival analysis showed a significant survival advantage of gene corrected versus untreated p47phox−/− mice (P < .001). Thus, stem-cell–directed ex vivo gene therapy is capable of restoring phagocyte oxidant-dependent host-defense function in this mouse model of a human immune-system disorder.

Long-Term Follow-Up of Patients Treated by Gene Therapy for X-Linked Chronic Granulomatous Disease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 194-194 ◽  
Author(s):  
Marion G. Ott ◽  
Manuel Grez ◽  
Stefan Stein ◽  
Ulrich Siler ◽  
Ulrike Koehl ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Chronic Granulomatous Disease ◽  
Ex Vivo ◽  
Therapeutic Option ◽  
High Morbidity ◽  
Granulomatous Disease ◽  
Phagocytic Cells

Abstract Chronic granulomatous disease (CGD) is a primary immunodeficiency in which phagocytic cells of affected patients have impaired antimicrobial activity due to a defect in the production of reactive oxygen species (ROS). CGD is caused by mutations in any one of four genes encoding for the subunits of the NADPH oxidase complex. Although curable by HSC transplantation, this strategy is usually limited only to patients with HLA-matched sibling or unrelated donors, as mismatched transplantation is associated with high morbidity and mortality due to graft failure and slow immune reconstitution. A therapeutic alternative for CGD patients is the genetic modification of autologous HSC. In January 2004 we initiated a Phase I/II clinical trial for X-CGD patients including conditioning with busulfan (8 mg/kg/total dose) prior to infusion of genetically modified HSC. G-CSF mobilized CD34+ cells from 2 adult patients (25 and 26 years) were transduced ex-vivo with a monocistronic gp91phox retroviral vector. Therapeutically significant gene marking levels were detected in neutrophils of both patients with up to 60% functionally corrected phagocytes 14 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking levels in B-cells has remained constant at a level of 20%, while gene marking in T-cells is below 5%. Gene marking in bone marrow was detected at levels between 30% and 40% one year after transplantation of gene modified cells. Killing assays in vitro have demonstrated antibacterial and antifungal activity in gene transduced phagocytes and both patients recovered of Staph. aureus and Aspergillus fumigatus infections after gene therapy. Our results suggest that gene therapy in combination with bone marrow conditioning is a therapeutic option for inherited diseases affecting the myeloid compartment and can be successfully used to treat CGD.

scholarly journals Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model

2021 ◽  
Vol 18 ◽  
pp. 347-354
Author(s):  
Masashi Noda ◽  
Kohei Tatsumi ◽  
Hideto Matsui ◽  
Yasunori Matsunari ◽  
Takeshi Sato ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Canine Model ◽  
Gene Transfer Techniques

scholarly journals 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 ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 41-48 ◽  
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

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.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

scholarly journals 723. In Vivo Expansion of MDS1/EVI1, PRDM16 and SETBP1 Integration Clones in Successful Chronic Granulomatous Disease (CGD) Gene Therapy Trial

2006 ◽  
Vol 13 ◽  
pp. S279
Author(s):  
Kerstin Schwarzwaelder ◽  
Manfred Schmidt ◽  
Marion G. Ott ◽  
Stefan Stein ◽  
Hanno Glimm ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Chronic Granulomatous Disease ◽  
Granulomatous Disease ◽  
Gene Therapy Trial ◽  
Therapy Trial

scholarly journals Keratinocyte Gene Transfer and Gene Therapy

1996 ◽  
Vol 7 (3) ◽  
pp. 204-221 ◽  
Author(s):  
J.A. Garlick ◽  
E.S. Fenjves
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Target Cells ◽  
Gene Products ◽  
Oral Keratinocytes ◽  
Systemic Delivery ◽  
Therapy Strategies

Gene therapy has moved beyond the pre-clinical stage to the treatment of a variety of inherited and acquired diseases. For such therapy to be successful, genes must be efficiently delivered to target cells and gene products must be expressed for prolonged periods of time without toxic effects to the host. This may be achieved by means of an in vivo strategy where genes are transferred directly into a host cell, or by means of an ex vivo approach through which cells are removed, cultured, targeted for gene delivery, and grafted back to the host. Several obstacles continue to delay safe and effective clinical application of gene therapy in a variety of target cells. The limited survival of transplanted cells, transient expression of transferred genes, and difficulties in targeting stem cells are technical issues requiring further investigation. Epidermal and oral keratinocytes are potential vehicles for gene therapy. Several features of these tissues can be utilized to achieve delivery of therapeutic gene products for local or systemic delivery. These qualities include: (1) the presence of stem cells; (2) the cell-, strata-, and site-specific regulation of keratinocyte gene expression; (3) tissue accessibility; and (4) secretory capacity. Such features can be exploited by the use of gene therapy strategies to facilitate: (1) identification, enrichment, and targeting of stem cells to ensure the continued presence of the transferred gene; (2) high-level and persistent transgene expression using keratinocyte-specific promoters; (3) tissue access needed for culture and grafting for ex vivo therapy and direct in vivo gene transfer; (4) secretion of transgene product for local or systemic delivery; and (5) monitoring of genetically modified tissue and removal if treatment termination is required. Optimal gene therapy strategies are being tested in a variety of tissues to treat dominant and recessive genetic disorders as well as acquired diseases such as neoplasia and infectious disease. This experience provides a basis for the application of such clinical studies to a spectrum of diseases effecting epidermal and oral keratinocytes. Gene therapy is in an early stage yet holds great promise for its ultimate clinical application.

scholarly journals Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4257-4265 ◽  
Author(s):  
Francesca Romana Santoni de Sio ◽  
Paolo Cascio ◽  
Anna Zingale ◽  
Mauro Gasparini ◽  
Luigi Naldini
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Cell Cycle Progression ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Proteasome Activity ◽  
Hematopoietic Stem ◽  
Vector Integration ◽  
Cytokine Stimulation

AbstractThe therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture, they display low permissivity to the vector, requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation, we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays, we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer, allowing the reach of very high levels of vector integration in their progeny in vivo. Thus, LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly, cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors, highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy.

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