scholarly journals 62. Gene Transfer in Murine X-Linked Chronic Granulomatous Disease Using an SFFV-Based Gamma-Retroviral Vector: Vector Integration Sites Are Influenced by Pre-Transplant Conditioning Regimen

2008 ◽  
Vol 16 ◽  
pp. S25
Keyword(s):  
Gene Transfer ◽  
Chronic Granulomatous Disease ◽  
Conditioning Regimen ◽  
Retroviral Vector ◽  
Granulomatous Disease ◽  
Vector Integration ◽  
Integration Sites

Insertional Activation of MDS1/EVI1, PRDM16 and SETBP1 in a Successful Chronic Granulomatous Disease (CGD) Gene Therapy Trial.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3274-3274 ◽  
Author(s):  
Kerstin Schwarzwaelder ◽  
Manfred Schmidt ◽  
Annette Deichmann ◽  
Marion G. Ott ◽  
Stefan Stein ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Chronic Granulomatous Disease ◽  
Retroviral Vector ◽  
Granulomatous Disease ◽  
Time Points ◽  
Gene Therapy Trial ◽  
Cell Clones ◽  
Integration Sites ◽  
Therapy Trial

Abstract The potential of gene therapy to correct genetic diseases of the lymphoid compartment has been demonstrated in ADA-SCID and X-linked SCID clinical gene therapy trials. The first successful correction of the myeloid compartment could be achieved in the latest chronic granulomatous disease (CGD) gene therapy trial. CD34+ bone marrow derived cells of 2 patients were transduced using a SFFV based retroviral vector encoding the therapeutic transgene gp91phox. After non-myeloablative conditioning the autologous cells were reinfused. 3 months post therapy the proportion of marked granulocytes was 20% in patient 1 and 10% in patient 2. 5 to 9 months after treatment the proportion of gp91phox expressing granulocytes expanded 4-fold in both patients. Until the latest time points analyzed, (P1: d820; P2: d560) the marking efficiency persisted at that level. In order to define the clonality of the corrected hematopoietic repopulation we accomplished linear amplification mediated PCR (LAM-PCR) on peripheral blood and bone marrow samples as well as sorted lymphoid and myeloid fractions derived from successive time points after therapy. To characterize the retroviral insertion site distribution, we carried out high throughput sequencing and mapping of the vector genome junctions. The hematopoietic repopulation in patient 1 was polyclonal up to day 542 after therapy. Subsequently the number of corrected cell clones and the activity of a predominant clone decreased up to 820 days post transplantation, when the patient succumbed to infectious complications. In this time frame, a different predominant clone appeared. The repopulation in patient 2 has been polyclonal until the latest time point analyzed. Identification of 435 integration sites from patient 1 and 330 insertion sites from patient 2 revealed the gene coding region of the zinc finger transcription factor homologues MDS1/EVI1 and PRDM16 as common integration sites (CIS) in both patients and the SETBP1 locus as a third CIS in patient 1. RT-PCR analysis demonstrated an activating influence of vector LTR on individual CIS genes. Our data show that prospectively studying insertions and stem cell contributions is feasible and that retroviral vector insertion may lead to an upregulation of genes causing an in vivo expansion of the affected cell clones, which can augment gene-corrected hematopoietic repopulation as an unexpected, thus far non-adverse side effect.

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.

scholarly journals Assessing the potential for AAV vector genotoxicity in a murine model

Blood ◽  
2011 ◽  
Vol 117 (12) ◽  
pp. 3311-3319 ◽  
Author(s):  
Hojun Li ◽  
Nirav Malani ◽  
Shari R. Hamilton ◽  
Alexander Schlachterman ◽  
Giulio Bussadori ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Normal Tissue ◽  
Expression Patterns ◽  
Tumor Formation ◽  
High Dose ◽  
Adjacent Normal Tissue ◽  
Aav Vector ◽  
Vector Integration ◽  
Integration Sites ◽  
Vector Dna

AbstractGene transfer using adeno-associated virus (AAV) vectors has great potential for treating human disease. Recently, questions have arisen about the safety of AAV vectors, specifically, whether integration of vector DNA in transduced cell genomes promotes tumor formation. This study addresses these questions with high-dose liver-directed AAV-mediated gene transfer in the adult mouse as a model (80 AAV-injected mice and 52 controls). After 18 months of follow-up, AAV-injected mice did not show a significantly higher rate of hepatocellular carcinoma compared with controls. Tumors in mice treated with AAV vectors did not have significantly different amounts of vector DNA compared with adjacent normal tissue. A novel high-throughput method for identifying AAV vector integration sites was developed and used to clone 1029 integrants. Integration patterns in tumor tissue and adjacent normal tissue were similar to each other, showing preferences for active genes, cytosine-phosphate-guanosine islands, and guanosine/cysteine-rich regions. Gene expression data showed that genes near integration sites did not show significant changes in expression patterns compared with genes more distal to integration sites. No integration events were identified as causing increased oncogene expression. Thus, we did not find evidence that AAV vectors cause insertional activation of oncogenes and subsequent tumor formation.

Gene transfer to primary chronic granulomatous disease monocytes

The Lancet ◽  
1995 ◽  
Vol 346 (8967) ◽  
pp. 92-93 ◽  
Author(s):  
A.J. Thrasher ◽  
C.M. Casimir ◽  
C. Kinnon ◽  
G. Morgan ◽  
R.J. Levinsky ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Chronic Granulomatous Disease ◽  
Granulomatous Disease

scholarly journals Results of allogeneic hematopoietic stem cell transplantation in patients with chronic granulomatous disease at the Russian Children’s Clinical Hospital

2020 ◽  
Vol 7 (2) ◽  
pp. 23-34
Author(s):  
E. B. Machneva ◽  
E. A. Pristanskova ◽  
L. V. Olkhova ◽  
A. V. Mezentseva ◽  
V. V. Konstantinova ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Chronic Granulomatous Disease ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Conditioning Regimen ◽  
Granulomatous Disease ◽  
Hematopoietic Stem

Relevance. Chronic granulomatous disease (CGD) belongs to the group of primary immunodeficiencies. Patients with CGD have an impaired quality of life, severe infections and inflammatory organ damage. Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment method for CGD. The authors of the article presented the experience of HSCT in patients with CGD in the Russian Children’s Clinical Hospital.Materials and methods. 20 (19 primary and 1 repeated) HSCT during the period from 2009 to 2020 were performed in nineteen patients with CGD. All patients had a long history of infections, three or more foci of chronic infection, 9 patients had a generalized BCG infection. Bone marrow (ВМ) from a related HLA-identical donor was the source of hematopoietic stem cells (HSC) for 4 (21 %) patients, peripheral blood stem cells (PBSC) for 2 (10.5 %). ВМ from a unrelated fully HLA-identical donor was performed in 9 (47.4 %) patients, PBSC – 2 (10.5 %). ВМ from a unrelated 9/10 HLA-compatible donor was performed in one (5.3 %) patient. In one case (5.3 %) the HSC source became PBSC from a unrelated 9/10 HLA-compatible donor after TcRαβ/CD19+ depletion. In 68.5 % (n = 13) cases the conditioning regimen included threosulfan, fludarabine, melphalan, and antithymocyte globulin. In 2 (10.5 %) patients, melphalan was excluded from the conditioning regimen; in 4 (21 %), it was replaced by thiotepa.Results. The overall survival (OS) was 88.9 ± 10.5 %, the event-free survival (EFS) was 88.1 ± 7.9 %, and there was no transplant mortality. Transplant rejections were observed in two patients who received HSC from a unrelated 9/10 HLA-compatible donor with a previous conditioning regimen that included only one alkylating agent. In 4 patients (21 %) there was a prolonged persistence of mixed chimerism after HSCT without clinical and laboratory signs of CGD. After successful transplantation all patients were cured of the infectious and inflammatory diseases characteristic of CGD.Conclusion. Results of HSCT in patients with CGD can be considered satisfactory, the OS and EFS are high. Failure of HSCT is associated with transplant rejection, which is most likely due to the donor and patient mismatch, as well as the use of conditioning modes with reduced intensity.

Retroviral Gene Therapy for X-Linked Chronic Granulomatous Disease: Results from Phase I/II Trial.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2349-2349 ◽  
Author(s):  
Joong Gon Kim ◽  
Hyo Seop Ahn ◽  
Hyoung Jin Kang ◽  
Sujeong Kim ◽  
Youngtae Hong ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Nadph Oxidase ◽  
Phase I ◽  
Chronic Granulomatous Disease ◽  
Viral Vectors ◽  
Conditioning Regimen ◽  
Transduction Efficiency ◽  
Granulomatous Disease ◽  
Gene Therapy Trial ◽  
Life Threatening

Abstract X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency disease caused by a defect in the gp91phox gene encoding one of the subunits of the NADPH oxidase complex. NADPH oxidase plays an important role in eradicating the pathogen engulfed by the phagocytes. Therefore, CGD patients suffer from recurring life-threatening infection by bacteria or fungi, and die before 30 in most cases. In an effort to treat this life-threatening disease, we initiated a phase I/II gene therapy trial in 2007. Two X-CGD patients were enrolled in the trial. The retroviral vector used for gene delivery was the MLV-based MT vector containing gp91 phox cDNA (Yu et al., Gene Ther2000; 7: 797, Hong et al., J Gene Med2004; 6: 724). Viral vectors have been produced from PG13 packaging cells in compliance with GMP. The clinical protocol was approved by the Korean FDA. G-CSF mobilized peripheral blood CD34+ cells were obtained from patients, and transduced in retronectin-coated gas-permeable bags containing SCGM media supplemented with SCF, FLT3L, TPO, and IL-3. The transduction efficiency was 10.5% for patient #1 and 28.5% for patient #2 when assessed by gp91 FACS analysis. Before receiving transduced cells, patients were treated with a conditioning regimen consisting of busulfan (3.2 mg/kg/day for 2 days) and fludarabine (40 mg/m2/day for 3 days). No adverse effects were observed from the use of busulfan and fludarabine. The percentage of superoxide-producing cells, as determined by DHR assay, was 6.4% and 14.5% at day 17, and decreased to less than 0.1% (after 1 year) and 0.4% (after 7 months). Thus far, abnormal cell expansion has not been observed.

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