240. In Vivo Expansion of Integration Clones Caused by Retroviral Activation of MDS1-EVI1, PRDM16 or SETBP1 in a Successful Clinical Chronic Granulomatous Disease (CGD) Gene Therapy Trial

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 gp91phox, 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.

Download Full-text

Silencing of proviral transgene expression by CpG methylation in a gene therapy trial for chronic granulomatous disease

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2007.10.075 ◽

2008 ◽

Vol 40 (2) ◽

pp. 283-284

Author(s):

Stephan Schultze-Strasser ◽

Rolf Kramer ◽

Carolin Preiss ◽

MG. Ott ◽

Stefan Stein ◽

...

Keyword(s):

Gene Therapy ◽

Chronic Granulomatous Disease ◽

Transgene Expression ◽

Cpg Methylation ◽

Granulomatous Disease ◽

Gene Therapy Trial ◽

Therapy Trial

Download Full-text

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

Blood ◽

10.1182/blood.v108.11.3274.3274 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3274-3274 ◽

Cited By ~ 1

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.

Download Full-text

Sustained Polyclonal Hematopoietic Repopulation after Successful SCID-X1 Gene Therapy by Means of a Non Random Integrating Pseudotyped Gammaretrovector.

Blood ◽

10.1182/blood.v104.11.290.290 ◽

2004 ◽

Vol 104 (11) ◽

pp. 290-290

Author(s):

Kerstin Schwarzwaelder ◽

Manfred Schmidt ◽

Steven Howe ◽

Claudia Prinz ◽

Manuela Wissler ◽

...

Keyword(s):

Gene Therapy ◽

Progenitor Cells ◽

Cell Clone ◽

Retroviral Vector ◽

Transduction Efficiency ◽

Chain Gene ◽

Gene Therapy Trial ◽

Integration Sites ◽

Therapy Trial

Abstract The integration of a retroviral vector as used in hematopoietic gene therapy trials produces a transition sequence from the vector DNA into the genomic DNA and may thus serve as a stable molecular marker unique for each cell clone. High sensitive linear amplification mediated PCR (LAM-PCR) allows the detection of specific retroviral integration sites. Thus it is possible to determine the clonal composition of the hematopoietic system in vivo (1). We could show that the hematopoietic repopulation in human SCID-X1 patients was derived from various, long-lived progenitor cell clones indicating retroviral transduction into pluripotent cells (manuscript submitted). In two cases of lymphoproliferative disorder after successful SCID-X1 gene therapy integration of the retroviral vector into the LMO-2 oncogene was probable the main reason for malignancy (2). The distribution of integration sites over the whole genome, the potential preference for integration at certain loci and which cells receive genetic correction and engraftment are therefore of considerable interest. Recently, another gene therapy trial has successfully corrected 4 infants suffering from SCID-X1. A GALV-pseudotyped MLV-based vector carrying the therapeutic common gamma chain gene was used for transduction of autologous CD34+ cells. The patients did not receive any conditioning therapy before transplantation. We analyzed lymphoid and myeloid DNA from all patients. The transduction efficiency of T lymphocytes and myeloid cells was up to 100 and 1 %, respectively. In vivo clonality analysis of CD3+ cells showed a polyclonal composition 1 to 2 years after transplantation. The myeloid repopulation also consisted of various different clones. These data may indicate transduction of pluripotent and long term active stem or progenitor cells. We here report on more than 300 sequenced integration sites of the patients, whereas 250 sequences could be assigned unequivocally to a unique locus. So far, no vector integration in the LMO-2 oncogene could be detected in this trial, and the patients do not reveal any other evidence for malignancy or clonal deformation of their stem cell compartment. We could show that integration of the mammalian gammaretroviral vector in this gene therapy trial is not random. Integration of the vector happens generally within or close to specific regions of genes. We found common integration sites (CIS) in RefSeq gene regions. The targeted RefSeq genes were classified according to the Gene Ontology database. Our data strongly support the presumption that curative gene therapeutic treatment requires a sustained polyclonal contribution of ex vivo manipulated stem and progenitor cells and provide an important insight into the integration manner of GALV-pseudotyped MLV-based vectors.

Download Full-text

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

Blood ◽

10.1182/blood.v112.11.2349.2349 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2349-2349 ◽

Cited By ~ 2

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.

Download Full-text

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

Blood ◽

10.1182/blood.v89.7.2268 ◽

1997 ◽

Vol 89 (7) ◽

pp. 2268-2275 ◽

Cited By ~ 101

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.

Download Full-text

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

Blood ◽

10.1182/blood.v106.11.194.194 ◽

2005 ◽

Vol 106 (11) ◽

pp. 194-194 ◽

Cited By ~ 1

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.

Download Full-text