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.

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

Blood ◽  
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.

scholarly journals Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve stable long-term correction of oxidase activity in peripheral blood neutrophils

Blood ◽  
2010 ◽  
Vol 115 (4) ◽  
pp. 783-791 ◽  
Author(s):  
Elizabeth M. Kang ◽  
Uimook Choi ◽  
Narda Theobald ◽  
Gilda Linton ◽  
Debra A. Long Priel ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Chronic Granulomatous Disease ◽  
Oxidase Activity ◽  
Severe Infection ◽  
Granulomatous Disease ◽  
Gene Therapy Trial ◽  
Blood Neutrophils ◽  
Severe Infections ◽  
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.

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

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

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

Myeloid Expansion after Insertional Activation of MDS1/EVI1, PRDM16 and SETBP1 in a Successful Chronic Granulomatous Gene Therapy Trial.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 198-198
Author(s):  
Kerstin Schwarzwaelder ◽  
Manfred Schmidt ◽  
Marion G. Ott ◽  
Stefan Stein ◽  
Hanno Glimm ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Side Effects ◽  
High Efficiency ◽  
Post Treatment ◽  
Multiple Time ◽  
Gene Therapy Trial ◽  
Vector Integration ◽  
Integration Sites ◽  
Therapy Trial

Abstract Successful gene therapy trials of ADA-SCID and SCID-X1 demonstrated the curative potential of oncoretroviral gene transfer. Integration of the retroviral vectors used in these studies has been thought to be a random process but severe side effects in gene therapy and in vitro studies revealed preferred insertion of these vectors mainly around transcription start sites. In SCID patients proliferation advantage of gene corrected cells was one reason for the success of the trials, whereas in the most recent chronic granulomatous disease (CGD) gene therapy trial corrected cells do not have any selective advantage therefore the two patients received mild busulfan treatment before transplantation. High efficiency transduction and conditioning have helped in the successful correction of the patients. Peripheral blood granulocytes show a stable expression (>10%) of the transgene (gp91phox) in patient 1 (15 months post treatment) as well as in patient 2 (11 months post treatment). We reasoned that, unlike T cells, which have the capability to proliferate independent of their bone marrow progenitors, granulocytes more directly reflect the influence of retrovirus insertion, and should therefore allow to closely monitor clonal fate in vivo and its potential relation to vector insertion. To study the clonality of the corrected myelopoiesis, the long term activity of individual cell clones, and the distribution of integration sites in active cells we carried out high sensitive LAM-PCR. The highly polyclonal composition of transduced cells forming myelopoiesis caused the sustained expression of gp91phox. Individual clones carrying the transgene could be detected at multiple time points. To define whether corrected cells have a proliferation advantage due to their vector integration we started large-scale sequencing and mapping of involved insertion sites. We here present >700 unique mappable integration sites of the two treated patients. The distribution of the SFFV based retroviral vector integration sites in this trial turned non random 5 months after transplantation. Corrected long-term myelopoiesis expanded 3- to 5- fold in the two patients due to activating common integration sites (CIS) in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1, suggesting that these genes influence regulation of normal long-term hematopoiesis in humans. Our data indicate that the therapeutic benefit in this trial was activated through insertional side effects, therefore our findings have important implications in novel gene therapy approaches.

scholarly journals High Titer Lentivector from a Stable Lenti-Producer Efficiently Corrects CD34+ Hematopoietic Stem Cells from Patients with p47phox-Deficient Chronic Granulomatous Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2036-2036
Author(s):  
Uimook Choi ◽  
Narda Theobald ◽  
Throm E Robert ◽  
John Gray ◽  
David J. Rawlings ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Chronic Granulomatous Disease ◽  
Lentiviral Vector ◽  
High Titer ◽  
Granulomatous Disease ◽  
Packaging Cell Line ◽  
Hematopoietic Stem ◽  
Nsg Mice

Abstract Chronic granulomatous disease (CGD) is an inherited immune deficiency due to mutations in the genes for the NADPH subunits (the genes for p47phox, p22phox, p67phox, p40phox autosomal chronic granulomatous disease), or gp91phox (X-linked chronic granulomatous disease). This results in a failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs), the major defect in chronic granulomatous disease causing recurrent infections and granulomatous complications. Hematopoietic stem cell transplantation (HSCT) with a suitable donor is potentially curative. However, in the absence of HLA-matched donor, gene therapy using autologous gene-corrected HSC offers potential for significant clinical benefit. To date, despite myeloid conditioning, gene therapy for CGD patients using gamma-retroviral vectors have achieved either minimal long-term gene marking and engraftment, or has been associated with insertional mutagenesis. In contrast, lentivector-mediated gene therapy has successfully treated patients with Wiskott-Aldrich syndrome and Metachromatic Leukodystrophy without any dysregulated clonal expansion. We used a lentivector construct which incorporates an MND internal promoter, a modified self-inactivating MoMuLV LTR U3 region with myeloproliferative sarcoma virus enhancer, and a 650bp single chicken b-globin insulator encoding codon-optimized p47phox gene. Mutations in p47phox accounts for the majority of AR-CGD. The production of large-scale, consistently-high-titer lentivector using a transient 4-plasmid transfection system however, is labor- and cost-prohibitive. To address this, we applied concatemeric array transfection of pCL20cW650 MND-p47-OPT into a stable packaging cell line (GPRTG) for HIV-based lentiviral vectors to create a stable producer of VSV-G pseudotyped pCL20cW650 MND-p47OP. The concatemer array of HIV lentiviral vector construct and bleomycin selectable gene cassette showed 10 copies of lentiviral vector in a stable producer line, capable of producing vector at 10^7 IU/ml. Hematopoietic CD34+ stem cells from p47phox- CGD were transduced with pCL20cW650 MND-p47-OPT vector (MOI 10) with 2 overnight transductions following 24 hours pre-activation with SCF, FLT-3L and TPO (100ng/ml). Following three weeks in vitro culture, non-transduced or transduced p47 CGD HSC versus normal HSC were 0%, 42% and 20% p47phox positive, respectively. To determine functional correction, PMA stimulated oxidant production was measured using the dihydrorhodamine assay, confirmation similar levels of oxidant generation in transduced patient cells compared with normal controls. More than 90% of CFU were vector positive, indicating a high level of gene marking. Transduced and control naïve p47phox-patient CD34+ HSC were transplanted into 20 immunodeficient Nodscid-gc deficient (NSG) mice, and at 13 weeks post-transplant the CD13+ human neutrophils arising in mouse bone marrow were assessed for p47phox expression. Over 40% CD13+ neutrophils expressed p47phox protein from NSG mice transplanted with transduced p47-patient CD34 HSC, compared with 74% or 0% in mice transplanted with normal CD34 or p47 patient naive CD34 cells respectively. Detailed histopathology of each transplanted mice confirmed the absence of vector insertion-related myeloid tumors, and deep sequencing of bone marrow CD45+ human cells from each mouse also demonstrated polyclonal distribution of vector integration sites. In conclusion, we provide preclinical data demonstrating the efficacy and safety of high titer VSVg-pseudotyped lentivector (CL20cW650 MND-p47-OPT) generated by our stable GPTRG p47 lenti-producer for correction of p47phox-deficient human CD34 HSC. Disclosures No relevant conflicts of interest to declare.

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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