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.

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.

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.

The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2839-2848 ◽  
Author(s):  
Hitoshi Hibino ◽  
Kenzaburo Tani ◽  
Kenji Ikebuchi ◽  
Ming-Shiuan Wu ◽  
Hajime Sugiyama ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Nonhuman Primate ◽  
Peripheral Blood ◽  
Common Marmoset ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
The Common

Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (<1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting hematopoietic stem cells.

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

The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2839-2848 ◽  
Author(s):  
Hitoshi Hibino ◽  
Kenzaburo Tani ◽  
Kenji Ikebuchi ◽  
Ming-Shiuan Wu ◽  
Hajime Sugiyama ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Nonhuman Primate ◽  
Peripheral Blood ◽  
Common Marmoset ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
The Common

Abstract Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (&lt;1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting hematopoietic stem cells.

scholarly journals a Diversity of Human Hematopoietic Differentiation Programs Identified through In Vivo Tracking of Hematopoiesis in Wiskott-Aldrich Syndrome Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3871-3871
Author(s):  
Emmanuelle Six ◽  
Laure Caccavelli ◽  
Arnaud Lecoules ◽  
Christopher L Nobles ◽  
France Male ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Blood Cell ◽  
Progenitor Cell ◽  
Conflicts Of Interest ◽  
Cell Clone ◽  
Hematopoietic Stem ◽  
Wiskott Aldrich Syndrome ◽  
New Findings ◽  
Integration Sites

Abstract The historical model of hematopoiesis, mainly derived from murine studies, is based on the existence of a single hematopoietic stem cell (HSC) capable of generating all blood cell lineages. However this model has been challenged by the proposal of four types of murine HSC, which differ by their relative contribution to the myeloid and lymphoid lineages. Here we have used data from a gene therapy trial to treat Wiskott-Aldrich syndrome (WAS) to explore hematopoiesis in humans. In the trial, the therapeutic vector (lentivirus) integrates into the genome at unique positions in each hematopoietic stem and progenitor cell (HSPC) and is consequently transmitted to all its progeny. Thus hematopoietic ontogeny in humans can be inferred by tracking the appearance of unique integration sites in fractionated blood cell populations. This provides a unique opportunity to model the developmental complexity of the human haematological system. Considerable effort over the last 15 years has been devoted to optimizing retroviral integration sites (RIS) analysis using ligation mediated PCR (LM-PCR), combined with acoustic shearing and high-throughput Illumina sequencing. Acoustic shearing enables more precise quantification of RIS abundance through the enumeration of the various sizes of shear fragments (SonicAbundance) containing a given RIS, which correspond to individual ancestor HSPC and its blood progeny. In four WAS patients treated by gene therapy, we have sorted peripheral blood samples for 5 cell types: myeloid (granulocytes and monocytes) and lymphoid subpopulations (T, B and NK cells), and analysed their RIS profile. Each RIS corresponds to a particular stem/progenitor cell clone, with a particular pattern defined by its presence or absence in each of the 5 lineages. These data are then use to reconstruct aspects of the hematopoietic hierarchy. In order to face the challenging issue of cell sorting contamination we have been using a stringent sort precision mode and we treat residual contamination explicitly in downstream statistical models. Using these approaches, we have characterized up to tens of thousands RIS per patients with a follow up of 4 years. In order to minimize biases due to sparse sampling, we concentrate on the more abundant RIS clones that can be more easily caught and are therefore analysed more reliably. We showed that a significant fraction of RIS clones are detected in a single lineage, while other RIS clones are characterized by different levels of contribution to the myeloid and lymphoid lineages, highlighting the heterogeneity of human HSC. Clones contributing to all 5 lineages are readily recovered but this study also unravels a diversity of inferred hematopoietic programs with various potentials contributing to human blood homeostasis. Longitudinal analysis of clonal dynamics is ongoing, with preliminary results highlighting the maintenance of this heterogeneity of HSPC over time. These new findings provide unique data on human hematopoiesis based on gene corrected WAS patients. Disclosures No relevant conflicts of interest to declare.

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