scholarly journals Dynamics of gene-modified progenitor cells analyzed by tracking retroviral integration sites in a human SCID-X1 gene therapy trial

Blood ◽  
2010 ◽  
Vol 115 (22) ◽  
pp. 4356-4366 ◽  
Author(s):  
Gary P. Wang ◽  
Charles C. Berry ◽  
Nirav Malani ◽  
Philippe Leboulch ◽  
Alain Fischer ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Integration Site ◽  
Severe Combined Immunodeficiency ◽  
Growth Control ◽  
Early Time ◽  
Cell Populations ◽  
Retroviral Integration ◽  
Gene Therapy Trial ◽  
Vector Integration ◽  
Integration Sites

Abstract X-linked severe-combined immunodeficiency (SCID-X1) has been treated by therapeutic gene transfer using gammaretroviral vectors, but insertional activation of proto-oncogenes contributed to leukemia in some patients. Here we report a longitudinal study of gene-corrected progenitor cell populations from 8 patients using 454 pyrosequencing to map vector integration sites, and extensive resampling to allow quantification of clonal abundance. The number of transduced cells infused into patients initially predicted the subsequent diversity of circulating cells. A capture-recapture analysis was used to estimate the size of the gene-corrected cell pool, revealing that less than 1/100th of the infused cells had long-term repopulating activity. Integration sites were clustered even at early time points, often near genes involved in growth control, and several patients harbored expanded cell clones with vectors integrated near the cancer-implicated genes CCND2 and HMGA2, but remain healthy. Integration site tracking also documented that chemotherapy for adverse events resulted in successful control. The longitudinal analysis emphasizes that key features of transduced cell populations—including diversity, integration site clustering, and expansion of some clones—were established early after transplantation. The approaches to sequencing and bioinformatics analysis reported here should be widely useful in assessing the outcome of gene therapy trials.

Gene Therapy for Severe Combined Immunodeficiency X1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 195-195 ◽  
Author(s):  
S. Hacein-Bey-Abina ◽  
M. Schmidt ◽  
F. Le Deist ◽  
A. Garrigue ◽  
A. Borkhardt ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cell ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Combined Immunodeficiency ◽  
Cell Repertoire ◽  
Retroviral Integration ◽  
Vector Integration ◽  
Clonal Proliferation ◽  
Integration Sites

Abstract We have previously reported that ex vivo retroviraly-mediated gc gene transfer into CD34 (+) bone marrow precursor cells led to the correction of the immunodeficiency in 9 out of 10 patients with X-linked severe combined immunodeficiency. Follow-up now reaches more that 6 years for the first 2 treated patients. Patients’immune function has been restored. The distribution of both TCR Vb family usage and TCR Vb CDR3 length still reveals a broadly diversified T cell repertoire. Moreover 6 years after treatment the thymus is still seeded by transduced progenitor cells as attested by the presence of TRECS in peripheral blood RTE. Among these patients, three (P4, P5 and P10) developed at 30 to 34 months after gene therapy a monoclonal T cell proliferation requiring a chemotherapy. P4 received also an allogenic HSCT from a MUD but died 26 months after the occurence of the lymphoproliferation. For P5 and P10, chemotherapy has led to an overall control of the clonal proliferation. These two patients are doing well and P5 is off treatment with a good immunological recovery. Genetic analysis of the blastic cells showed that in the two first cases the vector had integrated within or upstream of the LMO2 locus causing an insertional activation of LMO2 transcription. The last case revealed the involvement of several targeted sites, but their exact contribution to the lymphoproliferation is still under investigation. The repeated involvment of LMO2 as a site of vector integration in the proliferating T-cells points to an insertional activation of this gene as at least one of the causes of the oncogenic process. However, the long latency observed in all cases (> 30 months) suggests that additional “hits” have been required for overt desease. Synergy with gc expression and thereby induced proliferative signals (explaining occurrence in SCID-X1 patients only) is the most obvious hypothesis which we are trying to analyse in a mouse model. A deep analysis of retroviral integration patterns has been performed on patients’PBMCS by LAM-PCR to estimate the frequency of potentially harmful integration events and to assess the risk factors associated with the LTR’s strong enhancer effect of the MLV-based retroviral vector. 708 unique integration sites (IS) have been obtained from all analysed patients post-gene therapy and among them, 577 could be mapped unequivocally to the human genome. *Most of these insertions (63%) are located in the vicinity of 10kb or within the coding sequence of a known gene*. A significant peak of insertion frequency is related closely to the transcription strart site *among the 577 IS, 43 are common integration sites. Among the latter, we found out a high selection of genes involved in human oncogenic process.

Profiling Retroviral Integration Sites in Donor T-Lymphocytes for Suicide Gene Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1744-1744
Author(s):  
Frank A. Giordano ◽  
Stephanie Laufs ◽  
Katalin Z. Nagy ◽  
Boris Fehse ◽  
Agnes Hotz-Wagenblatt ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Gene Therapy ◽  
T Cells ◽  
T Lymphocytes ◽  
Homo Sapiens ◽  
Severe Combined Immunodeficiency ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Retroviral Integration ◽  
Integration Sites

Abstract Retroviral vectors encoding the herpes simplex thymidine kinase gene (HSV-Tk) have been employed to render T-lymphocytes (TLCs) sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused T cells have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Recent reports on insertional “genotoxicity” in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. Here we investigated retroviral integration sites in donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN of four donors in a clinical HSV-Tk study. A total of 114 retroviral integration sites were detected using highly sensitive and specific ligation-mediated PCR (LM-PCR). 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). The zinc ion binding group makes up 4 (resp. 6, minding that GTF2HII contains a C2H2 type and KIAA0427 a C-x8-C-x5-C-x3-H-type zinc finger) of them. Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Hit genes are currently examined more systematically in terms of function, e.g. involvement in signal transduction and transcription promoting processes. RISC (Retroviral Insertion estimate of Chromosomal Integration) scores and integration specific data will be submitted to a data warehouse, the collaborative RISC score database (CRSD). Such a systematic data collection similar to the IBMTR or EBMT databases will allow to recognize factors contributing to the safety, optimal transgene expression and persistence of transduced cells in the setting of allogenic matched donor transplantation.

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.

Long-Term Vector Integration Site Analysis Following Retroviral Mediated Gene Transfer to Hematopoietic Stem Cells for the Treatment of HIV Infection.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2350-2350
Author(s):  
Jun Hayakawa ◽  
Matthew Hsieh ◽  
Naoya Uchida ◽  
Kareem Washington ◽  
Oswald Phang ◽  
...  
Keyword(s):  
Integration Site ◽  
Genetically Modified ◽  
Late Phase ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Retroviral Integration ◽  
Vector Integration ◽  
Cd34 Cells ◽  
Integration Sites

Abstract We previously reported the efficacy of nonmyeloablative allogeneic transplantation in 2 HIV positive recipients, one of whom received retrovirus transduced hematopoietic stem cells to confer resistance to HIV (Blood. 2002; 99:698–701). Half of the donor cells were genetically modified with a Moloney murine leukemia virus (MoMLV) based HIV resistance vector containing a transdominant negative mutant Rev (TdRev) (2.58×10e8 cells) or a control vector MoMLV based vector encoding GP91phox (4.04×10e8 cells). Here we report an assessment of retroviral integration sites recovered out to 3 years post-transplantation. We identified 213 unique retroviral integration sites (RISs) from the patient’s peripheral blood samples myeloid and lymphoid cells from 1 to 36 months after reinfusion of genetically modified CD34+ cells by linear amplification-mediated PCR (LAM-PCR). While overall vector integration patterns were similar to that previously reported, only 3.75% of RISs were common among early (up to 3 months) and late samples (beyond 1 year). This low percentage of overlap offers further evidence that the early phase of hematopoiesis after transplantation derives primarily from short-term repopulating cells. Additionally, we identified 14 common integration sites (CISs). Interestingly, common integration sites were enriched among late samples; 14.9% of early RISs were CISs vs. 36.8% late. A total of 3 RISs were found near or within known oncogenes, but 2 (Integrin alpha 9 [ITGA9] and ADP-ribosylation factor-like 11 [ARL11]) were limited to early time points. An integration site near the MDS1 gene was detected in a late follow-up sample by LAM-PCR. We confirmed the integration site near the MDS1 gene by PCR with integration site-specific primers amplifying the region between the 3’-LTR of the provirus and the MDS1 locus. The MDS1 integration was not detected in early, but became detectable at all time points from 6 months to 3 years post transplant from both lymphoid and myeloid populations. Q-PCR using an integration specific Taqman probe was utilized to assess the level of clonal contribution to hematopoiesis from the clone containing the MDS1 RIS. The overall contribution of the MDS1 integrated clone remained stable during followup. Given an overall gene marking level of 0.001-0.01% with an MDS1 marking level estimated at 0.00001% in the follow up samples, the frequency of the MDS1 integrated clone is predicted to be 1/1000 marked LT-HSCs. We infused an estimated 1324 transduced LT-HSCs based upon cell dose, transduction efficiency and an estimated LT-HSC frequency of 5 per 10e3 CD34+ cells. The single integration in MDS1 in the context of non-LT-HSC limited hematopoiesis may thus account for the stability observed over time. In summary, the pattern of contribution by genetically modified cells is distinct between the early and late phase post transplantation and emphasizes the importance of long-term studies to assess the risk of integrating vectors. Additionally, the enrichment for CISs in the late phase supports the concept that integrations in the LT-HSCs favors genes that may be involved in “stemness”. Furthermore, integrations in or near putative oncogenes are likely insufficient alone as a cause of oncogenesis. Finally, LT-HSC dose may be an important determinant of the risk of integrating vectors in the context of HSC gene transfer.

Large Scale Analysis of Foamy Virus Vector Integration Sites in Human CD34+ Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 496-496 ◽  
Author(s):  
Grant D. Trobridge ◽  
Daniel G. Miller ◽  
Michael A. Jacobs ◽  
James M. Allen ◽  
Erik Olson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Large Scale ◽  
Integration Site ◽  
Human Fibroblasts ◽  
Transcription Start Sites ◽  
Vector Integration ◽  
Human Cd34 ◽  
Large Scale Analysis ◽  
Cd34 Cells ◽  
Integration Sites

Abstract The ability of retroviruses to efficiently integrate into the host cell’s genome has led to their use as gene delivery vehicles for gene therapy. However, integration in the genome can have adverse effects as observed in a gene therapy trial for X-linked SCID using an oncoretroviral vector. Recent studies have shown that an oncoretroviral vector integrated preferentially near transcription start sites and that a lentiviral vector integrated preferentially within genes. Foamy viruses are integrating retroviruses with many properties that distinguish them from onco- or lentiviruses, perhaps the most important characteristic for gene therapy being that they are non-pathogenic. We previously showed that foamy vectors efficiently transduce CD34+ SCID mouse-repopulating cells (SRCs) from human mobilized peripheral blood, demonstrating their potential for hematopoietic stem cell gene therapy. We present here the first large-scale analysis of foamy vector integration sites. Integration sites were determined by infecting human CD34+ cells or normal fibroblasts with a foamy vector carrying a bacterial origin of replication, then rescuing plasmids containing vector provirus-genomic junction sites in bacteria, and sequencing the foamy vector LTR-human genome junctions. Over 1900 unique integration sites in human CD34+ cells and 1000 unique sites in normal human fibroblasts were mapped using the human genome database. The foamy vector did not integrate preferentially into genes. The percentage of integration sites within Refseq genes in human CD34+ cells, human fibroblasts and randomly generated sites was 29, 23, and 32% respectively. Foamy vectors showed only a slight preference for integration within 1 kb 5′ or 3′ of Refseq transcription start sites. In summary, our data show that foamy vectors have a distinct integration site profile relative to oncoretroviral and lentiviral vectors. Future studies will be required to determine if the unique integration site preference of foamy vectors translates into a reduced risk for oncogenesis in gene therapy applications.

A ComParison of Retroviral Integration Sites in Donor T-Lymphocytes In Vivo and In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1403-1403
Author(s):  
Frank A. Giordano ◽  
Stephanie Laufs ◽  
Boris Fehse ◽  
K. Zsuzsanna Nagy ◽  
Agnes Hotz-Wagenblatt ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Insertional Mutagenesis ◽  
Homo Sapiens ◽  
Severe Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Retroviral Integration ◽  
Gene Therapy Trial ◽  
Integration Sites

Abstract Genetically modified T-Lymphocytes (TLCs) have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused TLCs have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Yet, reports on insertional mutagenesis in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. We investigated retroviral integration sites in vitro and in vivo. Therefore, donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN for a clinical HSV-Tk study were examined. TLCs of four different donors as well as whole blood samples of two patients transplanted with donor TLCs were analyzed either using highly sensitive and specific ligation-mediated PCR (LM-PCR). A total of 114 retroviral integration sites were detected in vitro. 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Current analyses are focusing at the in vivo pattern of retroviral integration in DNA of TLCs obtained from transplanted patient’s TLCs. Therefore we developed a new high sensitive PCR method (HS-PCR), an improved LM-PCR to even detect minimal quantities of transduced DNA.

scholarly journals DNA Palindromes with a Modest Arm Length of ≳20 Base Pairs Are a Significant Target for Recombinant Adeno-Associated Virus Vector Integration in the Liver, Muscles, and Heart in Mice

2007 ◽  
Vol 81 (20) ◽  
pp. 11290-11303 ◽  
Author(s):  
Katsuya Inagaki ◽  
Susanna M. Lewis ◽  
Xiaolin Wu ◽  
Congrong Ma ◽  
David J. Munroe ◽  
...  
Keyword(s):  
Mouse Liver ◽  
Integration Site ◽  
Marker Gene ◽  
Cpg Islands ◽  
Previous Method ◽  
Adeno Associated Virus ◽  
Transcription Start Sites ◽  
Vector Integration ◽  
Integration Sites

ABSTRACT Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of ≳20 bp (total length, ≳40 bp) are a significant target for rAAV integration. Up to ∼30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of ≳20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

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