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.

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.

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.

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.

scholarly journals Mouse Mammary Tumor Virus Integration Site Selection in Human and Mouse Genomes

2007 ◽  
Vol 82 (3) ◽  
pp. 1360-1367 ◽  
Author(s):  
Alexander Faschinger ◽  
Francoise Rouault ◽  
Johannes Sollner ◽  
Arno Lukas ◽  
Brian Salmons ◽  
...  
Keyword(s):  
Integration Site ◽  
Leukemia Virus ◽  
Transcription Start Sites ◽  
Mouse Mammary Tumor ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Tumor Virus ◽  
Mouse Cells ◽  
Human And Mouse ◽  
Integration Site Selection

ABSTRACT Based on integration site preferences, retroviruses can be placed into three groups. Viruses that comprise the first group, murine leukemia virus and foamy virus, integrate preferentially near transcription start sites. The second group, notably human immunodeficiency virus and simian immunodeficiency virus, preferentially targets transcription units. Avian sarcoma-leukosis virus (ASLV) and human T-cell leukemia virus (HTLV), forming the third group, show little preference for any genomic feature. We have previously shown that some human cells sustain mouse mammary tumor virus (MMTV) infection; therefore, we infected a susceptible human breast cell line, Hs578T, and, without introducing a species-specific bias, compared the MMTV integration profile to those of other retroviruses. Additionally, we infected a mouse cell line, NMuMG, and thus we could compare MMTV integration site selection in human and mouse cells. In total, we examined 468 unique MMTV integration sites. Irrespective of whether human or mouse cells were infected, no integration bias favoring transcription start sites was detected, a profile that is reminiscent of that of ASLV and HTLV. However, in contrast to ASLV and HTLV, not even a modest tendency in favor of integration within genes was observed. Similarly, repetitive sequences and genes that are frequently tagged by MMTV in mammary tumors were not preferentially targeted in cell culture either in mouse or in human cells; hence, we conclude that MMTV displays the most random dispersion of integration sites among retroviruses determined so far.

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.

Multiple Displacement Amplification Enables Large Scale Clonal Analysis after Retroviral Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5469-5469
Author(s):  
Stephanie Bleier ◽  
Patrick Maier ◽  
Frederik Wenz ◽  
W. Jens Zeller ◽  
Stephanie Laufs ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Large Scale ◽  
Integration Site ◽  
Multiple Displacement Amplification ◽  
Quantitative Real Time Pcr ◽  
Integration Site Analysis ◽  
Reliable Quantification

Abstract Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole genome amplification named multiple displacement amplification (MDA) with respect to the even and accurate representation of retrovirally transduced genomic DNA. We were able to show that MDA is a suitable method to subsequently specify engraftment efficiencies by quantitative real-time PCR as the retroviral integrations are amplified the same way and by the same probability as all other parts of the genome. We validated the method by analyzing a dilution series containing retrovirally transduced DNA and untransduced background DNA and retroviral integrations found in primary material from a retroviral transplantation model by quantitative real-time PCR. The representation of the portion of retroviral DNA in the amplified samples was 0.9-fold (range 0.2 – 2.1-fold) of the portion determined in the original genomic DNA. Furthermore, the succession of the combination of MDA and integration site analysis by ligation-mediated PCR showed an increase in the sensitivity of the method as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show that MDA enables large scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow up analysis in gene therapy studies even from smallest amounts of starting material.

Impact of Chemoselective Pressure on Integration Site Patterns of Lentivirally Transduced Human Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4622-4622
Author(s):  
Nadja Grund ◽  
Patrick Maier ◽  
Uwe Appelt ◽  
Heike Allgayer ◽  
Frederik Wenz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
In Silico ◽  
Integration Site ◽  
General Distribution ◽  
Cytostatic Drug ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Hematologic side effects of cancer chemotherapy like myelosuppression are frequently dose-limiting. Lentiviral gene therapy with cytostatic drug resistance gene transfer to human hematopoietic stem cells (CD34+) is a promising approach to overcome this problem. In this context it is of interest if chemotherapy mediated selection has an impact on lentiviral integration site patterns of transduced hematopoietic stem cells (CD34+). Concerning this issue, human CD34+ cells transduced with a lentiviral self-inactivating (SIN) vector encoding MGMTP140K (the O6-BG resistant mutant of O6-methylguanine- DNA methyltransferase) were in vitro treated with the alkylating agent BCNU. For integration site analysis LM-PCR was performed and integration patterns of the treated and untreated CD34+ cells were analyzed and compared with an in silico set of 106 random integrations. We found different integration preferences of the lentiviral vector between either the treated (82 integrations) or the untreated (30 integrations) CD34+ cells and the in silico set: both groups showed chromosomal preferences, a significant bias for integrations in genes (74,4% in the treated, respectively 70% in the untreated to 40% in the in silico group), especially by favouring introns, a random integration distribution regarding transcription start sites (TSS), and most importantly no significant differences concerning the number of integrations in or near cancer genes. Concerning all integration characteristics we could not find significant differences when comparing the untreated with the treated group. In conclusion, the general distribution of lentiviral integrations in either untreated or treated human CD34+ cells showed no distinct differences between both groups but significant differences compared to the in silico integration set. These results suggest that chemoselection of cells lentivirally overexpressing a specific chemoresistence gene might not influence the integration pattern. Therefore chemotherapy pressure seems not to hamper the safety of lentiviral vectors in gene transfer studies.

scholarly journals Longitudinal tracking reveals sustained polyclonal repopulation of human-HSPC in humanized mice despite vector integration bias

2020 ◽  
Author(s):  
Gajendra W. Suryawanshi ◽  
Hubert Arokium ◽  
Sanggu Kim ◽  
Wannisa Khamaikawin ◽  
Samantha Lin ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Biology ◽  
Integration Site ◽  
Fetal Liver ◽  
Small Animal ◽  
Humanized Mice ◽  
Vector Integration ◽  
Longitudinal Tracking ◽  
Stem And Progenitor Cells

AbstractClonal repopulation of human hemopoietic stem and progenitor cells (HSPC) in humanized mouse models remains only partially understood due to the lack of a quantitative clonal tracking technique for low sample volumes. Here, we present a low-volume vector integration site sequencing (LoVIS-Seq) assay that requires a mere 25μl mouse blood for quantitative clonal tracking of HSPC. Using LoVIS-Seq, we longitudinally tracked 897 VIS clones—providing a first-ever demonstration of clonal dynamics of both therapeutic and control vector-modified human cell populations simultaneously repopulating in humanized mice. Polyclonal repopulation of human cells became stable at 19 weeks post-transplant indicating faster clonal repopulation than observed in humans. Multi-omics data of human fetal liver HSPC revealed that in vivo repopulating clones have significant vector integration bias for H3K36me3-enriched regions. Despite this bias the repopulation remains normal, underscoring the safety of gene therapy vectors. LoVIS-Seq provides an efficient tool for exploring gene therapy and stem cell biology in small-animal models.

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