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.

scholarly journals Frequency and Spectrum of Genomic Integration of Recombinant Adeno-Associated Virus Serotype 8 Vector in Neonatal Mouse Liver

2008 ◽  
Vol 82 (19) ◽  
pp. 9513-9524 ◽  
Author(s):  
Katsuya Inagaki ◽  
Chuncheng Piao ◽  
Nicole M. Kotchey ◽  
Xiaolin Wu ◽  
Hiroyuki Nakai
Keyword(s):  
Insertional Mutagenesis ◽  
Neonatal Period ◽  
Integration Site ◽  
Adeno Associated Virus ◽  
Genomic Integration ◽  
Vector Integration ◽  
Multiple Organs ◽  
Vector Genome ◽  
Virus Serotype ◽  
Integration Sites

ABSTRACT Neonatal injection of recombinant adeno-associated virus serotype 8 (rAAV8) vectors results in widespread transduction in multiple organs and therefore holds promise in neonatal gene therapy. On the other hand, insertional mutagenesis causing liver cancer has been implicated in rAAV-mediated neonatal gene transfer. Here, to better understand rAAV integration in neonatal livers, we investigated the frequency and spectrum of genomic integration of rAAV8 vectors in the liver following intraperitoneal injection of 2.0 × 1011 vector genomes at birth. This dose was sufficient to transduce a majority of hepatocytes in the neonatal period. In the first approach, we injected mice with a β-galactosidase-expressing vector at birth and quantified rAAV integration events by taking advantage of liver regeneration in a chronic hepatitis animal model and following partial hepatectomy. In the second approach, we performed a new, quantitative rAAV vector genome rescue assay by which we identified rAAV integration sites and quantified integrations. As a result, we find that at least ∼0.05% of hepatocytes contained rAAV integration, while the average copy number of integrated double-stranded vector genome per cell in the liver was ∼0.2, suggesting concatemer integration. Twenty-three of 34 integrations (68%) occurred in genes, but none of them were near the mir-341 locus, the common rAAV integration site found in mouse hepatocellular carcinoma. Thus, rAAV8 vector integration occurs preferentially in genes at a frequency of 1 in approximately 103 hepatocytes when a majority of hepatocytes are once transduced in the neonatal period. Further studies are warranted to elucidate the relationship between vector dose and integration frequency or spectrum.

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 Supramolecular nanosubstrate–mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies

2020 ◽  
Vol 6 (43) ◽  
pp. eabb7107
Author(s):  
Peng Yang ◽  
Shih-Jie Chou ◽  
Jindian Li ◽  
Wenqiao Hui ◽  
Wenfei Liu ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Integration Site ◽  
Genetic Diseases ◽  
Proliferative Potential ◽  
Adeno Associated Virus ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Green Fluorescent ◽  
Virus Integration

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate–mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)–encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

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.

scholarly journals 532. Foamy Viral Vector Integration Sites in SCID-Repopulating Cells After MGMTP140K-Mediated In Vivo Selection

2015 ◽  
Vol 23 ◽  
pp. S213-S214
Author(s):  
Miles E. Olszko ◽  
Jennifer E. Adair ◽  
Ian Linde ◽  
Dustin T. Rae ◽  
Patty Trobridge ◽  
...  
Keyword(s):  
Viral Vector ◽  
Vector Integration ◽  
In Vivo Selection ◽  
Integration Sites

scholarly journals Clonal reconstruction from co-occurrence of vector integration sites allows accurate quantification of expanding clones in vivo

2021 ◽  
Author(s):  
Sebastian Wagner ◽  
Christoph Baldow ◽  
Andrea Calabria ◽  
Laura Rudilosso ◽  
Pierangela Gallina ◽  
...  
Keyword(s):  
Viral Vectors ◽  
R Package ◽  
Repeated Measurements ◽  
Preclinical Model ◽  
Gene Therapies ◽  
Vector Integration ◽  
Reconstruction Methods ◽  
Integration Sites ◽  
Safety Considerations

High transduction rates of viral vectors in gene therapies (GT) and experimental hematopoiesis ensure a high frequency of gene delivery, although multiple integration events can occur in the same cell. Therefore, tracing of integration sites (IS) leads to mis-quantification of the true clonal spectrum and limits safety considerations in GT. Hence, we use correlations between repeated measurements of IS abundances to estimate their mutual similarity and identify clusters of co-occurring IS, for which we assume a clonal origin. We evaluate the performance, robustness and specificity of our methodology using clonal simulations. The reconstruction methods, implemented and provided as an R-package, are further applied to experimental clonal mixes and to a preclinical model of hematopoietic GT. Our results demonstrate that clonal reconstruction from IS data allows to overcome systematic biases in the clonal quantification as an essential prerequisite for the assessment of safety and long-term efficacy of GT involving integrative vectors.

scholarly journals Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo

2007 ◽  
Vol 117 (8) ◽  
pp. 2241-2249 ◽  
Author(s):  
Kerstin Schwarzwaelder ◽  
Steven J. Howe ◽  
Manfred Schmidt ◽  
Martijn H. Brugman ◽  
Annette Deichmann ◽  
...  
Keyword(s):  
Integration Site ◽  
Site Distribution ◽  
Vector Integration

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.

scholarly journals Comprehensive Integration Site Analysis of Human Immunodeficiency Virus during In Vivo Infections Reveals Genomic Regions of Enrichment and Clonal Expansion

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2518-2518
Author(s):  
Kevin G Haworth ◽  
Lauren E Schefter ◽  
Zachary K. Norgaard ◽  
Jennifer E Adair ◽  
Hans-Peter Kiem
Keyword(s):  
Integration Site ◽  
Site Analysis ◽  
Genome Wide ◽  
Integration Site Analysis ◽  
Integration Sites ◽  
Infected Cells ◽  
Genomic Regions ◽  
Hiv Integration

Abstract BACKGROUND A key event in the lifecycle of Human Immunodeficiency Virus (HIV) is permanent integration into the infected cells genome. In addition to allowing long-term persistence of the virus, this results in a trackable mark carried in all infected cells. Active HIV replication represses cellular pathways, preventing further cell division. This would imply that any specific integration site (IS) which is clonally expanded either during active or repressed viral infection arises from either a dormant/inactive virus, or is perturbing local gene expression, leading to increased cell proliferation. Alternatively, a cell carrying HIV provirus could proliferate due to T-cell specific antigen stimulation. By analyzing the patterns of integration sites detected in cell cultures and tissue samples from animal models of HIV infection, we can better understand the basic virology of integration site selection and determine what may potentially drive infected cells to persist despite effective treatment regimens. METHODS Jurkat reporter cell lines or primary human CD4+ cells were cultured and infected with various strains of HIV including both CCR5 and CXCR4 tropic viruses. Infected cells were cultured up to 21 days post infection, then analyzed for HIV proviral integration sites by next-generation sequencing. For in vivo studies, NSG mice were infused with human CD34+ hematopoietic stem/progenitor cells, resulting in a reconstituted human immune system including high levels of CD4+ T cells capable of sustaining HIV infection. After 16 weeks post-challenge, tissues were collected and subjected to integration site analysis for HIV proviral DNA. Identified integration sites were mapped and compared across multiple parameters to identify chromosomal regions and associated genes enriched for integration events, as well as clonally expanded cells in vivo. RESULTS Genome-wide analysis of HIV integration sites reveals a remarkably similar chromosomal landscape both in tissue culture infection of Jurkat cells and in vivo infection data (Figure 1), as well as across multiple HIV strains. As previously observed, the majority of integrations occur near or within gene coding regions thought to be actively transcribed at time of infection. However, certain areas of the genome, and even unique genes, are enriched for IS in individual samples. In addition to these genomic regions of enrichment, we also observe specific clonal outgrowth of unique integration events in genes previously unidentified in the literature. Three genes in particular exhibit a significant increase of integration events during acute infection which are 3x higher than predicted by random chance alone. We also observe integration events in genes that have been documented by other labs in HIV+ clinical patient samples, however in our active infection models, we do not see those specific genes enriched or expanded. This could indicate that these genes play a role in persistence that is only present during anti-retroviral therapy which suppresses active replication. CONCLUSIONS We have cataloged the most extensive HIV IS library to date in both relevant tissue culture models and in vivo infection studies, including over 245,000 unique integration events and three different HIV strains commonly used in research. Genome-wide correlation studies reveal regions significantly enriched for HIV integrations and genes which repeatedly exhibit clonal outgrowth in multiple animals. These types of studies are now being applied to human patient samples to determine if latency and persistence of infection can be mapped to unique integration events or genes of interest. Such information may indicate when and how the latent HIV reservoir is seeded and what types of therapy or treatments are most effective at targeting and eliminating these populations. Circos plot comparing HIV integrations sites (IS) identified either during in vitro cell culture infections (black bars), or in vivo infection studies using humanized mice (red bars). The outer ring is composed of human chromosomes each of which are divided into 25kB fragment bins. Total number of unique integration sites identified in each bin is represented by the height of the histogram bars. The in vitro IS concentric ring scale represents increments of 25 outwards up to 250 while the in vivo IS scales inwards in increments of 2 up to 16. Figure 1 Comparison of in vitro vs in vivo HIV Integration Sites. Figure 1. Comparison of in vitro vs in vivo HIV Integration Sites. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership.

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.

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