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 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 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 Mutations altering acetylated residues in the CTD of HIV-1 integrase cause defects in proviral transcription at early times after integration of viral DNA

2020 ◽  
Vol 16 (12) ◽  
pp. e1009147
Author(s):  
Shelby Winans ◽  
Stephen P. Goff
Keyword(s):  
Life Cycle ◽  
Histone Modifications ◽  
Reverse Transcription ◽  
Integration Site ◽  
Viral Dna ◽  
Large Defect ◽  
Central Function ◽  
Site Distribution ◽  
Hiv 1

The central function of the retroviral integrase protein (IN) is to catalyze the integration of viral DNA into the host genome to form the provirus. The IN protein has also been reported to play a role in a number of other processes throughout the retroviral life cycle such as reverse transcription, nuclear import and particle morphogenesis. Studies have shown that HIV-1 IN is subject to multiple post-translational modifications (PTMs) including acetylation, phosphorylation and SUMOylation. However, the importance of these modifications during infection has been contentious. In this study we attempt to clarify the role of acetylation of HIV-1 IN during the retroviral life cycle. We show that conservative mutation of the known acetylated lysine residues has only a modest effect on reverse transcription and proviral integration efficiency in vivo. However, we observe a large defect in successful expression of proviral genes at early times after infection by an acetylation-deficient IN mutant that cannot be explained by delayed integration dynamics. We demonstrate that the difference between the expression of proviruses integrated by an acetylation mutant and WT IN is likely not due to altered integration site distribution but rather directly due to a lower rate of transcription. Further, the effect of the IN mutation on proviral gene expression is independent of the Tat protein or the LTR promoter. At early times after integration when the transcription defect is observed, the LTRs of proviruses integrated by the mutant IN have altered histone modifications as well as reduced IN protein occupancy. Over time as the transcription defect in the mutant virus diminishes, histone modifications on the WT and mutant proviral LTRs reach comparable levels. These results highlight an unexpected role for the IN protein in regulating proviral transcription at early times post-integration.

scholarly journals In vivo dynamics of human hematopoietic stem cells: novel concepts and future directions

2019 ◽  
Vol 3 (12) ◽  
pp. 1916-1924 ◽  
Author(s):  
Serena Scala ◽  
Alessandro Aiuti
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Integration Site ◽  
Transcriptome Profiling ◽  
Future Directions ◽  
Hematopoietic Stem ◽  
Vector Integration ◽  
Shed Light

Abstract Unveiling the mechanisms and the cellular dynamics at the basis of human hematopoietic homeostasis has been a main focus for the scientific community since the discovery of a pool of multipotent hematopoietic stem cells (HSCs) capable of sustaining the hematopoietic output throughout life and after transplantation. Recently, new works shed light on the (1) differentiation paths, (2) size and replication rate of human HSC population at steady state, and (3) role of the distinct subpopulations comprising the hematopoietic stem and progenitor cell reservoir after transplantation. These papers exploited cutting-edge technologies, including vector integration site clonal tracking, spontaneous mutations, and deep transcriptome profiling. Here we discuss the latest updates in human hematopoietic system biology and in vivo dynamics, highlighting novel concepts and common findings deriving from different approaches and the future directions of these studies. Taken together, this information contributed to partially resolving the complexity of the in vivo HSC behavior and has major implications for HSC transplantation and gene therapy as well as for the development of future therapies.

The Clonal Inventory of Gene Corrected Hematopoiesis in Three Successful Clinical Gene Therapy Trials

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3733-3733
Author(s):  
Kerstin Schwarzwaelder ◽  
Manfred Schmidt ◽  
Annette Deichmann ◽  
Steven J. Howe ◽  
Marion G. Ott ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Sanger Sequencing ◽  
Integration Site ◽  
Cellular Gene ◽  
Post Transplantation ◽  
Cellular Gene Expression ◽  
Site Distribution ◽  
Clinical Gene Therapy

Abstract To enhance the safety and efficacy of future gene therapy trials using integrating vector systems it is necessary to analyse the clonality of the genetically modified cell pool. The comparative analyses of integration site distribution and cellular gene expression will further reveal causal mechanisms of in vivo clone selection. We followed the repopulation clonality of 21 patients which participated in 3 successful clinical gene therapy trials via linear amplification mediated PCR (LAM PCR). We identified the integration sites (IS) of pre and post transplantation samples by Sanger sequencing and accomplished RNA analyses. The comparative results from all trials showed that vector integration is favoured in gene coding regions, in particular transcriptional start sites. In both X-SCID trials significantly more post transplantation IS were located in or in the vicinity of genes encoding proteins with kinase or transferase activity. In pre transplantation samples no uniform gene class was overrepresented. In both trials we detected common insertion sites mainly post transplantation and the effect was more pronounced in the trial where 4 patients developed vector induced leukemia. Notably, we detected no significant differences regarding the IS distribution in leukemic versus non leukemic patients. The gene corrected repopulation of patient 1 and 2 of the X-CGD trial was polyclonal until 542 and 777 days after transplantation, respectively. 5 months after therapy dominant clones appeared. In patient 1, between 616 and 820 days post transplantation (post mortem time point) the number of participating clones and the contribution of a dominant clone decreased while the contribution of another dominant clone increased. In both patients the integrated vector induced the upregulation of the genes MDS1/EVI1, PRDM16 or SETBP1 and thus led to the in vivo expansion of affected cell clones. From these trials we sequenced >2000 unique IS by Sanger sequencing and several thousand via pyrosequencing (datamining is ongoing). Our data show that the integration site distribution was non random, that the integrated vector influenced the cellular gene expression which caused subtle to massive changes in the repopulation clonality and that it will soon be possible to define the clonal inventory of patients using next generation sequencing technologies.

Clonal Analysis of Hematopoietic Stem Cells after Serial Transplantation of Animals Receiving MGMTP140K Gene Transduced Cells Following Chemotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1182-1182
Author(s):  
Stephanie Laufs ◽  
Ursula Sorg ◽  
Veronika Kleff ◽  
Laila Gao ◽  
Michael Flasshove ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Integration Site ◽  
Hematopoietic Stem ◽  
Vector Integration ◽  
Serial Transplantation ◽  
Selection Of

Abstract Gene transfer of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) into hematopoietic stem cells has been shown to protect hematopoiesis from the toxic side effects of O6-guanine alkylating drugs such as BCNU, ACNU or temozolomide (TMZ). In addition, MGMT gene transfer allows efficient in vivo selection of transduced hematopoietic stem cells and enrichment of genetically corrected cells in the context of gene therapy for monogenetic diseases. We here have analysed the long-term effect of MGMT gene transfer on the hematopoietic stem cell compartment using an in vivo murine transplantation/gene therapy model and a retroviral vector carrying the gene for MGMTP140K, a mutant resistant to the wtMGMT-specific inhibitor O6-benzylguanine (BG). Serial transplants were performed and primary, secondary as well as tertiary recipients were treated with combined BG/ACNU, BG/BCNU or BG/TMZ chemotherapy at doses myeloablative in non-MGMT-protected hematopoiesis. Serial transplantation was performed with 1.8 – 3.0 x 106 mononuclear bone marrow cells and 2 to 3 animals were transplanted per primary or secondary animal. While initial gene transfer efficiency was low (1–5% of cells engrafted at week four) chemotherapy resulted in efficient selection of transduced cells in primary animals (70–90% transgene expression in peripheral blood). Secondary and tertiary recipients showed 40–80% transgene expression even before CTX. Efficient stem cell engraftment and protection from CTX was demonstrated in > 90% of secondary animals, while tertiary recipients clearly demonstrated compromised engraftment and a substantial number of animals did not survive CTX treatment. Retroviral vector integration site analysis to study the clonality of hematopoiesis of stem cells by ligation mediated PCR (LM-PCR) was performed in the serially transplanted mice. In three mice of the secondary transplantation cohort we detected 3, 0, and 6 clones, respectively. In three mice of the tertiary transplantation cohort 7, 2, and 2 clones, respectively, were found. Thus, an exhaustion of transduced hematopoiesis following regenerative stress by high dose chemotherapy was not evident. Of the total 20 detected clones one could not be mapped to the mouse genome, while the others could be blasted against the mouse genome (assembly 2004, http://genome.ucsc.edu/; >99.5% identity). It turned out that 5 of 8 integrations landed in RefSeq in the tertiary transplantation cohort, while 3 of 8 integrations occurred in RefSeq genes in the secondary transplantation cohort. This suggests that clones profit from the transcription machinery of their integration site. Thus, our LM-PCR results indicate that the multiclonality of hematopoiesis is conserved after serial transplants which may be considered a safety feature for drug-resistance gene therapy. Furthermore, vector integration in highly resistant stem cells is favored in actively transcribed genomic regions.

scholarly journals Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing

2020 ◽  
Author(s):  
Joost van Haasteren ◽  
Altar M Munis ◽  
Deborah R Gill ◽  
Stephen C Hyde
Keyword(s):  
Long Range ◽  
Insertional Mutagenesis ◽  
Lentiviral Vector ◽  
Integration Site ◽  
Low Cost ◽  
Safety Evaluation ◽  
Read Length ◽  
Chromosomal Dna ◽  
Wide Range

Abstract The gene and cell therapy fields are advancing rapidly, with a potential to treat and cure a wide range of diseases, and lentivirus-based gene transfer agents are the vector of choice for many investigators. Early cases of insertional mutagenesis caused by gammaretroviral vectors highlighted that integration site (IS) analysis was a major safety and quality control checkpoint for lentiviral applications. The methods established to detect lentiviral integrations using next-generation sequencing (NGS) are limited by short read length, inadvertent PCR bias, low yield, or lengthy protocols. Here, we describe a new method to sequence IS using Amplification-free Integration Site sequencing (AFIS-Seq). AFIS-Seq is based on amplification-free, Cas9-mediated enrichment of high-molecular-weight chromosomal DNA suitable for long-range Nanopore MinION sequencing. This accessible and low-cost approach generates long reads enabling IS mapping with high certainty within a single day. We demonstrate proof-of-concept by mapping IS of lentiviral vectors in a variety of cell models and report up to 1600-fold enrichment of the signal. This method can be further extended to sequencing of Cas9-mediated integration of genes and to in vivo analysis of IS. AFIS-Seq uses long-read sequencing to facilitate safety evaluation of preclinical lentiviral vector gene therapies by providing IS analysis with improved confidence.

scholarly journals Single-dose MGTA-145/plerixafor leads to efficient mobilization and in vivo transduction of HSCs with thalassemia correction in mice

2021 ◽  
Vol 5 (5) ◽  
pp. 1239-1249
Author(s):  
Chang Li ◽  
Kevin A. Goncalves ◽  
Tamás Raskó ◽  
Amit Pande ◽  
Sucheol Gil ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Mononuclear Cells ◽  
Vector System ◽  
Hemopoietic Stem Cell ◽  
Peripheral Blood Mononuclear ◽  
Gene Therapy Approach ◽  
Vector Integration ◽  
Genome Wide ◽  
Gfp Marking

Abstract We have developed an in vivo hemopoietic stem cell (HSC) gene therapy approach without the need for myelosuppressive conditioning and autologous HSC transplantation. It involves HSC mobilization and IV injection of a helper-dependent adenovirus HDAd5/35++ vector system. The current mobilization regimen consists of granulocyte colony-stimulating factor (G-CSF) injections over a 4-day period, followed by the administration of plerixafor/AMD3100. We tested a simpler, 2-hour, G-CSF–free mobilization regimen using truncated GRO-β (MGTA-145; a CXCR2 agonist) and plerixafor in the context of in vivo HSC transduction in mice. The MGTA-145+plerixafor combination resulted in robust mobilization of HSCs. Importantly, compared with G-CSF+plerixafor, MGTA-145+plerixafor led to significantly less leukocytosis and no elevation of serum interleukin-6 levels and was thus likely to be less toxic. With both mobilization regimens, after in vivo selection with O6-benzylguanine (O6BG)/BCNU, stable GFP marking was achieved in >90% of peripheral blood mononuclear cells. Genome-wide analysis showed random, multiclonal vector integration. In vivo HSC transduction after mobilization with MGTA-145+plerixafor in a mouse model for thalassemia resulted in >95% human γ-globin+ erythrocytes at a level of 36% of mouse β-globin. Phenotypic analyses showed a complete correction of thalassemia. The γ-globin marking percentage and level were maintained in secondary recipients, further demonstrating that MGTA145+plerixafor mobilizes long-term repopulating HSCs. Our study indicates that brief exposure to MGTA-145+plerixafor may be advantageous as a mobilization regimen for in vivo HSC gene therapy applications across diseases, including thalassemia and sickle cell disease.

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