scholarly journals Linear Amplification Mediated PCR for the retrieval of lentiviral vector integration sites from tumors induced by insertional mutagenesis.

2013 ◽  
Author(s):  
Marco Ranzani ◽  
Marco Ranzani ◽  
Stefano Annunziato ◽  
Fabrizio Benedicenti ◽  
Pierangela Gallina ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Lentiviral Vector ◽  
Linear Amplification ◽  
Vector Integration ◽  
Integration Sites

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.

scholarly journals Distribution of Lentiviral Vector Integration Sites in Mice Following Therapeutic Gene Transfer to Treat β-thalassemia

2011 ◽  
Vol 19 (7) ◽  
pp. 1273-1286 ◽  
Author(s):  
Keshet Ronen ◽  
Olivier Negre ◽  
Shannah Roth ◽  
Charlotte Colomb ◽  
Nirav Malani ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Lentiviral Vector ◽  
Therapeutic Gene ◽  
Vector Integration ◽  
Integration Sites

Lentiviral vector integration sites in human NOD/SCID repopulating cells

2006 ◽  
Vol 8 (10) ◽  
pp. 1197-1207 ◽  
Author(s):  
Stephanie Laufs ◽  
Guillermo Guenechea ◽  
Africa Gonzalez-Murillo ◽  
K. Zsuzsanna Nagy ◽  
M. Luz Lozano ◽  
...  
Keyword(s):  
Lentiviral Vector ◽  
Vector Integration ◽  
Integration Sites

scholarly journals Vector Integration and Efficacy of CD19-Directed CAR T Cell Therapy in Acute Lymphoblastic Leukemia (ALL) and Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4548-4548
Author(s):  
Christopher L Nobles ◽  
John K Everett ◽  
Shantan Reddy ◽  
Joseph A Fraietta ◽  
David L. Porter ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Lentiviral Vector ◽  
Advisory Committees ◽  
Vector Integration ◽  
Car T ◽  
Engineered T Cells ◽  
Integration Sites ◽  
Equity Ownership

Abstract CD19-specific Chimeric Antigen Receptor (CTL019)-engineered T-cells provide a breakthrough for personalized cancer therapy. An anti-CD19 CAR gene with 41BB costimulatory domain is delivered into patient T-cells ex vivo using a lentiviral vector, expanded in culture and then reinfused into patients. While dramatically successful for some treatment-refractory cancers, a significant proportion of patients do not experience therapeutic levels of CAR T cell expansion - thus it is important to investigate factors driving successful expansion in responders in more detail. Here we have analyzed sites of lentiviral vector integration in CAR T cells from trials to ALL and CLL, comparing successful and unsuccessful therapy in longitudinal data sets for 40 subjects. The location of each integrated vector marks a cell lineage uniquely allowing the fate mapping of individual CAR-engineered T cells in the infusion product and after adoptive transfer. We found that 81.4% of integrations had occurred in annotated transcription units which is consistent with previous reports for lentiviral vector integration sites. Relatively larger and more diverse populations of CAR-modified T-cells were associated with improved outcome (Chao1 index, p=0.043). Population sizes were also significantly more diverse in the infusion product compared with day 28 post-infusion, and more diverse at this time point when comparing responders with non-responders, or even partial responders with non-responders (p<0.05). Thus, the population size measured in the infusion product and one month after infusion forecasts the patient's response to CTL019. Vector integration can also modify activity of nearby genes, as we recently reported for an integration event in the DNA methylcytosine dioxygenase gene TET2, where clonal CTL019 expansion was associated with successful therapy (Fraietta et. al., Nature, 2018). Insertional mutagenesis was evaluated here over five criteria, including i) clonal expansion after infusion, ii) increasing frequency of unique integration sites per gene after infusion, iii) development of orientation bias, iv) long-term persistence, and v) accumulation of integration site clusters. Our analysis disclosed genes and cell pathways, including apoptosis and epigenetics, associated with superior cell proliferation and persistence. These data thus provide multiple approaches for improvement of the anti-leukemia activity of CAR T cells. Disclosures Fraietta: Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Porter:Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board; Genentech: Other: Spouse employment. Frey:Servier Consultancy: Consultancy; Novartis: Consultancy. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Siegel:Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Tmunity: Research Funding; Parker Foundation: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees. Melenhorst:Shanghai UNICAR Therapy, Inc: Consultancy; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding.

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.

Mutagenesis via Insertional or Restriction Enzyme-Mediated Integration (REMI) as a Tool to Tag Pathogenicity Related Genes in Plant Pathogenic Fungi

1999 ◽  
Vol 380 (7-8) ◽  
pp. 855-864 ◽  
Author(s):  
F. J. Maier ◽  
W. Schäfer
Keyword(s):  
Restriction Enzyme ◽  
Molecular Basis ◽  
Insertional Mutagenesis ◽  
Transformation Efficiency ◽  
Pathogenic Fungi ◽  
Transformation Protocol ◽  
Vector Integration ◽  
Future Developments ◽  
Genetically Determined ◽  
Remi Mutagenesis

Abstract Random insertional mutagenesis is a powerful tool to investigate the molecular basis of most genetically determined processes, for example in pathogenic fungi. An improved version of this method is the insertional mutagenesis via restriction enzyme mediated integration (REMI). Transformation efficiency and mode of vector integration are species dependent and further influenced by vector conformation, restriction enzyme activity, and transformation protocol. An overview is given, covering the mutants and already identified genes obtained after REMI mutagenesis. An outlook describes the future developments in the field.

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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