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

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

10.1101/2021.08.10.455805 ◽

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.

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Ub-ISAP: a streamlined UNIX pipeline for mining unique viral vector integration sites from next generation sequencing data

BMC Bioinformatics ◽

10.1186/s12859-017-1719-4 ◽

2017 ◽

Vol 18 (1) ◽

Cited By ~ 2

Author(s):

Atul Kamboj ◽

Claus V. Hallwirth ◽

Ian E. Alexander ◽

Geoffrey B. McCowage ◽

Belinda Kramer

Keyword(s):

Next Generation Sequencing ◽

Viral Vector ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Vector Integration ◽

Integration Sites ◽

Generation Sequencing

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Bioinformatic Clonality Analysis of Next-Generation Sequencing-Derived Viral Vector Integration Sites

Human Gene Therapy Methods ◽

10.1089/hgtb.2011.219 ◽

2012 ◽

Vol 23 (2) ◽

pp. 111-118 ◽

Cited By ~ 36

Author(s):

Anne Arens ◽

Jens-Uwe Appelt ◽

Cynthia C. Bartholomae ◽

Richard Gabriel ◽

Anna Paruzynski ◽

...

Keyword(s):

Next Generation Sequencing ◽

Viral Vector ◽

Next Generation ◽

Vector Integration ◽

Clonality Analysis ◽

Integration Sites ◽

Generation Sequencing

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Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

Blood ◽

10.1182/blood-2008-09-176412 ◽

2009 ◽

Vol 113 (21) ◽

pp. 5094-5103 ◽

Cited By ~ 23

Author(s):

Brian C. Beard ◽

Reeteka Sud ◽

Kirsten A. Keyser ◽

Christina Ironside ◽

Tobias Neff ◽

...

Keyword(s):

Animal Studies ◽

Large Animal ◽

Hematopoietic Stem ◽

Transcription Start Sites ◽

Leukocyte Antigen ◽

In Vivo Selection ◽

Integration Sites ◽

Methylguanine Methyltransferase

Abstract Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n = 157) and after (n = 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of MGMTP140K gene-modified cells after in vivo selection in dog leukocyte antigen (DLA)–matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs more than 800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.

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Enhanced Stability and Controlled Delivery of MOF Encapsulated Vaccines and Their Immunogenic Response In Vivo

10.26434/chemrxiv.7335122.v1 ◽

2018 ◽

Author(s):

Michael Luzuriaga ◽

Raymond P. Welch ◽

Madushani Dharmawardana ◽

Candace Benjamin ◽

Shaobo Li ◽

...

Keyword(s):

Viral Vector ◽

Controlled Delivery ◽

Conformational Structure ◽

Spectroscopy Analysis ◽

Zeolitic Imidazolate Framework ◽

Structural Conformation ◽

Depth Study ◽

Viral Nanoparticle

<div><div><div><p>Vaccines have an innate tendency to lose their structural conformation upon environmental and chemical stressors. A loss in conformation reduces the therapeutic ability to prevent the spread of a pathogen. Herein, we report an in-depth study of zeolitic imidazolate framework-8 (ZIF-8) and its ability to provide protection for a model viral vector against dena- turing conditions. The immunoassay and spectroscopy analysis together demonstrate enhanced thermal and chemical stability to the conformational structure of the encapsulated viral nanoparticle. The long-term biological activity of this virus-ZIF composite was investigated in animal models to further elucidate the integrity of the encapsulated virus, the bio-safety, and immunogenicity of the overall composite. Additionally, histological analysis found no observable tissue damage in the skin or vital organs in mice, following multiple subcutaneous administrations. This study shows that ZIF-based protein composites are strong candidates for improved preservation of proteinaceous drugs, are biocompatible, and capable of controlling the release and adsorption of drugs in vivo.</p></div></div></div>

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Faculty Opinions recommendation of In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1166488.627478 ◽

2009 ◽

Author(s):

Vivian Cody

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Dihydrofolate Reductase ◽

Human Embryonic Stem Cell ◽

Embryonic Stem ◽

In Vivo Selection ◽

Selection Of

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The In Vivo Selection Method in Breast Cancer Metastasis

International Journal of Molecular Sciences ◽

10.3390/ijms22041886 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1886

Author(s):

Jun Nakayama ◽

Yuxuan Han ◽

Yuka Kuroiwa ◽

Kazushi Azuma ◽

Yusuke Yamamoto ◽

...

Keyword(s):

Cancer Cells ◽

Cell Lines ◽

Cancer Metastasis ◽

Metastatic Cancer ◽

Expression Patterns ◽

Selection Method ◽

Gene Signatures ◽

In Vivo Selection ◽

Metastatic Cancer Cells

Metastasis is a complex event in cancer progression and causes most deaths from cancer. Repeated transplantation of metastatic cancer cells derived from transplanted murine organs can be used to select the population of highly metastatic cancer cells; this method is called as in vivo selection. The in vivo selection method and highly metastatic cancer cell lines have contributed to reveal the molecular mechanisms of cancer metastasis. Here, we present an overview of the methodology for the in vivo selection method. Recent comparative analysis of the transplantation methods for metastasis have revealed the divergence of metastasis gene signatures. Even cancer cells that metastasize to the same organ show various metastatic cascades and gene expression patterns by changing the transplantation method for the in vivo selection. These findings suggest that the selection of metastasis models for the study of metastasis gene signatures has the potential to influence research results. The study of novel gene signatures that are identified from novel highly metastatic cell lines and patient-derived xenografts (PDXs) will be helpful for understanding the novel mechanisms of metastasis.

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Performance of ectomycorrhizal alders exposed to specific Canadian oil sands tailing stressors under in vivo bipartite symbiotic conditions

Canadian Journal of Microbiology ◽

10.1139/cjm-2015-0703 ◽

2016 ◽

Vol 62 (7) ◽

pp. 543-549 ◽

Cited By ~ 4

Author(s):

Martin Beaudoin-Nadeau ◽

André Gagné ◽

Cyntia Bissonnette ◽

Pier-Anne Bélanger ◽

J. André Fortin ◽

...

Keyword(s):

Oil Sands ◽

Seedling Survival ◽

Naphthenic Acids ◽

Paxillus Involutus ◽

Alnus Incana ◽

In Vivo Selection ◽

Alnus Viridis ◽

Leaf Necrosis ◽

Different Strains

Canadian oil sands tailings are predominately sodic residues contaminated by hydrocarbons such as naphthenic acids. These conditions are harsh for plant development. In this study, we evaluated the effect of inoculating roots of Alnus viridis ssp. crispa and Alnus incana ssp. rugosa with ectomycorrhizal fungi in the presence of tailings compounds. Seedlings were inoculated with 7 different strains of Paxillus involutus and Alpova diplophloeus and were grown under different treatments of NaCl, Na2SO4, and naphthenic acids in a growth chamber. Afterwards, seedling survival, height, dry biomass, leaf necrosis, and root mycorrhization rate were measured. Paxillus involutus Mai was the most successful strain in enhancing alder survival, health, and growth. Seedlings inoculated with this strain displayed a 25% increase in survival rate, 2-fold greater biomass, and 2-fold less leaf necrosis compared with controls. Contrary to our expectations, A. diplophloeus was not as effective as P. involutus in improving seedling fitness, likely because it did not form ectomycorrhizae on roots of either alder species. High intraspecific variation characterized strains of P. involutus in their ability to stimulate alder height and growth and to minimize leaf necrosis. We conclude that in vivo selection under bipartite symbiotic conditions is essential to select effective strains that will be of use for the revegetation and reclamation of derelict lands.

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