VISPA2: a scalable pipeline for high-throughput identification and annotation of vector integration sites

Spinozzi, Giulio; Calabria, Andrea; Brasca, Stefano; Beretta, Stefano; Merelli, Ivan; Milanesi, Luciano; Montini, Eugenio

doi:10.1186/s12859-017-1937-9

Cited by 25 publications

(33 citation statements)

References 38 publications

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“… 3 , 4 To identify the vector ISs, the sequences of the PCR products generated by MiSeq Illumina sequencing were mapped on the mouse genome by a dedicated bioinformatics pipeline. 41 Overall, we mapped 1,673 ISs from in vitro cultures, 3,080 from PB (at 16 weeks), and 1,738 BM cells (at 12 weeks). Tables S2 A and S2B summarize the number of ISs identified for each sample.…”

Section: Resultsmentioning

confidence: 99%

Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-Thalassemia

Lidonnici

Paleari

Tiboni

et al. 2018

Molecular Therapy - Methods & Clinical Development

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Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for β-thalassemia, we performed in vitro and in vivo studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes. Because these vectors did not show significant advantages, GLOBE lentiviral vector (LV) was elected for further safety characterization. To support the use of hematopoietic stem cells (HSCs) transduced by GLOBE LV for the treatment of β-thalassemia, we conducted toxicology, tumorigenicity, and biodistribution studies in compliance with the OECD Principles of Good Laboratory Practice. We demonstrated a lack of toxicity and tumorigenic potential associated with GLOBE LV-transduced cells. Vector integration site (IS) studies demonstrated that both murine and human transduced HSCs retain self-renewal capacity and generate new blood cell progeny in the absence of clonal dominance. Moreover, IS analysis showed an absence of enrichment in cancer-related genes, and the genes targeted by GLOBE LV in human HSCs are well known sites of integration, as seen in other lentiviral gene therapy trials, and have not been associated with clonal expansion. Taken together, these integrated studies provide safety data supporting the clinical application of GLOBE-mediated gene therapy for β-thalassemia.

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Section: Resultsmentioning

confidence: 99%

Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-Thalassemia

Lidonnici

Paleari

Tiboni

et al. 2018

Molecular Therapy - Methods & Clinical Development

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“…The genomic DNA portions flanking the LV insertion sites were retrieved by state-ofthe-art LAM (17) and a sonication-based linkermediated PCR method, 18 sequenced by Illumina technology, and mapped on the human genome (version hg19) by a dedicated bioinformatics pipeline. 19 Overall, the study mapped >2,700 integration sites from in vitro cultured HSPCs transduced with or without CsA and BM cells of mice transplanted with HSPCs transduced in the presence (n = 6) or absence of CsA (n = 6; Fig. 2A).…”

Section: Impact Of One-hit Csa and Rapa-based Protocols On Gene Transmentioning

confidence: 88%

“…The vector integration sites were mapped on the human genome (version hg19) using VISPA2, a bioinformatics pipeline designed to generate a the list of insertion sites and the nearest annotated RefSeq gene. 19 Filtering and normalization procedures to remove potential PCR contaminations with other independent studies and clonal abundance measurements were performed, as described previously. 1,2 Common insertion sites (CIS) were identified by the kernel convolution framework method developed by De Ridder et al 20 Enrichment analysis for ontological classes among the vector targeted genes was performed by the Genomic Regions Enrichment of Annotations Tool.…”

Section: Integration Site Mapping and Bioinformatics Analysismentioning

confidence: 99%

Assessing the Impact of Cyclosporin A on Lentiviral Transduction and Preservation of Human Hematopoietic Stem Cells in Clinically RelevantEx VivoGene Therapy Settings

et al. 2019

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Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to lentiviral vector (LV) transduction without compromising their biological properties remains critical for broad-range implementation of gene therapy as a treatment option for several inherited diseases. This study demonstrates that the use of one-hit ex vivo LV transduction protocols based on either cyclosporin A (CsA) or rapamycin enable as efficient gene transfer as the current two-hit clinical standard into bone marrow–derived CD34+ cells while better preserving their engraftment capacity in vivo. CsA was additive with another enhancer of transduction, prostaglandin E2, suggesting that tailored enhancer combinations may be applied to overcome multiple blocks to transduction simultaneously in HSPC. Interestingly, besides enhancing LV transduction, CsA also significantly reduced HSPC proliferation, preserving the quiescent G0 fraction and the more primitive multipotent progenitors, thereby yielding the highest engraftment levels in vivo. Importantly, no alterations in the vector integration profiles could be detected between CsA and control transduced HSPC. Overall, the present findings contribute to the development of more efficient and sustainable LV gene therapy protocols, underscoring the benefits of scaling down required vector doses, as well as shortening the HSPC ex vivo culture time.

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“…Currently, limited tools has been specifically designed for transgene integration and rearrangement studies or release their codes. We compared the performance of transgeneR with VISPA2 [10]. Using the same…”

Section: Comparison With Independent Toolsmentioning

confidence: 99%

“…Next generation sequencing has revolutionized genomic/gene expression studies and its utility in GM animals and crops is anticipated to increase. Attempts have been made towards this goal, such as Target Loci Amplification (TLA) [8], VISPA2 [9,10], CONTRAILS [11] and analysis pipelines [12]. Among them, TLA is an integrated platform by combining experimental targeted locus amplification and sequencing technologies to study the transgene events.…”

Section: Introductionmentioning

confidence: 99%

TransgeneR: a one-stop tool for transgene integration and rearrangement discovery using sequencing data

Meng

2018

Preprint

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Genetically modified organisms are widely used in lifescience research, agriculture and in commercial products.However, in most cases, the genetic modification in the host genome is often less well characterized with respect to integration location, copy number and host gene expression. The application of next generation sequencing technologies has enabled the characterization of transgene events but still limited by the lack of computational tools. We present a one-stop R tool, transgeneR,

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VISPA2: a scalable pipeline for high-throughput identification and annotation of vector integration sites

Cited by 25 publications

References 38 publications

Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-Thalassemia

Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-Thalassemia

Assessing the Impact of Cyclosporin A on Lentiviral Transduction and Preservation of Human Hematopoietic Stem Cells in Clinically RelevantEx VivoGene Therapy Settings

TransgeneR: a one-stop tool for transgene integration and rearrangement discovery using sequencing data

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