Lentivirus vectors are effective for treatment of genetic disease. However, safety associated with vector related genotoxicity is of concern and currently available models are not reliably predictive of safety in humans. We have developed hInGeTox as the first human in vitro platform that uses induced pluripotent stem cells and their hepatocyte like cell derivatives to better understand vector-host interactions that relate vectors to their potential genotoxicity. Using lentiviral vectors carrying the eGFP expression cassette under SFFV promoter activity, that only differ by their LTR and SIN configuration, we characterised vector host interactions potentially implicated in genotoxicity. To do this, lentiviral infected cells were subjected to an array of assays and data from these was used for multi-omics analyses of vector effects on cells at early and late harvest time points. Data on the integration sites of lentiviral vectors in cancer genes and differential expression levels of these genes, showed that both vector configurations are capable of activating cancer genes. Through IS tracking in bulk infected cell populations, we also saw an increase in the viral sequence count in cancer genes present over time which were differentially regulated. RNASeq also showed each vector had potential to generate fusion transcripts with the human genome suggestive of gene splicing or vector mediated readthrough from the internal SFFV promoter. Initially, after infection, both vector configurations were associated with differential expression of genes associated cytokine production, however, after culturing over time there were differences in differential expression in cells infected by each LV. This was marked in particular by the expression of genes involved in the response to DNA damage in cells transduced by the SIN vector, suggesting effects likely to prevent tumour development, in contrast to the expression of genes involved in methylation, characteristic of tumour development, in cells transduced by the LTR vector. Both sets of lentiviral infected cells were also found associated with differential expression of MECOM and LMO2 genes known to be associated with clonal dominance, supporting their potential genotoxicity. Alignment of transcriptomic signatures from iPSC and HLC infected cultures with known cancer gene signatures showed the LTR vector with a higher cancer score than the SIN vector over time in iPSC and also in HLC, which further suggests higher genotoxic potential by the LTR configuration lentivirus. By application of hInGeTox to cells infected with LV at the pre-clinical stage of development, we hope that hInGeTox can act as a useful pre-clinical tool to identify lentivirus-host interactions that may be considered contributory to genotoxicity to improve safer lentiviral vector design for gene therapy.

Abstract Lentivirus vectors are effective for treatment of genetic disease and cancer, however, vector related insertional mutagenesis related genotoxicity is of concern and currently available safety models are not reliably predictive of safety in humans. We have developed hInGeTox as the first human in vitro platform that uses induced pluripotent stem cells and their hepatocyte like derivatives to further understand LV host interaction for vector safety evaluation and design. To characterise LV for genotoxic association, we used LTR and SIN configuration LV infected cells for a multi-omics analysis on data that included LV integration sites in cancer genes and their associated differential expression, clonal tracking of IS, novel vector/host fusion transcripts and methylated cancer genes with altered gene expression after infection. We present hInGeTox as a useful pre-clinical tool to identify lentivirus contributory factors mediating genotoxicity to use for improving LV design to provide gene therapy.

Lentiviral vectors (LV) are attractive for permanent and effective gene therapy. However, integration into the host genome can cause insertional mutagenesis highlighting the importance of understanding of LV integration. Insertion site (IS) tethering is believed to involve cellular proteins such as PSIP1/LEDGF/p75, which binds to the virus pre-integration complexes (PICs) helping to target the virus genome. Transcription factors (TF) that bind both the vector LTR and host genome are also suspected influential to this. To determine the role of TF in the tethering process, we mapped predicted transcription factor binding sites (pTFBS) near to IS chosen by HIV-1 LV using a narrow 20 bp window in infected human induced pluripotent stem cells (iPSCs) and their hepatocyte-like cell (HLC) derivatives. We then aligned the pTFBS with these sequences found in the LTRs of native and self-inactivated LTRs. We found significant enrichment of these sequences for pTFBS essential to HIV-1 life cycle and virus survival. These same sites also appear in HIV-1 patient IS and in mice infected with HIV-1 based LV. This in silco data analysis suggests pTFBS present in the virus LTR and IS sites selected by HIV-1 LV are important to virus survival and propagation.

Integrating viral gene transfer vectors are commonly used gene delivery tools in clinical gene therapy trials providing stable integration and continuous gene expression of the transgene in the treated host cell. However, integration of the reverse-transcribed vector DNA into the host genome is a potentially mutagenic event that may directly contribute to unwanted side effects. A comprehensive and accurate analysis of the integration site (IS) repertoire is indispensable to study clonality in transduced cells obtained from patients undergoing gene therapy and to identify potential in vivo selection of affected cell clones. To date, next-generation sequencing (NGS) of vector-genome junctions allows sophisticated studies on the integration repertoire in vitro and in vivo. We have explored the use of the Illumina MiSeq Personal Sequencer platform to sequence vector ISs amplified by non-restrictive linear amplification-mediated PCR (nrLAM-PCR) and LAM-PCR. MiSeq-based high-quality IS sequence retrieval is accomplished by the introduction of a double-barcode strategy that substantially minimizes the frequency of IS sequence collisions compared to the conventionally used single-barcode protocol. Here, we present an updated protocol of (nr)LAM-PCR for the analysis of lentiviral IS using a double-barcode system and followed by deep sequencing using the MiSeq device.

Human Gene TherapyVol. 25, No. 6 Pioneer PerspectiveVector Integration and TumorigenesisChristof von Kalle, Annette Deichmann, and Manfred SchmidtChristof von KalleNationales Centrum für Tumorerkrankungen, 69120 Heidelberg, Germany.Abteilung für Translationale Onkologie, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany.Search for more papers by this author, Annette DeichmannNationales Centrum für Tumorerkrankungen, 69120 Heidelberg, Germany.Abteilung für Translationale Onkologie, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany.Search for more papers by this author, and Manfred SchmidtNationales Centrum für Tumorerkrankungen, 69120 Heidelberg, Germany.Abteilung für Translationale Onkologie, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany.Search for more papers by this authorPublished Online:20 Jun 2014https://doi.org/10.1089/hum.2014.2525AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookXLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byGermline T cell receptor exchange results in physiological T cell development and function1 February 2023 | Nature Communications, Vol. 14, No. 1A review on CRISPR/Cas: a versatile tool for cancer screening, diagnosis, and clinic treatment26 May 2023 | Functional & Integrative Genomics, Vol. 23, No. 2CRISPR-Cas System: The Current and Emerging Translational Landscape7 April 2023 | Cells, Vol. 12, No. 8Current advances of CRISPR-Cas technology in cell therapyCell Insight, Vol. 1, No. 6Combination therapy with CAR T cells and oncolytic viruses: a new era in cancer immunotherapy22 June 2021 | Cancer Gene Therapy, Vol. 29, No. 6A review of the underlying genetics and emerging therapies for canine cardiomyopathiesJournal of Veterinary Cardiology, Vol. 40Genetical engineering for NK and T cell immunotherapy with CRISPR/Cas9 technology: Implications and challengesCellular Immunology, Vol. 369T-Cell Dysfunction as a Limitation of Adoptive Immunotherapy: Current Concepts and Mitigation Strategies3 February 2021 | Cancers, Vol. 13, No. 4Nanotechnology Promotes Genetic and Functional Modifications of Therapeutic T Cells Against Cancer20 February 2020 | Advanced Science, Vol. 7, No. 10Gene Therapy Clinical TrialsHighly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors19 November 2019 | Nature Communications, Vol. 10, No. 1Production of CAR T-cells by GMP-grade lentiviral vectors: latest advances and future prospects17 July 2019 | Critical Reviews in Clinical Laboratory Sciences, Vol. 56, No. 6At the end of the beginning: immunotherapies as living drugs15 July 2019 | Nature Immunology, Vol. 20, No. 8Imaging of CAR T-Cells in Cancer Patients: Paving the Way to Treatment Monitoring and Outcome Prediction3 May 2019 | Journal of Nuclear Medicine, Vol. 60, No. 7Approaches to treat immune hot, altered and cold tumours with combination immunotherapies4 January 2019 | Nature Reviews Drug Discovery, Vol. 18, No. 3A Plasmid-Expressed CRISPR/Cas9 System Suppresses Replication of HSV Type I in a Vero Cell Culture18 April 2019 | Molecular Biology, Vol. 53, No. 1Gene editing in T cell therapyJournal of Genetics and Genomics, Vol. 44, No. 9Chimeric antigen receptor T-cells for B-cell malignanciesTranslational Research, Vol. 187Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection22 February 2017 | Nature, Vol. 543, No. 7643 Volume 25Issue 6Jun 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Christof von Kalle, Annette Deichmann, and Manfred Schmidt.Vector Integration and Tumorigenesis.Human Gene Therapy.Jun 2014.475-481.http://doi.org/10.1089/hum.2014.2525Published in Volume: 25 Issue 6: June 20, 2014PDF download