The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy
Eugenio Montini, Daniela Cesana, Manfred Schmidt, Francesca Sanvito, Cynthia C. Bartholomae, Marco Ranzani, Fabrizio Benedicenti, Lucia Sergi Sergi, Alessandro Ambrosi, Maurilio Ponzoni, Claudio Doglioni, Clelia Di Serio, Christof von Kalle, Luigi Naldini
Eugenio Montini, Daniela Cesana, Manfred Schmidt, Francesca Sanvito, Cynthia C. Bartholomae, Marco Ranzani, Fabrizio Benedicenti, Lucia Sergi Sergi, Alessandro Ambrosi, Maurilio Ponzoni, Claudio Doglioni, Clelia Di Serio, Christof von Kalle, Luigi Naldini
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Research Article

The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy

Abstract

γ-Retroviral vectors (γRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between γRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of γRVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as γRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by γRVs. This integration-site bias was intrinsic to γRVs, as it was also observed for SIN γRVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.

Authors

Eugenio Montini, Daniela Cesana, Manfred Schmidt, Francesca Sanvito, Cynthia C. Bartholomae, Marco Ranzani, Fabrizio Benedicenti, Lucia Sergi Sergi, Alessandro Ambrosi, Maurilio Ponzoni, Claudio Doglioni, Clelia Di Serio, Christof von Kalle, Luigi Naldini

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Figure 5

Gene expression analysis at LV.SF.LTR integration sites in tumors.

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Gene expression analysis at LV.SF.LTR integration sites in tumors. (A–C)...
(A–C) Expression of the indicated genes was measured by Q–RT-PCR on tumor-infiltrated BM or spleen cDNA (see also Supplemental Table 6). Expression data for primary and serially transplanted tumors with an integrated vector near the tested gene (INT) and phenotype-matched tumors with integrated vector in different sites or without integrations (No INT) are plotted. Each point is the fold change relative to matched-type tumor-infiltrated BM or spleen from the mock group (control level = 1); the horizontal bar represents the average. P value of the Mann-Whitney test comparison between the samples is indicated. P < 0.05 is considered significant. Genomic region targeted by the vector (vector position and orientation are represented by arrows) is shown below each set of expression data. Genes above the thick horizontal bar (chromosome) are transcribed from left to right; those below the chromosome are transcribed in the opposite direction. (A) Tgtp, which encodes for an interferon-inducible T cell–specific GTPase and whose TSS maps 530 bp from the vector integration, was overexpressed in both tumor-infiltrated BM and spleen of 2 primary and 4 secondary transplanted mice bearing the same integration; the expression of other genes surrounding the integration was not altered (see details in Supplemental Table 6). (B) Another integration from the same groups of mice mapped within the Sos1 (37) oncogene, leading to its significant overexpression. (C) Vector integration occurred within the Eps15 (38) oncogene, leading to its overexpression in tumors of 1 primary and 2 secondary transplanted mice.