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 6

Oncogenic LV/Braf chimeric transcripts in an LV.SF.LTR tumor.

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Oncogenic LV/Braf chimeric transcripts in an LV.SF.LTR tumor. (A) Ge...
(A) Genomic position of an LV.SF.LTR integration in a myeloid tumor targeting intron 11 of Braf. Chromosome (Chr) number and coordinates are indicated on top. The genomic interval covering exons 11 to 14 (gray boxes) is depicted. The position of the LV.SF.LTR integration (black box; LTR direction is indicated by the gray arrow) clusters with 20 SB integrations from sarcomas (39) in a narrow 4-kb region within introns 11 and 12. (B) RT-PCR using primers complementary to LV LTR and exon 22 of Braf on cDNA from the tumor described in A amplified a 1500-bp product. RT+, tumor cDNA; RT–, tumor RNA processed without reverse transcriptase; M molecular size markers. (C) The sequence of the RT-PCR product in B aligns to LV and to Braf exons. Black bars, amplified cDNA sequence; dashed lines, splicing events; F and R arrows, primers used for cDNA amplification; 3′UTR, 3′ untranslated region of Braf; SD, LV 5′ splice donor site. The cDNA sequence was LV specific up to the splice donor site (HIV) fused to the correct splice junction of exon 13 of Braf (boxed); exon 12 appears to be skipped. The first putative starting ATG codon in exon 13 is in the correct frame to produce a truncated Braf protein (indicated).