Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations

Daniela Cesana, Jacopo Sgualdino, Laura Rudilosso, Stefania Merella, Luigi Naldini, Eugenio Montini

Research output: Contribution to journalArticlepeer-review

Abstract

Gamma-retroviral/lentiviral vectors (γRV/LV) with self-inactivating (SIN) long terminal repeats (LTRs) and internal moderate cellular promoters pose a reduced risk of insertional mutagenesis when compared with vectors with active LTRs. Yet, in a recent LV-based clinical trial for β-thalassemia, vector integration within the HMGA2 gene induced the formation of an aberrantly spliced mRNA form that appeared to cause clonal dominance. Using a method that we developed, cDNA linear amplification-mediated PCR, in combination with high-throughput sequencing, we conducted a whole transcriptome analysis of chimeric LV-cellular fusion transcripts in transduced human lymphoblastoid cells and primary hematopoietic stem/progenitor cells. We observed a surprising abundance of read-through transcription originating outside and inside the provirus and identified the vector sequences contributing to the aberrant splicing process. We found that SIN LV has a sharply reduced propensity to engage in aberrant splicing compared with that of vectors carrying active LTRs. Moreover, by recoding the identified vector splice sites, we reduced residual read-through transcription and demonstrated an effective strategy for improving vectors. Characterization of the mechanisms and genetic features underlying vector-induced aberrant splicing will enable the generation of safer vectors, with low impact on the cellular transcriptome.

Original languageEnglish
Pages (from-to)1667-1676
Number of pages10
JournalJournal of Clinical Investigation
Volume122
Issue number5
DOIs
Publication statusPublished - May 1 2012

ASJC Scopus subject areas

  • Medicine(all)

Fingerprint Dive into the research topics of 'Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations'. Together they form a unique fingerprint.

Cite this