Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts
Arianna Moiani, … , Giuliana Ferrari, Fulvio Mavilio
Arianna Moiani, … , Giuliana Ferrari, Fulvio Mavilio
Published April 23, 2012
Citation Information: J Clin Invest. 2012;122(5):1653-1666. https://doi.org/10.1172/JCI61852.
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Research Article

Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts

Abstract

Retroviral vectors integrate in genes and regulatory elements and may cause transcriptional deregulation of gene expression in target cells. Integration into transcribed genes also has the potential to deregulate gene expression at the posttranscriptional level by interfering with splicing and polyadenylation of primary transcripts. To examine the impact of retroviral vector integration on transcript splicing, we transduced primary human cells or cultured cells with HIV-derived vectors carrying a reporter gene or a human β-globin gene under the control of a reduced-size locus-control region (LCR). Cells were randomly cloned and integration sites were determined in individual clones. We identified aberrantly spliced, chimeric transcripts in more than half of the targeted genes in all cell types. Chimeric transcripts were generated through the use of constitutive and cryptic splice sites in the HIV 5ι long terminal repeat and gag gene as well as in the β-globin gene and LCR. Compared with constitutively spliced transcripts, most aberrant transcripts accumulated at a low level, at least in part as a consequence of nonsense-mediated mRNA degradation. A limited set of cryptic splice sites caused the majority of aberrant splicing events, providing a strategy for recoding lentiviral vector backbones and transgenes to reduce their potential posttranscriptional genotoxicity.

Authors

Arianna Moiani, Ylenia Paleari, Daniela Sartori, Riccardo Mezzadra, Annarita Miccio, Claudia Cattoglio, Fabienne Cocchiarella, Maria Rosa Lidonnici, Giuliana Ferrari, Fulvio Mavilio

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Figure 2

5′RACE and RT-PCR analysis of aberrantly spliced transcripts in clones of Jurkat and SupT1 T cells, primary T cells, HaCaT keratinocytes, and myeloid HEL cells.

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5′RACE and RT-PCR analysis of aberrantly spliced transcripts in clones o...
(A) Schematic structure of the integrated provirus. The “expression cassette” represents the IRES-GFP splice trap in Jurkat and SupT1 cells, the CMV-GFP cassette in transduced T cells, the K14-GFP cassette in HaCaT cells, and the β-globin gene in reverse orientation in HEL cells (see Figure 1). E(n) indicates the exon upstream of the lentiviral integration, and E(n+1) indicates the downstream exon. SD1, gag major SD site; SA7, gag major SA site. Arrows indicate the vector-specific primer used to reverse transcribe the mRNAs into cDNAs (Lenti-RT), the upstream exon-specific forward primer (E-for), and the vector-specific reverse primer (Lenti-rev) used in the RT-PCR reaction. (B) 5′ RACE and RT-PCR products in representative SupT1/Jurkat, HaCaT, T lymphocytes, and HEL clones stained by ethidium bromide on 1% agarose gels. Transcripts were amplified using the Lenti-rev primer and a primer specific for the upstream exon of the gene identified on top of each lane. Molecular weight markers (sizes in bp) are indicated on the left of each gel.