Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts
Arianna Moiani, Ylenia Paleari, Daniela Sartori, Riccardo Mezzadra, Annarita Miccio, Claudia Cattoglio, Fabienne Cocchiarella, Maria Rosa Lidonnici, Giuliana Ferrari, Fulvio Mavilio
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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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 6

RT-PCR analysis of aberrantly spliced transcripts in random, unselected clones of HEL cells transduced with the GLOBE vector.

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RT-PCR analysis of aberrantly spliced transcripts in random, unselected ...
(A) Schematic structure of the GLOBE provirus integrated between exons E(n) and E(n+1) in reverse transcriptional orientation. SD, SD site; SA, SA site. E-for, E-rev, and Globin-rev primers are indicated by arrows. The β-globin HS3 and HS2 LCR elements; promoter; I, II, and III exons; and polyadenylation signal are indicated. (B) Schematic view of the families of chimeric transcripts generated by alternative splicing to the β-globin first intron constitutive SA site (type 4) or to the β-globin promoter (type 5) and HS3 (type 6 and 7) cryptic SA sites, as identified from sequencing of the PCR products. Exons are indicated by continuous lines, spliced sequences are indicated by dotted lines. (C) Mapping of the cryptic SA (red triangles) and SD (blue triangles) sites identified in the HS3 element and the 5′ UTR of the β-globin gene. The dinucleotides at the beginning or end of a spliced sequence are indicated in blue and red, respectively. The frequency of SD and SA site usage in all the sequenced transcripts is reported in Table 3.