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.
View: Text | PDF
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

×

Figure 4

Semiquantitative PCR analysis of wild-type and aberrantly spliced transcripts from the HCFC2, PLEKHA7, and DOCK5 genes in HaCaT clones transduced with the K14-GFP vector.

Options: View larger image (or click on image) Download as PowerPoint
Semiquantitative PCR analysis of wild-type and aberrantly spliced transc...
(A) cDNAs were prepared using random hexamer primers from Poly(A)+ RNA. Wild-type transcripts were amplified using the E-for and E-rev primers (arrows) annealing to the exons upstream and downstream of the provirus. Fusion transcripts were amplified using the E-for and Lenti-rev primers. (B) PCR reactions were arrested at 24, 28, and 33 cycles and run on 1% agarose gels in the following order (from left): wild-type transcript amplified from the HaCaT clone; wild-type transcript amplified from a HaCaT bulk culture; fusion transcript(s) in the HaCaT clone; and GAPDH transcript in the HaCaT clone, used for signal normalization. The GAPDH transcript in the HaCaT bulk culture was run on each gel but shown only once at the right of all panels. Transcripts were ranked in 4 arbitrary classes of relative abundance, i.e., low, when fusion transcripts were detected 8 PCR cycles later than wild-type transcripts; intermediate, when fusion transcripts were detected 4 PCR cycles later than wild-type transcripts; and high, when chimeric and wild-type transcripts were detected after the same number of PCR cycles. A fusion transcript was classified as rare (data not shown) when it was undetectable after 33 PCR cycles starting from RNA reverse transcribed with random hexamers, although it was detected and sequenced using RNA reverse transcribed with the vector-specific Lenti-RT primer (Figure 2).