Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations
Daniela Cesana, … , Luigi Naldini, Eugenio Montini
Daniela Cesana, … , Luigi Naldini, Eugenio Montini
Published May 1, 2012; First published April 23, 2012
Citation Information: J Clin Invest. 2012;122(5):1667-1676. https://doi.org/10.1172/JCI62189.
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Category: Research Article

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

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.

Authors

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

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Figure 1

cLAM-PCR procedure for the retrieval of LV cellular fusion transcripts.

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cLAM-PCR procedure for the retrieval of LV cellular fusion transcripts. ...
(A) Scheme of the experimental procedure for cLAM-PCR. Total mRNA is retrotranscribed into double-stranded cDNA (ds cDNA) using oligo-dT primers. Linear PCR uses a biotinylated primer located upstream/downstream of a known LV splice site, allowing extension into vector or an unknown cellular portion of a chimeric transcript. Single-stranded product is purified by streptavidin-coupled magnetic beads, double stranded using Klenow enzyme, and cut using restriction enzymes (REs). A linker cassette compatible with the restriction enzyme cut is ligated, and 2 sequential nested PCRs are performed. The final PCR products are then sequenced. ss cDNA, single-stranded cDNA. (B) FACS plots showing the percentage of GFP+ in JY cells and CD34+ HSPCs after SIN.LV.PGK transduction. The VCN and the MOI are indicated. Numbers in the graph indicate the percentage of GFP+ cells. (C) Representative band pattern of cLAM-PCR performed on mRNA from SIN.LV.PGK-transduced cells. Retrotranscribed mRNA (RT+) and negative controls (RT–) were used. By sequencing, bands in retrotranscribed mRNA samples corresponded to aberrant transcripts or unspliced internal control sequences. Rare faint bands in negative controls corresponded to oligonucleotide dimers or concatemers. H2O, negative PCR control from the linear amplification reaction to the second exponential phase. M, marker.