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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 April 23, 2012
Citation Information: J Clin Invest. 2012;122(5):1667-1676. https://doi.org/10.1172/JCI62189.
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Research Article Article has an altmetric score of 8

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

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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 4

Representation of aberrant splicing events within the LV backbone and quantification of transcription levels of LV backbone portions.

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Representation of aberrant splicing events within the LV backbone and qu...
(A) Schematic representation of the position of the recoded splice sites within the LV backbone. The different mutations were distributed in 3 different vector constructs (indicated as MutSD, Mut1_13, and Mut14_15). RRE, rev-responsive element; wPRE, woodchuck hepatitis posttranscriptional regulatory element. (B) Titers of the 3 different recoded vectors. The titer is defined as number of transducing units per milliliter (TU/ml) of vector preparation. (C) Representation of the positions of the 4 TaqMan primer sets on SIN.LV.PGK and LV.SF.LTR vectors. U3RU5 recognizes the portion from the LV.LTR to the SD1, encompassing the cryptic splice acceptor SA1; LV.FUSION recognizes the internally spliced transcript (SD1 to SA2); SA-PPT recognizes the sequence downstream of the canonical splice acceptor SA2, encompassing the cryptic donor SD4; and GFP recognizes the GFP transgene sequence. (D–G) RT-qPCR results of transcription levels of different LV backbone portions performed on JY cells transduced with SIN.LV.PGK or LV.SF.LTR at MOI 0.1 (white bars) or MOI 10 (black bars). ΔCt values were obtained using β2 microglobulin (B2M) as normalizer to measure the relative expression levels with respect to the housekeeping cellular gene (vs. B2M). ΔCt values obtained using GFP as normalizer to measure the relative expression levels with respect to transgene expression (vs. GFP). (H) ΔCt values obtained using GFP as normalizer from JY cells transduced with SIN.LV.PGK. The recoded vectors are indicated. Probe sets used are indicated. Statistical evaluation was performed by 1-way ANOVA with Bonferroni’s correction (*P < 0.001; **P < 0.0001).

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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