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J Clin Invest. 2012 May;122(5):1667-76. doi: 10.1172/JCI62189. Epub 2012 Apr 23.

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

Author information

1
San Raffaele Telethon Institute for Gene Therapy and Division of Regenerative Medicine, Stem Cells and Gene Therapy, Milan, Italy.

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.

PMID:
22523064
PMCID:
PMC3336994
DOI:
10.1172/JCI62189
[Indexed for MEDLINE]
Free PMC Article

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