title: 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.affiliation: San Raffaele Telethon Institute for Gene Therapy and Division of Regenerative Medicine, Stem Cells and Gene Therapy, Milan, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Cesana
authors.forename: Jacopo
authors.initials: J
authors.lastname: Sgualdino
authors.forename: Laura
authors.initials: L
authors.lastname: Rudilosso
authors.forename: Stefania
authors.initials: S
authors.lastname: Merella
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
authors.forename: Eugenio
authors.initials: E
authors.lastname: Montini
mesh_terms: D017398:Alternative Splicing; D001483:Base Sequence; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D020043:Genes, Essential; D006412:Hematopoietic Stem Cells / Q000821:virology; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000502:physiology*; D016180:Lentivirus Infections / Q000235:genetics*; D016254:Mutagenesis, Insertional; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D061251:Primary Cell Culture; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020079:Terminal Repeat Sequences; D059467:Transcriptome; D016662:Virus Integration
chemical_list: D050506:Mutant Chimeric Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger