title: Alternative splicing caused by lentiviral integration in the human genome.
abstract: Gene transfer vectors derived from murine oncoretroviruses or human lentiviruses are widely used in human gene therapy. Integration of these vectors in the human genome may, however, have genotoxic effects, caused by deregulation of gene expression at the transcriptional or posttranscriptional level. In particular, integration of lentiviral vectors within transcribed genes has a significant potential to affect their expression by interfering with splicing and polyadenylation of primary transcripts. Aberrant splicing is caused by the usage of both constitutive and cryptic splice sites located in the retroviral backbone as well as in the gene expression cassettes. We describe a set of simple methods that allow the identification of chimeric transcripts generated by the insertion of a lentiviral vector within genes and the evaluation of their relative abundance. Identification of the splice sites, either constitutive or cryptic, that are frequently used by the cell splicing machinery within a given vector provides a useful resource to attempt recoding of the vector with the objective of reducing its potential genotoxicity in a clinical context.
authors.affiliation: Division of Genetics and Cell Biology, Istituto Scientifico H. San Raffaele, Milan, Italy.
authors.forename: Arianna
authors.initials: A
authors.lastname: Moiani
authors.forename: Fulvio
authors.initials: F
authors.lastname: Mavilio
mesh_terms: D017398:Alternative Splicing; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002874:Chromosome Mapping; D004249:DNA Damage; D004252:DNA Mutational Analysis; D018014:Gene Transfer Techniques; D005814:Genes, Viral; D015316:Genetic Therapy / Q000009:adverse effects*; D015894:Genome, Human; D006801:Humans; D016254:Mutagenesis, Insertional; D050506:Mutant Chimeric Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000302:isolation & purification; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016662:Virus Integration
chemical_list: D050506:Mutant Chimeric Proteins; D012333:RNA, Messenger