title: Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter.
abstract: Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors.
authors.affiliation: Molecular Biology Interdepartmental PhD Program, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Aaron R
authors.identifier: http://orcid.org/0000-0003-4588-2513
authors.initials: AR
authors.lastname: Cooper
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Georgia R
authors.initials: GR
authors.lastname: Lill
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Eric H
authors.initials: EH
authors.lastname: Gschweng
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA Department of Pediatrics, University of California, Los Angeles, Los Angeles, CA 90095, USA Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA dkohn1@mednet.ucla.edu.
authors.forename: Donald B
authors.identifier: http://orcid.org/0000-0003-1840-6087
authors.initials: DB
authors.lastname: Kohn
mesh_terms: D004742:Enhancer Elements, Genetic; D005091:Exons; D015870:Gene Expression; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007438:Introns; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics*; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D025822:Ubiquitin C / Q000235:genetics*
chemical_list: C094814:EEF1A1 protein, human; D020648:Peptide Elongation Factor 1; D025822:Ubiquitin C