title: The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors.
abstract: The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression from a variety of viral vectors, although the precise mechanism is not known. WPRE is most effective when placed downstream of the transgene, proximal to the polyadenylation signal. We hypothesized that WPRE likely reduces viral mRNA readthrough transcription by improving transcript termination, which in turn would increase viral titers and expression. Using a Cre-lox-mediated plasmid-based assay, we found significant readthrough transcription from gamma-retroviral vector (RV) long terminal repeat (wt RV-LTR) and RV LTR with a self-inactivating deletion (SIN RV-LTR). WPRE, when placed upstream of the RV LTRs, significantly reduced readthrough transcription. Readthrough, present at much lower levels with the SIN HIV-1 LV-LTR, was also reduced with WPRE. When placed in RV vectors, WPRE increased total RV genomic mRNA; and increased viral titers from transiently transfected 293T cells and stable PG13 producer cells by 7- to 15-fold. The mechanism of increased titers and expression was not due to increased nuclear mRNA export, increased rate of viral transcription or a significant increase in viral mRNA half-life. Our results showed that WPRE improved vector genomic transcript termination to increase titers and expression from RVs.
authors.affiliation: Division of Hematology-Oncology, Department of Pediatrics and Pathology, Keck School of Medicine University of Southern California, Childrens Hospital Los Angeles, Los Angeles, CA, USA.
authors.forename: T
authors.initials: T
authors.lastname: Higashimoto
authors.forename: F
authors.initials: F
authors.lastname: Urbinati
authors.forename: A
authors.initials: A
authors.lastname: Perumbeti
authors.forename: G
authors.initials: G
authors.lastname: Jiang
authors.forename: A
authors.initials: A
authors.lastname: Zarzuela
authors.forename: L-J
authors.initials: LJ
authors.lastname: Chang
authors.forename: D B
authors.initials: DB
authors.lastname: Kohn
authors.forename: P
authors.initials: P
authors.lastname: Malik
mesh_terms: D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics*; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D016364:Reading Frames; D050436:Regulatory Elements, Transcriptional; D012190:Retroviridae / Q000235:genetics*; D015302:Simian Immunodeficiency Virus / Q000235:genetics; D020079:Terminal Repeat Sequences; D014158:Transcription, Genetic; D014162:Transfection; D019076:Transgenes