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 g-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.

Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS

“…When placed in the 3 0 untranslated region of gene transfer cassettes, the WPRE enhances the expression of the transgene by increasing both nuclear and cytoplasmic mRNA levels, early during the biogenesis of RNA transcripts. 2,6 Although the WPRE is coupled in part to the CRM1-dependent export machinery, 7 its posttranscriptional effects do not result from increased RNA export or from an increased rate of transcription or longer half-life of RNA species but rather from improved 3 0 end transcript processing. The WPRE seems to increase the amount of polyadenylated transcripts and clearly augments the size of the polyA tail of RNA.…”

“…When placed upstream of the 3 0 LTR, the WPRE improves transcript termination and therefore can significantly reduce transcript read-through, particularly with Moloney-derived vectors. 6,8 Probably in part through this effect, the WPRE can also augment the titers of viral preparations although results are variable and the exact mechanisms underlying this effect remain to be determined. 6,8 The magnitude of the effects of WPRE vary upon the specific context of the promoter, transgene and cell type, and this has been shown both for its effects on transgene expression 4 or viral titers.…”

“…6,8 Probably in part through this effect, the WPRE can also augment the titers of viral preparations although results are variable and the exact mechanisms underlying this effect remain to be determined. 6,8 The magnitude of the effects of WPRE vary upon the specific context of the promoter, transgene and cell type, and this has been shown both for its effects on transgene expression 4 or viral titers. 3,6,8 For this reason, the effects of this 3 0 regulatory element need to be evaluated in specific contexts.…”

“…6,8 The magnitude of the effects of WPRE vary upon the specific context of the promoter, transgene and cell type, and this has been shown both for its effects on transgene expression 4 or viral titers. 3,6,8 For this reason, the effects of this 3 0 regulatory element need to be evaluated in specific contexts.…”

Evolving Lessons on Nanomaterial-Coated Viral Vectors for Local and Systemic Gene Therapy

“…To address the safety concerns associated with the generation of replicationcompetent LV, packaging systems have been modified by removing nonessential viral genes and separating functional viral components into separate expression plasmids [80]. To increase the transduction efficacy of LVs, additional elements, such as a lentiviral central polypurine tract, WPRE or PRE variants devoid of the X protein open reading frame, are routinely being introduced into lentiviral systems [81][82][83]. For example, incorporation of WPRE into a lentiviral system has been shown to increase lentiviral titer and transgene expression by improving the half-life, export and polyadenylation of mRNA [82].…”