Enhanced Expression of Transgenes from Adeno-Associated Virus Vectors with the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element: Implications for Gene Therapy

Abstract

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) evolved to stimulate the expression of intronless viral messages. To determine whether this ability to enhance expression could be useful in nonviral and heterologou s viral gene delivery systems, we analyzed the ability of the WPRE to elevate the expression of a cDNA encoding the green fluorescent protein (GFP) in these contexts. We find that the WPRE can stimulate the expression of GFP when the gene is delivered by transfection or transduction with recombinant adeno-associated virus (AAV). Enhancement occurred both during transient expression and when the gene is stably incorporated into the genome of target cells. This enhancement required that the WPRE be located in cis within the GFP message, and was observed in both transformed cell lines and primary human fibroblasts. These results demonstrate that the WPRE will be an effective tool for increasing the long-term expression of transgenes in gene therapy. OVERVIEW SUMMARYThe optimization of transgene expression at the posttranscriptional level will enhance the capacity of vector delivery systems to produce therapeutic results in target tissues. In this study, the woodchuck hepatitis virus posttranscriptional regulatory elem ent (WPRE) is shown to enhance the expression of an eGFP reporter gene in both transient and stable contexts, when delivered either by transfection or by transduction with recombinant AAV. This enhancement of transgene expression is observed both in cell lines and in primary cells. Inclusion of the WPRE in transgenic messages will better the prospects for therapeutic levels of transgene expression using currently existing gene delivery technologies.

Intravenous administration of an AAV-2 vector for the expression of factor IX in mice and a dog model of hemophilia B

“…Use of the promoter alone in the construct rAAV-LSPhFIX resulted in very low human FIX expression in C57Bl/6 mice in the absence of these cis-acting regulatory sequences. Addition of the WPRE element increased gene expression approximately 25-fold, in agreement with previous publications, [22][23] while addition of the b-globin IVS-II intron sequence enhanced this level approximately 85-fold. Interestingly, the combination of both WPRE and intron in the same vector yielded no significant increase in gene expression in comparison to the vector containing only the intron, perhaps signifying a redundancy in these two elements in their ability to Average cFIX values are given for dog G10 between days 11 and 113.…”

“…In comparison, cell culture analysis of expression levels from the two vectors showed that while the LSP promoter showed a liver-specific expression pattern, expression from the MFG promoter/intron was higher even in those liver-derived cell lines. This divergence between cell culture expression and in vivo expression levels has been observed in a number of other vector systems including hydrodynamic-based gene delivery, 20 retroviral 27 and rAAV vectors, 23 and underscores the need for careful interpretation of vector design. FIX expression following i.v.…”

“…PREs such as that derived from the woodchuck hepatitis virus 21 have also been incorporated into transgene expression cassettes to optimize protein production. The woodchuck post-transcriptional regulatory element (WPRE) increases transgene expression from retroviral, 22 and AAV 23 vectors through mechanisms that affect mRNA processing, including the enhancement of polyadenylation, mRNA export and the inhibition of splicing. 24,25 Here, we investigate a sideby-side comparison of these regulatory elements and show that an intron has the greatest impact on transgene expression in these vectors.…”

Hypothalamic pro-opiomelanocortin gene delivery ameliorates obesity and glucose intolerance in aged rats

“…The woodchuck hepatitis virus post-transcriptional regulatory element is placed downstream of the Pomc transgene to enhance its expression [28] (Fig. 1).…”

Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord

“…Utilizing recombinant adeno-associated viral BDNF/WPRE (rAAV/BDNF/WPRE)-mediated overexpression of BDNF 26,27 near the spinal lumbar dorsal horn after partial nerve injury, we hypothesized that chronic spinal BDNF over-expression would alleviate chronic neuropathic pain. This would provide an alternate method to cell transplants that supply the antinociceptive agent.…”