828. Optimizing the Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE) for Safety and Function in Lentiviral Vectors

We have been developing a gene transfer system based on the non-primate lentivirus equine infectious anemia virus (EIAV) for human gene transfer. Previously, we reported that recombinant EIAV vectors pseudotyped with the G-glycoprotein of the vesicular stomatitis virus (VSV) are able to efficiently transduce muscle, hematopoietic, and neuronal cells. The vector has been optimized for vector titers and trans-gene expression in transduced cells by incorporating various cis-acting elements (EIAV Rev responsive element (RRE), Mason Pfizer monkey transport element (CTE), post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE)) into the vector. The optimal vector configuration resulted in placement of the RRE upstream of the internal promoter and the WPRE into the 3-prime untranslated region downstream of the reporter gene. Although this configuration of vector yields the highest viral titers and transgene protein expression in target cells, safety concerns have been raised regarding the potential oncogenic activity of the truncated WHV X protein encoded in this element (Kingsman et al, Gene Therapy 12, 3, 2005). The WPRE used in our vectors is a 900 bp tripartite element described by Donello et al (J Virol, 78, 5085, 1998). The gamma and the alpha sub-elements provide the majority of the post-transcriptional effects on localization of viral RNA with some contribution from the beta sub-element which also encodes the WHV X protein, the function of which is still not clearly understood. To test whether the X protein is being expressed from our constructs, we fused a luciferase reporter gene in frame with the X protein. Luciferase activity was observed in transduced cells, which clearly suggests that the truncated X protein is being expressed. We are determining the levels of expression in primary human hepatocytes and in transduced mouse liver. We have constructed a minimal WPRE sequence of 513 bp that retains its complete cis-acting function. This element contains a 32 amino acid portion of protein X. We are currently making mutations in the protein X promoter which is a 21 nucleotide domain immediately upstream of the ATG codon of protein X and mutating the start codon and X ORF to prevent any aberrant X protein expression in target cells.