1006. Effect on Expression of Including Binding Sites for the ELAV-Like Proteins HuD and HuR in Transgene Cassettes

ELAV-like (embryonic lethal abnormal vision) proteins are RNA- binding proteins first described in Drosophila. ELAV proteins are necessary for neuronal differentiation and maintenance. The human ELAV-like family includes HuB, HuC, Hel-N1, HuD and HuR and consists of three RNA-recognition motifs through which they bind with high affinity and specificity to target mRNAs with AU-rich elements. HuR is expressed in all cells, while HuD is considered neural specific. To optimize vectors for clinical application, we initially evaluated the expression patterns of several viral and mammalian promoters (Zheng and Baum, Mol. Ther., 2005). Since ELAV-like proteins can regulate mRNA stability and/or translation, we hypothesized that inclusion of binding sites for some ELAV-like proteins in expression cassettes could improve transgene expression. To test this, we selected two such binding sites (from p53 and vascular endothelial growth factor [VEGF]) for HuR, and seven binding sites for HuD (from c-fos, IL-3, c-myc-1, c-myc-2, tau, adenovirus IVa2 and growth-associated protein 43 [GAP-43]). Binding sites were inserted into the pACEF1a-luc-bGH expression plasmid, which includes the human elongation factor 1a (EF1a) promoter, luciferase cDNA (luc) and the bovine growth hormone poly A (bGH) in the expression cassette, between the luciferase cDNA and bGH poly A. The 293 and A5 (rat epithelial) cell lines were transfected with plasmids in vitro with LipofectamineTM 2000. We compared expression from these plasmids to that from expression plasmids containing the common SV40 poly A and either the EF1a or cytomegalovirus (CMV) promoter (pACEF1a-luc and pACCMV-luc; i.e., without any ELAV-like protein binding sites). In 293 cells, 24 or 48 hours post-transfection, pACCMV-luc resulted in 3 - 4 fold higher expression than pACEF1a-luc-bGH without ELAV-like protein binding sites. Addition of the p53 binding site to pACEF1a-luc-bGH increased luciferase expression 20 - 30% in 293 cells. The other ELAV-like protein binding sites either had no effect or decreased luciferase expression in 293 cells. In A5 cells, however, pACEF1a- luc-bGH containing the p53 binding site had the highest luciferase activity 24 or 48 hours post-transfection, markedly higher (2 - 3 fold) than that seen with either pACCMV-luc or pACEF1a-luc. pACEF1a-luc-bGH with binding sites from c-myc-2, VEGF and IL-3 also led to increased luciferase expression (45 - 120%) in A5 cells 48 hours post-transfection. These data support our hypothesis, especially in the A5 rat epithelial cell line. The pACEF1a-luc-bGH plasmids containing p53, c-myc-2, VEGF and IL-3 binding sites will next be tested to see if their beneficial effects are realized in vivo.