We have also observed that liver eGFP expression was lower in rats receiving vectors containing miR142-T compared to those injected with AAV2/8-TBG-eGFP (Fig. S2F and S2B respectively). We speculate Cisplatin DNA Synthesis inhibitor that the addition of the miR142-T sequence to the eGFP transcript may reduce its stability. Inclusion of the WPRE sequence in the 3�� UTR of the transgene expression cassette has been reported to increase expression levels in the context of various viral vectors and cell types [36]�C[51]. However, concerns have been raised regarding the inclusion of WPRE in viral vectors because its sequence overlaps with that of the WHV X protein (WHX), a transcriptional activator implicated in the development of liver tumors [52], [53].

To avoid expression of WHX-derived polypeptides, a version of WPRE mutated in both the WHX promoter and the translation start sequence has been generated which has an efficacy in transgene expression enhancement similar to that of the wild-type WPRE sequence [51], [54]. We tested the impact of this mutant WPRE variant (WPRE-m, bp 1094�C1636, GenBank “type”:”entrez-nucleotide”,”attrs”:”text”:”J04514″,”term_id”:”336146″,”term_text”:”J04514″J04514 and [51]), on transgene expression levels both in the human HepG2 hepatoma cell line and in the rat liver. HepG2 cells were transfected with expression plasmids encoding eGFP or feline ARSB (fARSB) under the control of the TBG promoter, containing or not the WPRE-m element (pAAV-TBG-eGFP, pAAV-TBG-eGFP-WPRE-m, pAAV-TBG-fARSB, pAAV-TBG-fARSB-WPRE-m). Similar levels of eGFP [eGFP (OD): 274��0.

4 for pAAV-TBG-eGFP and 301��18 for pAAV-TBG-eGFP-WPRE-m transfected cells] or normalized ARSB activity [ARSB(nmol/mg/h)/eGFP(OD): 0.36��0.04 for pAAV-TBG-fARSB and 0.37��0.05 for pAAV-TBG-fARSB-WPRE-m transfected cells] were measured independently of the WPRE-m inclusion. We then injected rats at P30 with AAV2/8-TBG-eGFP vectors including or not the WPRE-m variant and assessed transgene expression levels in livers collected at P90. We analyzed both the presence of eGFP positive cells on cryo-sections by fluorescence microscopy and eGFP levels of expression in liver lysates by Western blot with anti-eGFP antibodies (Fig. 4A and B, Fig. S2E and Fig. S5A). Despite the presence of similar amounts of AAV vector genomes (Fig. 4C and Fig S5B), the levels of eGFP expression appeared lower in livers receiving the vectors with than without WPRE-m (Fig.

4A and B, Fig. S2E and Fig. S5A). To exclude that the differences observed in eGFP expression levels were due to the quality of viral vector preparations, three independent experiments using AV-951 3 different preps of vector for each construct were performed and showed similar results (Fig. 4A and B, Fig. S2E and Fig. S5A). Figure 4 Inclusion of the WPRE-m variant is associated with decreased hepatocyte transduction levels in rats.