355. Minicircles Show Improved Hepatic Expression of Their Transgene from a Natural Endogenous Promoter and Are Lost Upon Partial Hepatectomy Due to the Episomal Nature of the Vector

Abstract

We have previously showed the successful treatment of phenylketonuria (PKU) in the PKU mouse model (C57Bl/6-Pahenu2) by using non-viral naked DNA minicircle (MC) vectors, which are devoid of any viral or bacterial sequences, upon liver-directed phenylalanine hydroxylase (Pah) gene transfer via hydrodynamic vein injection (Viecelli et al., Hepatology 2014). Here we improved the efficacy of transgene expression and investigated the fate of MC vectors in mouse liver. We found that the therapeutic doses of MCs could be significantly lowered by using a codon-optimized murine Pah cDNA in combination with a truncated 5’-intron. Moreover, when using the natural or endogenous 3.6 kb murine Pah-promoter to drive the Pah transgene, vector doses could again be lowered compared to two other “minimal” liver-specific promoters, a synthetic hybrid enhancer/promoter (P3) or the classical CBA (modified cytomegalovirus enhancer/chicken β-actin) promoter. Following 70% partial hepatectomy, MC-vector-treated PKU mice showed normal liver regeneration and blood L-Phe concentration increased to pretreatment levels, indicating that treated PKU mice had lost therapeutic MC vectors during liver regeneration. These results corroborate previous observations that MC-DNA do not integrate - or only at a very low frequency that cannot be detected by our assays - and thus express their transgene as episomal vectors. In conclusion, MC-vectors which do not have a defined size-limitation, offer a favorable safety profile due to their non-integrating behavior in combination with a (large) natural or endogenous promoter, and at the same time have the potential for long-term gene-therapy of liver defects.