1039. Mechanistic Insights into the Persistence of Non-Viral Mediated Gene Transfer In Vivo

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Non-viral vectors offer great potential for clinical application in gene therapy trials. These vectors have several advantages over the viral-based gene therapy approaches, including non-toxicity and ease of production. However, the ability to achieve reproducible, persistent, and therapeutic levels of gene expression in vivo by non-viral gene transfer approaches has been problematic because of the relatively transient nature of transgene expression in vivo. This may limit the use of these vectors in the clinic. Understanding the mechanisms required for persistent gene expression will be helpful for vector design and eventual clinical implementation. We have suggested that covalent linkage of bacterial DNA to the expression cassette plays a critical role in transcriptional silencing of the transgenes in vivo. To gain an insight into the role of the covalent linkage of plasmid DNA to the expression cassette and transcriptional repression, and whether this silencing effect could be alleviated by altering the molecular structure of vector DNAs in vivo, we generated a scheme for converting routine plasmids into a purified expression cassette, free of bacterial DNA after gene transfer in vivo. To do this, we co-injected the I-SCeI cDNA together with a plasmid reporter containing either the human alpha-1-antitrypsin (hAAT) or the human clotting factor IX (hFIX) expression cassette flanked by two I-SCeI sites. Two weeks after DNA administration, mice injected with the reporter gene alone or with the irrelevant control plasmid showed very low serum levels of hAAT and hFIX, which remained low throughout the length of the experiment. However, animals that expressed I-SCeI had a 5-10 fold increase in serum hAAT and hFIX that persisted for at least 8 months (length of study). In addition, expression of I-SCeI resulted in cleavage and excision of the expression cassettes from plasmid backbone, forming mostly circles devoid of bacterial DNA sequences, as established by a battery of different Southern blot and PCR analyses (in vivo and in vitro) in both C57Bl6 and scid mice. In contrast, only the input parental circular plasmid DNA band was detected in mice injected either with the reporter gene alone or an I-SCeI plasmid with the hAAT reporter plasmid lacking the I-SCeI sites. This study presents further independent evidence that removing the covalent linkage between plasmid and transgene sequences leads to a marked increase and persistence of transgene expression. Thus, unraveling the mechanisms by which the covalent linkage of bacterial DNA to the expression cassette is connected to gene silencing is fundamental to establish the mechanism of transcriptional regulation in mammalian systems and will be important for the development of versatile non-viral vectors that can be used to achieve persistent gene expression in different cell types.