DEVELOPMENT OF S/MAR MINICIRLES VECTOR FOR PERSISTENT EXPRESSION IN VIVO.

An ideal vector for gene therapy must fulfil the following requirements: non-toxicity, mitotic stability and persistent therapeutic levels of transgene expression. Viral vectors are widely used due to their ability to sustain prolonged expression. Their potentially tumorigenic effects are a limiting factor for in vivo applications. Non-viral vectors, which can be designed to be free from viral sequences, are a promising alternative for gene transfer although they often produce transient transgene expression. This limitation of this vector type is primarily due to bacterial sequences they contain and these have proven to be toxic for the mammalian cells as they contain a high number of unmethylated CpG motifs. We have developed a new prototype gene expression vector devoid of any bacterial sequence. This minicircle vector comprises entirely mammalian sequences. Following hydrodynamic delivery into murine liver we have shown that this construct is able to sustain persistent expression of the luciferase marker gene in vivo for more than 25 days. In contrast, expression from an original expression plasmid (from which the minicircle is derived) which harbours these extraneous bacterial sequences dropped significantly to 10% of its initial expression after 25 days. The inclusion of an S/MAR element into the minicircle sequence not only produced increasing levels of expression following administration but after 25 days showed sustained levels which were twice as high as those found immediately following administration. These promising results demonstrate the utility of minimally sized non-viral vectors in combination with S/MAR motifs for persistent, non toxic therapeutic gene transfer.