Replicating minicircles: Generation of nonviral episomes for the efficient modification of dividing cells

Abstract

Nonviral replicating circular episomes are a rather new addition to the field of mammalian expression vectors. After their establishment, which conventionally requires an initial phase under selection pressure, these entities utilize the replication apparatus of the host cell to replicate in accord with the cell cycle. The requirements of a selection agent, the gradual inactivation by cellular defense mechanisms, and a limited cloning capacity (up to 5 kb could be realized for the prototype) have remained the critical parameters. Here we introduce a site-specific recombination-based strategy that permits the excision of prokaryotic vector parts after the parental construct has been amplified as a plasmid. The remaining 4 kb 'minicircle' consists of only one active transcription unit and a scaffold/matrix attachment region (S/MAR). In contrast to the parent plasmid vector it can be established in the absence of selection, it is not subject to epigenetic silencing and it replicates stably without a sign of integration. In further contrast to available minicircles that are maintained only in non-dividing tissues our minicircle represents the first example that is suited for the modification of dividing cells and tissues due to its association with the nuclear matrix and its authentic segregation.