pCOR: a new design of plasmid vectors for nonviral gene therapy

A totally redesigned host/vector system with improved : properties in terms of safety has been developed. The pCOR plasmids are narrow-host range plas mid vectors for nonviral gene therapy. These plasmids contain a conditional origin of replication and must be propagated in a specifically engineered E. coli host strain, greatly reducing the potential for propagation in the environment or in treated patients. The pCOR backbone has several features that increase safety in terms of dissemination and selection: (1) the origi n of replication requires a plasmid-specific initiator protein, pi protein, encoded by the pir gene limiting its host range to bacterial strains that produce this transacting protein; (2) the plasmid's selectable marker is no t an antibiotic resistance gene but a gene encoding a bacterial suppressor tRNA. Optimized E. coli hosts supporting pCOR replication and selection wer e constructed. High yields of supercoiled pCOR monomers were obtained (100 mg/l) through fed-batch fermentation. pCOR vectors carrying the luciferase reporter gene gave high levels of luciferase activity when injected into mu rine skeletal muscle.