Another important factor is the size of the plasmid. From a regulatory, manufacturing, and therapeutic point of view, it is evident that all unnecessary DNA sequences should be removed from the vector.
As a general rule, the plasmid should be designed to be as small as possible. Since a large plasmid exerts a metabolic load onto the E. coli host, it will reduce the cell’s resources for plasmid replication, leading to a decreased pDNA yield.
From a therapeutic point of view, a small plasmid penetrates cellular hurdles more efficiently and shows increased expression rates. The size of the plasmid, however, may be predetermined by the therapeutic approach. Plasmids coding for one gene typically have about 5 kbp (monocistronic), while such coding for multiple genes may equal 8 kbp or larger (polycistronic). In contrast, rather small plasmids are obtained when a gene-silencing approach—based on RNAi—is pursued. Plasmids coding for expressed interfering RNA (eiRNA) rarely exceed 3.5 kbp in size.
The selection marker of the plasmid is another factor to be considered. Most commonly, plasmids code for an antibiotic-resistance gene (e.g., against kanamycin) to allow selection of plasmid-carrying clones. The therapeutic coadministration of a prokaryotic resistance gene and the use of antibiotics during manufacturing are problematic from a regulatory point of view. Therefore, concepts for antibiotic-free plasmid selection based on operator-repressor titration or the supplementation of an auxotrophic gene have been developed.
Boehringer Ingelheim Austria has developed a host-vector system for antibiotic-free plasmid selection based on the natural ColE1/pUC ori.