Delivery of a PCR amplified DNA fragment into cells: A model for using synthetic genes for gene therapy

Synthetic genes offer many potential advantages over conventional plasmid DNA, such as simplicity in purification, absence of endotoxin contamination, and more importantly, flexibility in chemical modifications to render them specific properties. We have used PCR amplified fragments as a model to test the feasibility of using synthetic genes for gene therapy. The CAT reporter gene driven by the CMV promoter (CMV-CAT), ie a nuclear expression system, or by the bacteriophage T7 promoter (T7-CAT), ie a cytoplasmic expression system, was used to evaluate this concept. The expression efficiency of both plasmids (pUCCMV-CAT and pT7-CAT) and their corresponding PCR fragments (fCMV-CAT and fT7-CAT) were compared on a molar basis. Limited expression of CAT was found with fCMV-CAT. However, fT7-CAT consistently gave a CAT activity comparable to that of pT7-CAT. When fT7-CAT was codelivered with pCMV/T7-T7pol (a self-amplifying T7 RNA polymerase autogene), high CAT activity could be detected up to 9 days. This expression was much longer than the duration of expression with a nuclear expression system. These encouraging results imply that gene therapy with synthetic genes could be both feasible and efficient.