Design of PCR‐amplified DNA fragments for in vivo gene delivery: Size‐dependency on stability and transgene expression

Abstract

PCR-amplified DNA fragments can be more efficient and safer vectors than conventional plasmid DNA because of their smaller size and fewer numbers of immunostimulatory cytosine-phosphate-guanine (CpG) motifs. In the present study, the expression unit of plasmid DNA encoding farnesylated enhanced green fluorescent protein (EGFPF; pEGFP-F) or firefly luciferase (pLuc) was amplified by polymerase chain reaction (PCR) to obtain DNA fragments (EGFPF-mini, Luc-mini). EGFPF-mini was as effective as pEGFP-F on the basis of the number of EGFPF-expressing cells after intravenous injection into mice by the hydrodynamics-based procedure. Then, the effects of the length of DNA fragments on transgene expression were examined using luciferase-expressing DNA preparations. Luc-mini preparations showed high levels of luciferase activity in cultured cells as well as in mouse liver, even although the levels did not exceed that of pLuc. An elongation of the DNA fragment on either side of the minimal expression unit was effective in increasing the transgene expression and the stability against nucleases. PCR-amplified DNA fragments showed a sustained luciferase activity in mouse liver compared with pLuc, indicating that they are effective in achieving a prolonged expression. Their stabilization against nucleases will further increase the potential of such short, structure-controlled and synthetic DNA fragments for in vivo gene delivery.