The duration of transgene expression from plasmid DNAs is important for gene therapy with nonviral vectors. In the present study, various cytosine-phosphate-guanine (CpG)-free and CpG-containing transcription regulatory sequences were introduced into plasmid DNAs with a CpG-free backbone. The transgenes encoding mouse secreted alkaline phosphatase and Gaussia princeps luciferase, which are both apparently non-immunogenic, were used as reporters. The plasmid DNAs were injected by the hydrodynamics-based method, and the expression was monitored for 28 d. All transcription regulatory sequences achieved long-term expression, with different expression levels depending on the sequences themselves. These results suggested that durable transgene expression at the proper level can be achieved with plasmid DNAs containing the CpG-free backbone.
Background: Durable transgene expression from plasmid DNAs is the key to gene therapy with non-viral vectors. A comparison of the durability of transgene expression from plasmid DNAs with the CpG-free and -containing backbones is important.
Methods:We constructed plasmid DNAs with the CpG-containing backbone, various transcription regulatory sequences with and without CpG, and the gene encoding Gaussia princeps luciferase, which is apparently non-immunogenic. The tail vein hydrodynamics-based method was used for plasmid injection into mice, and the luciferase activity in serum was tracked for 28 days.
Results:The plasmid DNAs containing the albumin promoter [with or without the cytomegalovirus (CMV) enhancer] and the elongation factor (EF)1α promoter plus the CMV enhancer exhibited long-term luciferase expression. The expression from the plasmid DNA containing the albumin promoter without the CMV enhancer was maintained for at least 24 weeks and was similar to that from the corresponding CpG-free plasmid DNA.
Conclusions:The results obtained in the present study suggest that special sequences/systems are unnecessary for durable transgene expression from plasmid DNAs when the proper transcription regulatory sequences are used.
K E Y W O R D Shydrodynamics-based method, non-viral vector, plasmid DNA, transgene expression
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