The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression.
BACKGROUND
Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance.


METHODS
The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells.


CONCLUSIONS
In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors.
Azzoni
Adriano R
AR
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, 1049-001 Lisboa, Portugal.
Ribeiro
Sofia C
SC
Monteiro
Gabriel A
GA
Prazeres
Duarte M F
DM
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004720:Endonucleases / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011061:Poly A / Q000494:pharmacology*; D026723:Polyadenylation; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000502:physiology; D019076:Transgenes / Q000235:genetics*
D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D011061:Poly A; D004720:Endonucleases