The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression
The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression
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DOI：
10.1002/jgm.1031
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发表时间：
2007-05-01
影响因子：
3.5
通讯作者：
Prazeres, Duarte M. F.
中科院分区：
文献类型：
--
作者：
Azzoni, Adriano R.;Ribeiro, Sofia C.;Prazeres, Duarte M. F.
背景 质粒 (pDNA) 的有效递送和表达是使用非病毒载体进行基因治疗和 DNA 疫苗接种的主要问题。除了使用佐剂之外，pDNA 载体本身还可以设计成最大限度地提高在富含核酸酶的环境中的存活率。先前已证明聚腺苷酸化序列中富含同嘌呤的区域对于 pDNA 抗性尤其重要。 方法 研究了模型 pDNA 载体 (pVAX1GFP) 的聚 A 序列修饰对核酸酶抗性和转基因表达的影响。研究了四种多聚 A 序列：牛生长激素 (BGH)、突变体 BGH、SV40 和合成多聚 A。通过与哺乳动物核酸酶体外孵育来评估质粒抗性（半衰期）。通过量化 CHO、杂交瘤和 HeLa 细胞中的 pDNA、mRNA 和 GFP 表达，研究了转基因表达的影响。 结果和结论 体外和细胞培养研究表明，含有 SV40 和合成聚 A 序列的质粒在核酸酶抗性方面表现出显着改善（半衰期增加多达两倍）。然而，RT-PCR 分析表明，mRNA 稳态水平的显着降低是转基因表达降低的原因，并且在使用抗性更强的质粒时检测到 CHO 和杂交瘤细胞的转染水平。有趣的是，HeLa 细胞的转染表明，poly A 效率和质粒抗性都会显着干扰转基因表达。结果强烈表明，poly A 的选择不仅对于 mRNA 成熟/稳定性很重要，而且对于 pDNA 抗性也很重要，因此在 pDNA 载体的设计和评估中应予以考虑。版权所有 (C) 2007 John Wiley & Sons, Ltd.
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.Methodology 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), mutantBGH, 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.Results and 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. Copyright (C) 2007 John Wiley & Sons, Ltd.