Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields.
Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields.
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DOI：
10.1002/jgm.1499
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发表时间：
2010-10
影响因子：
3.5
通讯作者：
Williams, James A.
中科院分区：
文献类型：
--
作者：
Carnes, Aaron E.;Luke, Jeremy M.;Vincent, Justin M.;Anderson, Sheryl;Schukar, Angela;Hodgson, Clague P.;Williams, James A.
出于安全考虑，监管机构建议从基于质粒dna的基因药物中去除抗生素耐药性标记和非必需序列。在本研究中，我们分析了影响细菌生产和哺乳动物转基因表达的无抗生素（AF）载体设计标准。研究了CMV-HTLV-I R RNA Pol II启动子（蛋白转基因）和小鼠U6 RNA Pol III启动子（RNA转基因）载体的设计。通过诱导补料分批发酵评价质粒产量。转染人HEK293细胞后，分别用荧光法和RT-PCR法测定RNA Pol ii定向EGFP和RNA Pol iii定向RNA的表达。蔗糖选择性最小化蛋白和治疗性RNA表达载体设计结合了基于RNA的AF选择与高产发酵制造（bbb10 1000 mg/L质粒DNA）和高水平的体内表达编码产物。AF选择标记也成功地应用于将现有的卡那霉素耐药DNA疫苗质粒gWIZ和pVAX1转化为AF载体，证明了对现有载体进行改造的一般效用。确定了高产质粒发酵的最小载体尺寸。研究了质粒二聚体的稳定发酵策略，提高了载体效力，发酵产量可达1,740 mg/L。我们报道了高效的蛋白或RNA（如短发夹RNA或microRNA）产物的AF基因药物表达载体的开发。这些AF表达载体经过优化，超过了新确定的高拷贝质粒复制的大小阈值，并且比其他载体直接具有更高的转基因表达水平。
For safety considerations, regulatory agencies recommend elimination of antibiotic resistance markers and nonessential sequences from plasmid DNA-based gene medicines. In the present study we analyzed antibiotic-free (AF) vector design criteria impacting bacterial production and mammalian transgene expression. Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed EGFP and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction (RT-PCR), respectively, after transfection of human HEK293 cells. Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1,000 mg/L plasmid DNA) and high level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1,740 mg/L was developed. We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors.
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影响因子：
16
作者：
Williams, James A.;Carnes, Aaron E.;Hodgson, Clague P.
通讯作者：
Hodgson, Clague P.
影响因子：
5.5
作者：
Luke J;Carnes AE;Hodgson CP;Williams JA
通讯作者：
Williams JA
影响因子：
14.9
作者：
Hassani Z;François JC;Alfama G;Dubois GM;Paris M;Giovannangeli C;Demeneix BA
通讯作者：
Demeneix BA
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
5.5
作者：
Williams, Jarnes A.;Luke, Jeremy;Hodgson, Clague
通讯作者：
Hodgson, Clague