Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo
Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo
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DOI：
10.1089/hum.2005.16.126
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发表时间：
2005-01-01
影响因子：
4.2
通讯作者：
Kay, MA
中科院分区：
文献类型：
--
作者：
Chen, ZY;He, CY;Kay, MA
我们以前已经表明，不含质粒细菌DNA序列的小环DNA载体能够在体内持续高水平表达转基因。微环在细菌中由含有诱导型噬菌体phiC31整合酶基因和侧接有attB和attP位点的治疗性表达盒的亲本质粒产生。attB和attP之间的phiC31介导的分子内重组导致形成两个环状DNA分子，一个含有真核表达盒（小环），另一个含有质粒细菌DNA骨架（BB）。先前，通过限制性酶消化步骤和在氯化铯中的超离心从质粒BB中纯化小环。我们现在已经将核酸内切酶I-SceI基因连同其识别位点一起包括在小环产生质粒中，以允许质粒BB在细菌中线性化和降解。然后可以通过常规质粒纯化程序如一步亲和柱分离小环。通过对我们以前的策略进行额外的修改，我们可以制备编码4-kb人因子IX表达盒的小环，从1升细菌培养物中制备高达1.8mg纯度为97%的小环。高产量、简单纯化以及稳健和持久的转基因表达使得这些载体对于基因治疗应用是可行的。
We have shown previously that minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage phiC31 integrase gene and a therapeutic expression cassette flanked with attB and attP sites. The phiC31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic expression cassette ( minicircle), and the other the plasmid bacterial DNA backbone ( BB). Previously, the minicircle was purified away from the plasmid BB by a restriction enzyme digestion step and ultracentrifugation in cesium chloride. We have now included the endonuclease I-SceI gene together with its recognition site in the minicircle-producing plasmid to allow the linearization and degradation of the plasmid BB in bacteria. The minicircle can then be isolated by routine plasmid purification procedures such as a one-step affinity column. With additional modifications to our previous strategy, we can prepare a minicircle encoding a 4-kb human factor IX expression cassette, up to 1.8 mg of minicircle with 97% purity was prepared from a 1 liter bacterial culture. The high yield, simple purification, and robust and persistent transgene expression make these vectors viable for gene therapy applications.