Laboratory of Molecular Virology and Gene Therapy, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
PLoS One. 2013 Aug 1;8(8):e69879. doi: 10.1371/journal.pone.0069879. Print 2013.
Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9) Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.
传统的非病毒基因转移使用含有抗生素抗性基因、顺式作用的细菌序列元件和原核甲基化模式的细菌质粒 DNA，这些可能会对体内转基因的表达和载体稳定性产生不利影响。在这里，我们描述了一种新型的真核载体 DNA 的复制形式，它仅由腺相关病毒 (AAV) 反向末端重复序列侧翼的表达盒组成。广泛的结构分析表明，这种源自 AAV 的载体 DNA 由线性、双链分子组成，具有共价封闭末端（称为闭合末端、线性双链体或“CELiD”DNA）。CELiD 载体在 Sf9 昆虫细胞中产生，需要 AAV rep 基因表达进行扩增。从稳定转染 ITR 侧翼转基因的昆虫细胞系中产生的 CELiD DNA 数量超过每 5×10(9) Sf9 细胞 60mg，而从转导相同数量的亲本 Sf9 细胞的重组杆状病毒感染中获得的 CELiD DNA 数量为 1-15mg。在小鼠中，系统递送的 CELiD DNA 导致肝脏中长期、稳定的转基因表达。CELiD 载体代表了一种新型的真核替代细菌质粒 DNA 的选择。
