A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells.
A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells.
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发表时间：
1996-05
期刊：
影响因子：
5.1
通讯作者：
S. Gottschalk;J. Sparrow;J. Hauer;M. Mims;F. Leland;S. Woo;L. Smith
中科院分区：
文献类型：
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作者：
S. Gottschalk;J. Sparrow;J. Hauer;M. Mims;F. Leland;S. Woo;L. Smith
为了开发用于治疗疾病的非病毒基因传递系统，我们的策略是用模拟病毒蛋白功能的短合成肽构建 DNA 复合物。我们设计并合成了两种模拟病毒功能的肽 - DNA 缩合剂 YKAK(8)WK 和两亲性、pH 依赖性内体释放剂 GLFEALLELLESLWELLLEA。活性基因传递复合物是通过涉及带相反电荷的静电相互作用的自发自组装过程逐步构建的。为了组装具有不同总净电荷的DNA-肽复合物，仅考虑DNA磷酸盐的负电荷、YKAK(8)WK的10个ε-氨基的正电荷和GLFEALLELLESLWELLLEA的5个γ-羧基的负电荷。第一步，带负电荷的DNA与过量的YKAK(8)WK快速混合，形成带正电荷的DNA-YKAK(8)WK复合物，这几乎不产生基因转移。在第二步中，为了形成活性复合物，阳离子DNA复合物通过静电相互作用快速混合并自发掺入。使用 CMV-luc、YKAK(8)WK 和 GLFEALLELLESLWELLLEA 的这些复合物进行转染，在多种细胞系中获得高水平的基因表达。这些简单的 DNA 复合物仅包含三种分子定义的成分，具有基因传递的一般用途，并且可以在基因治疗的某些应用中替代病毒载体和阳离子脂质。
To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptides that mimic the functions of viral proteins. We have designed and synthesized two peptides which emulate viral functions - a DNA condensing agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasing agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was constructed step-wise through a spontaneous self-assembly process involving oppositely charged, electrostatic interactions. To assemble DNA-peptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amino groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, negatively charged DNA was rapidly-mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gene transfer. In the second step and to form the active complex,the cationic DNA complex was rapidly mixed with spontaneously incorporated through electrostatic interactions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high-levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three molecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some applications in gene therapy.