Gene transfer with synthetic virus-like particles via the integrin-mediated endocytosis pathway
Gene transfer with synthetic virus-like particles via the integrin-mediated endocytosis pathway
复制标题
DOI：
10.1038/sj.gt.3300783
复制
发表时间：
1999-01-01
期刊：
影响因子：
5.1
通讯作者：
Behr, JP
中科院分区：
文献类型：
--
作者：
Erbacher, P;Remy, JS;Behr, JP
含DNA的阳离子颗粒与细胞表面阴离子蛋白聚糖之间的相互作用是进入培养细胞的有效手段。然而，治疗性体内基因递送水平需要与不那么普遍存在的分子结合。为了追踪腺病毒，将硫醇衍生的聚乙烯亚胺（PEI）通过二硫桥与整合素结合肽CYGGRGDTP偶联。最广泛缀合的衍生物（5.5%的PEI胺官能团）显示出用于全身基因递送的感兴趣的物理性质。在过量PEI-RGD的存在下，质粒DNA浓缩成30-100 nm环形颗粒的相当均匀的群体，如在T50 mM盐中的电子显微镜图像所揭示的。由于突出的两性离子肽残基的屏蔽效应，它们的表面电荷接近中性。与PEI相比，整合素表达上皮细胞（HeLa）和成纤维细胞（MRC 5）的转染效率增加了10至100倍，甚至在血清中也是如此。当靶向肽序列（RGD/RGE）中的谷氨酸被谷氨酸取代时，这种大的增强因子丢失，证实了整合素参与转染。因此，PEI-RGD/DNA复合物与腺病毒共享组成特性，例如大小和中心保护的DNA核心，以及“早期”特性，即由整合素介导的细胞进入和酸触发的内体逃逸。
The interaction between cationic DNA-containing particles and cell surface anionic proteoglycans is an efficient means of entering cultured cells. Therapeutic in vivo gene delivery levels, however, require binding to less ubiquitous molecules. In an effort to follow adenovirus, thiol-derivatized polyethylenimine (PEI) was conjugated to the integrin-binding peptide CYGGRGDTP via a disulfide bridge. The most extensively conjugated derivative (5.5% of the PEI amine functions) showed physical properties of interest for systemic gene delivery. In the presence of excess PEI-RGD, plasmid DNA was condensed into a rather homogeneous population of 30-100 nm toroidal particles as revealed by electron microscopy images in T50 mM salt. Their surface charge was close to neutrality as a consequence of the shielding effect of the prominent zwitterionic peptide residues. Transfection efficiency of integrin-expressing epithelial (HeLa) and fibroblast (MRC5) cells was increased by 10- to 100-fold as compared with PEI, even in serum. This large enhancement factor was lost when asparlic acid was replaced by glutamic acid in the targeted peptide sequence (RGD/RGE), confirming the involvement of integrins in transfection. PEI-RGD/DNA complexes thus share with adenovirus constitutive properties such as size and a centrally protected DNA core, and 'early' properties, ie cell entry mediated by integrins and acid-triggered endosome escape.