Grosse Stéphanie, Aron Yolande, Thévenot Guiti, François Dominique, Monsigny Michel, Fajac Isabelle
Laboratoire de Physiologie Respiratoire, IFR Alfred Jost, CHU Cochin, AP-HP-Université Paris 5, Paris, France.
J Gene Med. 2005 Oct;7(10):1275-86. doi: 10.1002/jgm.772.
Although polycations are among the most efficient nonviral vectors for gene transfer, the gene expression they allow is still too low for in vivo applications. To engineer more potent polycationic vectors, the factors governing the intracellular trafficking of a plasmid complexed with current polycations need to be identified.
The trafficking of plasmid DNA complexed to glycosylated polylysines or polyethylenimine (PEI) derivatives was studied by electron microscopy of human airway epithelial cells. The cellular processing of complexes varied with their size and the polycation derivative used: large complexes (> 200 nm) made with all polycationic vectors studied were internalized by macropinocytosis. In contrast, intermediate (100-200 nm) ligand-coupled polylysine and PEI complexes primarily entered through clathrin-coated pits. Complexes were then found in endosomal vesicles, accumulated in lysosomes or vesicles near the nucleus and their nuclear entry was limited. For the population of small complexes (< or = 100 nm) obtained with PEI derivatives, they were internalized through caveolae and pursued a traffic pattern of potocytosis to the endoplasmic reticulum where their fate remains unclear. Finally, some complexes exited the cells either by regurgitation when PEI derivatives were used or through an exosome-like pathway for glycosylated-polylysine complexes.
The different pathways of complex trafficking observed in relation with complex size imply the development and study of vectors forming complexes with definite size. Moreover, the complex exit we describe may contribute to the well-established short-term efficiency of gene transfer based on synthetic vectors. It favors the engineering of vectors allowing repeated treatment.
尽管聚阳离子是基因转移中最有效的非病毒载体之一，但它们所允许的基因表达水平对于体内应用而言仍然过低。为了构建更有效的聚阳离子载体，需要确定与当前聚阳离子复合的质粒在细胞内运输的调控因素。
通过对人气道上皮细胞进行电子显微镜观察，研究了与糖基化聚赖氨酸或聚乙烯亚胺（PEI）衍生物复合的质粒DNA的运输情况。复合物的细胞处理过程因其大小和所使用的聚阳离子衍生物而异：用所有研究的聚阳离子载体制备的大复合物（>200nm）通过巨胞饮作用内化。相比之下，中等大小（100-至200nm）的配体偶联聚赖氨酸和PEI复合物主要通过网格蛋白包被小窝进入。复合物随后出现在内体囊泡中，积聚在溶酶体或细胞核附近的囊泡中，并且它们进入细胞核受到限制。对于用PEI衍生物获得的小复合物群体（≤100nm），它们通过小窝内化，并沿着胞饮作用的运输途径到达内质网，其命运尚不清楚。最后，当使用PEI衍生物时，一些复合物通过反流从细胞中排出，而糖基化聚赖氨酸复合物则通过类似外泌体的途径排出。
观察到的与复合物大小相关的不同复合物运输途径意味着需要开发和研究形成具有确定大小复合物的载体。此外，我们所描述的复合物排出可能有助于基于合成载体的成熟的短期基因转移效率。这有利于构建允许重复治疗的载体。
