Collins Louise, Fabre John W
Department of Clinical Sciences, Institute of Liver Studies, Guy's King's and St Thomas' School of Medicine, King's College Hospital, Bessemer Road, London SE5 9PJ, UK.
J Gene Med. 2004 Feb;6(2):185-94. doi: 10.1002/jgm.482.
Efficient and non-toxic gene delivery, preferably with non-viral DNA vectors readily transferable to clinical practice, is generally regarded as a major limitation for gene therapy.
A 31 amino acid, integrin-targeted bifunctional synthetic peptide (polylysine-molossin), and two (Lys)(16)-containing control peptides, were assessed for ex vivo gene delivery to the rabbit cornea. Critical physical properties of polylysine-molossin/DNA complexes were evaluated and both chloroquine and a 20 amino acid fusogenic peptide were used to promote endocytic exit.
Polylysine-molossin/DNA complexes and (Lys)(16)/DNA complexes at 10 microg/ml of DNA were much smaller and much more positively charged in non-ionic isotonic medium (5% dextrose or 5% dextrose buffered to pH 7.4 in 10 mM Tris) when compared with culture medium or phosphate-buffered saline (PBS). Addition of the fusogenic peptide (net charge -5) reversed the positive charge of complexes in PBS, and reduced the strong positive charge of polylysine-molossin/DNA complexes in dextrose. Polylysine-molossin/DNA complexes in 5% dextrose were much more effective for gene delivery to the cornea when compared with complexes in culture medium, and the fusogenic peptide was much more effective than chloroquine for promoting gene delivery. The optimal DNA/polylysine-molossin/fusogenic peptide w/w ratio was 1 : 3 : 2 at 10 microg/ml of DNA. Essentially 100% of corneal endothelial cells were transfected under these optimal conditions, without any evidence of toxicity. Integrin-targeting did not contribute significantly to gene delivery in this system.
This DNA vector system, consisting entirely of synthetic peptides, is ideally suited for clinical applications of gene therapy of the corneal endothelium.
高效且无毒的基因递送，最好是使用易于转化为临床实践的非病毒DNA载体，通常被认为是基因治疗的主要限制因素。
评估一种31个氨基酸的整合素靶向双功能合成肽（聚赖氨酸 - 莫洛辛）以及两种含（Lys）16的对照肽对兔角膜的离体基因递送效果。评估聚赖氨酸 - 莫洛辛/DNA复合物的关键物理性质，并使用氯喹和一种20个氨基酸的融合肽来促进内吞逃逸。
与培养基或磷酸盐缓冲盐水（PBS）相比，在非离子等渗介质（5%葡萄糖或在10 mM Tris中缓冲至pH 7.4的5%葡萄糖）中，10 μg/ml DNA的聚赖氨酸 - 莫洛辛/DNA复合物和（Lys）16/DNA复合物要小得多且带更多正电荷。添加融合肽（净电荷 -5）可逆转PBS中复合物的正电荷，并降低葡萄糖中聚赖氨酸 - 莫洛辛/DNA复合物的强正电荷。与培养基中的复合物相比，5%葡萄糖中的聚赖氨酸 - 莫洛辛/DNA复合物对角膜的基因递送更有效，并且融合肽在促进基因递送方面比氯喹更有效。在10 μg/ml DNA时，最佳的DNA/聚赖氨酸 - 莫洛辛/融合肽重量比为1 : 3 : 2。在这些最佳条件下，基本上100%的角膜内皮细胞被转染，且没有任何毒性迹象。在该系统中，整合素靶向对基因递送没有显著贡献。
这个完全由合成肽组成的DNA载体系统非常适合角膜内皮基因治疗的临床应用。
