Kim Na Hyung, Provoda Chester, Lee Kyung-Dall
Department of Pharmaceutical Sciences, Center for Molecular Drug Targeting, University of Michigan , Ann Arbor, Michigan 48109, United States.
Mol Pharm. 2015 Feb 2;12(2):342-50. doi: 10.1021/mp5004543. Epub 2015 Jan 22.
To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems.
为提高基因递送效率以实现有效的基因治疗，载体携带能够促进克服导致DNA从细胞外转运至最终细胞核区室的各个步骤中的障碍的功能组件至关重要。在本研究中，我们设计了基因工程融合蛋白作为一个平台，以在一个嵌合蛋白中整合多种功能。在此测试的这种嵌合体的原型包含两个结构域：一个与DNA结合；另一个能够促进DNA从内体逃逸。融合蛋白由单核细胞增生李斯特菌的内体溶解成孔蛋白李斯特菌溶血素O（LLO）和DNA凝聚多肽鱼精蛋白（PN）的22个氨基酸序列单独或成对组成：LLO-PN和LLO-PNPN。我们证明，在使用培养细胞进行的体外实验中，掺入少量LLO-PN融合蛋白可显著提高鱼精蛋白凝聚DNA的基因递送效率，而LLO-PNPN则能进一步提高效率。此外，阴离子脂质体与阳离子LLO-PNPN/鱼精蛋白/DNA复合物结合，产生净负表面电荷，在有血清存在的情况下，可提高体外转染效率。在小鼠中进行的最初一组少量数据表明，观察到的基因表达增强也适用于体内基因递送。这项研究表明，在非病毒载体中掺入具有多种功能组件的重组融合蛋白，如LLO-鱼精蛋白融合蛋白，对于各种非病毒基因递送系统来说是一种有前景的策略。
