Development of Helical Polypeptides for Non-Viral Gene Delivery Systems

描述（由申请人提供）：由于其广泛的潜力，基因治疗在过去的20年里得到了大量的研究。然而，一种临床可行的基因治疗方法尚未实现。基因治疗转化发展的最大障碍是缺乏安全有效的方法将遗传信息传递到目标细胞和组织。虽然病毒是目前传递遗传信息最有效的方式，但它们也会带来严重的健康风险——包括免疫原性和致癌性——这在之前的临床试验中已经表现出来。阳离子聚合物和脂质有可能成为非免疫原性和非致癌的递送载体，但许多可用的材料相对效率较低。即使是效率最高的脂质和聚合物的效率也比病毒低几个数量级，通常需要使用微克的DNA来实现转基因表达，这与仅含有picgrams遗传物质的病毒悬浮液所产生的效果相当。为了提高非病毒载体的效率，它们必须被设计成能够克服所有基因传递载体共同的细胞外屏障，以及一旦传递载体到达目标细胞时遇到的第二组细胞内屏障。具体来说，1)载体必须与细胞结合，2)被内化，3)从内吞囊泡中逃逸到细胞质中，4)穿过细胞质进入核膜，5)迁移到细胞核中，6)释放DNA。病毒非常高效，因为它们进化出了特定的功能来应对这些挑战。然而，目前的合成材料缺乏有效地将DNA从细胞外环境护送到细胞核中所必需的部分或全部功能。有了这个R21应用程序，我们建议开发一种活性阳离子？-螺旋模板用于构建基因传递文库。由于螺旋结构域在许多膜破坏材料中频繁出现，我们对在整个文库中保留螺旋结构感兴趣。我们相信，将这种二级结构作为库的核心特征，将产生能够有效地从内吞囊泡中逃逸的材料。通过微妙地改变接枝到螺旋骨架上的侧链的亲疏水平衡，我们将生成具有各种DNA结合强度的材料。通过这种方式，我们希望发现具有DNA结合强度和内溶性的适当平衡的聚合物，以产生有效的基因传递。

DESCRIPTION (provided by applicant): Because of its broad potential, gene therapy has been heavily investigated over the past 20 years. However, a clinically viable gene therapy treatment has yet to be realized. The single greatest impediment to the translational development of gene therapy is the lack of safe and efficient means to deliver genetic information to target cells and tissues. While viruses are currently the most efficient way to deliver genetic information, they also pose serious health risks-including immunogenicity and oncogenicity-which have manifested themselves in prior clinical trials. Cationic polymers and lipids have the potential to be non-immunogenic and non-oncogenic delivery vehicles, but many of the available materials are relatively inefficient. Even the most efficient lipids and polymers are orders of magnitude less efficient than viruses, often necessitating the use of micrograms of DNA to achieve transgene expression comparable to that resulting from a virus suspension containing only picograms of genetic material. In order to improve the efficiency of non-viral vectors, they must be designed to overcome extracellular barriers common to all gene delivery vehicles as well as a second set of intracellular barriers encountered once the delivery vehicle reaches the cells of interest. Specifically, 1) the vector must bind to the cell, 2) be internalized, 3) escape from endocytic vesicles into the cytoplasm, 4) move through the cytosol to the nuclear envelope, 5) migrate into the nucleus and 6) release the DNA. Viruses are very efficient because they have evolved specific functions for meeting each of these challenges. Current synthetic materials, however, lack some or all of the functions necessary for efficiently escorting the DNA from outside the extracellular environment into the nucleus. With this R21 application, we propose to develop a reactive cationic ?-helical template for the construction of a library of materials for gene delivery. We are interested in retaining helical architecture throughout the library due to the frequent occurrence of helical domains in many membrane disruptive materials. We believe having this secondary structure as the core feature of the library will yield materials which are able to effectively escape from endocytic vesicles. By subtly changing the hydrophilic/hydrophobic balance of side chains grafted onto the helical backbone, we will generate materials which have a variety of DNA binding strengths. In this manner, we hope to discover polymers with the appropriate balance of DNA binding strength and endosomolytic properties to yield efficient gene delivery. PUBLIC HEALTH RELEVANCE: This R21 application addresses endosomal escape-one of the most important barriers to efficient non-viral gene delivery-through the development of a reactive cationic ?-helical template for the construction of a library of materials for non-viral gene delivery. The cationic helical architecture is believed to aid endosomolysis while subtle changes in the hydrophilic/hydrophobic balance of side chains grafted onto the helical template will allow the identification of polymers with the appropriate balance of DNA binding strength and endosomolytic properties to yield efficient gene delivery.