CAREER: Acid-Transforming Polypeptides as Stimuli-responsive, Efficient, Biocompatible, and Tunable Nonviral Gene Carriers

ID：MPS/DMR/BMAT（7623）0956091 PI：Kwon，Young Jik ORG：加州大学欧文分校标题：职业：酸转化多肽作为刺激响应，高效，生物相容性和可调的非病毒基因载体智力优势：合成基因载体（或非病毒载体），有效地将基因传递到所需的生物靶点，具有高生物相容性和多功能性，是一种在基础和翻译基因治疗研究中需求很高的技术。 与病毒载体相比，非病毒载体的细胞外和细胞内性能仍然相对较差，病毒载体虽然被证明非常有效，但通常具有免疫原性并且难以修饰以改变其结构和功能。 拟议的CAREER研究将合成新型的刺激转化多肽，这些多肽在中性pH下将核酸与阳离子臂复合，但在细胞隔室中水解后，这些多肽转化为中性的天然多肽，从而使复合物不稳定并释放核酸。具体而言，该项目将研究新型生物材料，其中包含缩酮化丝氨酸（kSer）残基，这些残基类似于赖氨酸（Lys），但在酸水解时转化为丝氨酸（Ser）。 kSer残基将与赖氨酸残基、聚乙二醇（PEG）和其它功能性多肽一起沿着使用，以产生通用聚合物非病毒载体。初步的体外研究表明，聚（kSer-Lys）/DNA聚合复合物和PEG-聚（kSer）/DNA胶束显着提高基因传递效率，通过几个改进的细胞内途径。PI建议通过更好地理解关键的细胞外和细胞内基因递送途径来开发这些新的基于酸转化多肽（ATPP）的基因载体。 他将（1）研究ATPP与融合和核定位信号肽的杂交，以改善靶向，（2）探索ATPP与功能肽和/或PEG之间的刺激敏感性连接的影响，这些连接可以被这些刺激切断以改善功效，以及（3）优化含ATPP的聚合物以改善DNA复合，细胞内化，细胞内靶向，DNA释放，更广泛的重要性：有效地递送治疗性基因，特别是使用非病毒方法，不仅在临床上（例如，基因治疗）而且在生物现象的遗传操纵（例如，将体细胞重编程为诱导干细胞）。从这项研究中开发的新型生物材料将提供给本科生和高中生进行实践研究体验和夏季研究计划。该项目将激发本科生的创造性和独立思考，并给他们一个机会来测试他们的假设与PI。该外展计划旨在激发参与高中生对科学和工程职业的兴趣，特别是那些来自附近城市地区代表性不足的群体的学生。 拟议的研究将通过本科生直接参与职业研究和外展研究计划，转化为课堂和其他领域。一门新的本科课程将介绍使用刺激响应聚合物生物材料进行基因传递的基本概念;然后将挑战学生在职业研究中发展创造性的想法。具有最具创新性建议的学生将被邀请到PI的实验室测试他们的假设，并将协助高中参与者参加一个关于使用酸转化多肽进行基因传递的夏季研究计划。这个推广计划将从PI的讲座中发展出来，并与社会经济贫困地区当地高中的实践实验相结合。最积极的高中生将参加夏季研究计划，然后在本科生导师的指导下开发一个科学博览会项目。这些教育活动的影响将通过学生的出版物和演讲以及他们对科学和工程专业的选择来评估。

ID: MPS/DMR/BMAT(7623) 0956091 PI: Kwon, Young Jik ORG: University of California-IrvineTitle: CAREER: Acid-Transforming Polypeptides as Stimuli-Responsive, Efficient, Biocompatible, and Tunable Nonviral Gene CarriersINTELLECTUAL MERIT: Synthetic gene carriers (or nonviral vectors) that efficiently deliver genes to desired biological targets with high biocompatibility and versatility are a technology in high demand in basic and translational gene therapy research. The extra- and intracellular performance of nonviral vectors remains relatively poor compared to viral vectors, which, though proven to be very efficient, are often immunogenic and difficult to modify in order to vary their structures and functions. The proposed CAREER research will synthesize novel, stimuli-transforming polypeptides that complex nucleic acids with a cationic arm under neutral pH, but which, upon hydrolysis in a cellular compartment, are converted to neutral, naturally occurring polypeptides, thus destabilizing the complex and releasing nucleic acids. Specifically, the project will investigate novel biomaterials that incorporate ketalized serine (kSer) residues, which are analogous to lysine (Lys) but which transform into serine (Ser) upon acid-hydrolysis. The kSer residues will be used along with lysine residues, polyethylene glycol (PEG) and other functional polypeptides to create versatile polymeric non-viral vectors. Preliminary in vitro studies have shown that poly(kSer-Lys)/DNA polyplexes and PEG-poly(kSer)/DNA micelles significantly enhanced gene delivery efficiency via several improved intracellular pathways. The PI proposes to develop these novel acid-transforming polypeptide (ATPP)-based gene carriers by better understanding key extra- and intracellular gene delivery pathways. He will (1) investigate hybridization of ATPPs with fusogenic and nuclear localization signal peptides in order to improve targeting, (2) explore the effects of stimuli-sensitive linkages between ATPPs and functional peptides and/or PEG that can be severed by such stimuli to improve efficacy, and (3) optimize the ATPP-containing polymers to improve DNA complexation, cellular internalization, intracellular targeting, DNA release, and transfection.BROADER IMPACTS: Efficiently delivering therapeutic genes, especially using nonviral methods, is of great interest not only in the clinic (e.g., gene therapy) but also in the genetic manipulation of biological phenomena (e.g., reprogramming somatic cells to be induced stem cells) in biocompatible and versatile ways. The novel biomaterials developed from this research will be available to undergraduate and high school students for a hands-on research experience and for a summer research program. The proposed project will motivate undergraduate students to think creatively and independently and give them a chance to test their hypotheses with the PI. The outreach program aims to stimulate interest in scientific and engineering careers among participating high school students, particularly those from underrepresented groups in nearby urban areas. The proposed research will be translated to classrooms and beyond through undergraduate students' direct participation in the CAREER research and an outreach research program. A new undergraduate course will introduce basic concepts of gene delivery using stimuli-responsive polymeric biomaterials; it will then challenge students to develop creative ideas on the CAREER research. Students with the most innovative suggestions will be invited to test their hypotheses in the PI's laboratory and will assist high school participants in a summer research program on gene delivery using the acid-transforming polypeptides. This outreach program will grow out of the PI's lectures in combination with hands-on experiments at local high schools in socio-economically disadvantaged areas. The most motivated high school students will participate in a summer research program, followed by developing a science fair project under the mentorship of the undergraduate student mentors. Impacts of these educational activities will be assessed by students' publications and presentations and by their choice of professional careers in science and engineering.