CAREER: Investigation of DNA Transport into the Cell's Nucleus

职业：研究 DNA 转运至细胞核的过程

• 批准号: 0954360
• 负责人: Yang Yun
• 金额: $ 45万
• 依托单位: University of Akron
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0954360&HistoricalAwards=false
• 关键词: CAREER; Investigation; DNA; Transport; into

0954360YunOnce considered a promising field of biomedical research, the enthusiasm for gene therapy has been tempered by the near lack of clinical successes and by the potential toxicity associated with viral gene carriers. Non-viral gene therapies are safer alternatives, but their effectiveness has been limited by a low expression of therapeutic proteins. Although many cellular barriers to transporting DNA into the cell's cytoplasm have been surmounted with the advancement of non-viral gene therapy, a major challenge that has impeded its usefulness for clinical applications is the shuttling of DNA from the cytoplasm, through the nuclear pores, and into the nucleus. Overall, this mechanism is poorly understood, and the coupling of an active shuttling agent, such as a nuclear localization signal, with DNA has yielded inconsistent and disputable results. These outcomes could possibly be attributed to the complexing of a positively charged polymer, such as linear polyethylenimine, to DNA (DNA complex) that has been previously coupled with a nuclear localization signal in order to facilitate the transport of DNA into the cell's cytoplasm. While the linear polyethylenimine is vital for the initial stages of gene delivery, it may impede the nuclear penetration since most DNA complexes are too large to fit into the nuclear pores, which are approximately 20 nanometers in diameter. Thus, the goal of this CAREER proposal is to investigate the relationship of nuclear entry with the structural morphologies and the dimensions of DNA complexes. Previous studies have not addressed this topic and have therefore missed an important opportunity to improve the efficacy of non-viral gene therapy. DNA complexes have been shown to form a variety of structures, including rod, toroid, and globular conformations. Since most toroid and globular conformations of DNA complexes are larger than 20 nanometers, it is hypothesized that they are limited in their ability to gain entry into the nucleus through the nuclear pores. Thus, this proposal involves the nanoscale engineering of DNA complexes in order to control their sizes and/or to direct their structural conformations into rods using different complexing processes. Resolving this problem could be the transformative breakthrough that accelerates the field of non-viral gene therapy from basic research to clinical applications. If successful, non-viral delivery systems could be as effective as viruses. The educational activities include the integration of research with teaching at all educational levels, recruitment of undergraduate and graduate students, and outreach to local high schools. The key components of this plan are indentifying and recruiting K-12 students that are the first generation in their family to attend college and are under-represented in the field of biomedical engineering. Since these students will be recruited from economically depressed areas of Northeast Ohio, the summer mentoring program includes paid research positions that are designed to enhance the student's prospects for college acceptance and to help their competition for scholarships. In addition, high school teachers will also be recruited to conduct research. Ideally, these teachers will be paired with their own students to form the basis of a research team that will also include graduate and undergraduate students that are funded by this award. The graduate students will undergo engineering and leadership training and will be given opportunities in the mentorship of both K-12 and undergraduate students. This proposal also seeks to enhance the broader impact activities by the distribution of educational materials and by the development of a graduate level course that is designed specifically for high school teachers. It is hoped that the proposed educational activities would foster interest in students that have never considered biomedical engineering as a viable career path.

基因疗法一度被认为是生物医学研究的一个很有前途的领域，但由于临床上几乎没有成功的病例，以及病毒基因携带者的潜在毒性，人们对基因疗法的热情有所减弱。非病毒基因疗法是更安全的替代方案，但其有效性受到治疗性蛋白质低表达的限制。尽管随着非病毒基因治疗的发展，将DNA转运到细胞的细胞质中的许多细胞障碍已经被克服，但是阻碍其用于临床应用的主要挑战是DNA从细胞质穿梭通过核孔并进入细胞核。总的来说，这种机制是知之甚少，和耦合的一个积极的穿梭剂，如核定位信号，与DNA产生了不一致和有争议的结果。这些结果可能归因于带正电荷的聚合物（例如线性聚乙烯亚胺）与DNA（DNA复合物）的复合，所述DNA先前已经与核定位信号偶联以促进DNA转运到细胞的细胞质中。虽然线性聚乙烯亚胺对于基因递送的初始阶段至关重要，但它可能会阻碍核渗透，因为大多数DNA复合物太大而无法装入直径约为20纳米的核孔中。因此，本CAREER提案的目标是研究核进入与DNA复合物的结构形态和尺寸的关系。以前的研究没有解决这个问题，因此错过了一个重要的机会，以提高非病毒基因治疗的疗效。DNA复合物已被证明可以形成多种结构，包括棒状、环状和球状构象。由于DNA复合物的大多数环形和球形构象大于20纳米，因此假设它们通过核孔进入细胞核的能力有限。因此，该提议涉及DNA复合物的纳米级工程，以控制它们的尺寸和/或使用不同的复合过程将它们的结构构象引导到棒中。解决这个问题可能是加速非病毒基因治疗从基础研究到临床应用的变革性突破。如果成功的话，非病毒传递系统可能和病毒一样有效。教育活动包括在各级教育中将研究与教学结合起来，招收本科生和研究生，并向当地高中推广。该计划的关键组成部分是识别和招募K-12学生，他们是家庭中第一代上大学的学生，在生物医学工程领域代表性不足。由于这些学生将从俄亥俄州东北部经济不景气的地区招募，夏季辅导计划包括有偿研究职位，旨在提高学生的大学录取前景，并帮助他们竞争奖学金。此外，还将招募高中教师进行研究。理想情况下，这些教师将与自己的学生配对，形成一个研究团队的基础，该团队还将包括由该奖项资助的研究生和本科生。研究生将接受工程和领导力培训，并将获得K-12和本科生的导师机会。这项建议还力求通过分发教育材料和专门为高中教师开设研究生课程来加强更广泛的影响活动。希望拟议的教育活动将培养学生的兴趣，从来没有考虑生物医学工程作为一个可行的职业道路。