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

曾被认为是生物医学研究的一个很有前途的领域，由于几乎没有临床成功，以及与病毒基因携带者相关的潜在毒性，人们对基因治疗的热情有所减弱。非病毒基因疗法是更安全的选择，但其有效性受到治疗性蛋白低表达的限制。尽管随着非病毒基因治疗的发展，许多将DNA运送到细胞质的细胞障碍已经被克服，但阻碍其临床应用的一个主要挑战是DNA从细胞质、穿过核孔和进入细胞核的穿梭。总体而言，人们对这一机制知之甚少，而活跃的穿梭物质，如核定位信号，与DNA的偶联产生了不一致和有争议的结果。这些结果可能归因于正电聚合物，如线性聚乙烯亚胺，与DNA(DNA复合体)的络合，DNA复合体先前已与核定位信号偶联，以促进DNA向细胞质的运输。虽然线性聚乙烯亚胺在基因传递的最初阶段是至关重要的，但它可能会阻碍核渗透，因为大多数DNA复合体太大，无法装入直径约20纳米的核孔中。因此，这份职业计划的目标是研究核进入与DNA复合体的结构形态和尺寸之间的关系。以前的研究没有涉及这一主题，因此错过了提高非病毒基因治疗效果的重要机会。DNA复合体可以形成多种结构，包括杆状、环状和球状构象。由于DNA复合体的环状和球状构象大多大于20纳米，因此推测它们通过核孔进入细胞核的能力有限。因此，这项建议涉及DNA复合体的纳米工程，以控制其大小和/或使用不同的络合过程将其结构构象定向为棒状。这一问题的解决可能是加速非病毒基因治疗领域从基础研究到临床应用的变革性突破。如果成功，非病毒递送系统可能与病毒一样有效。教育活动包括在所有教育级别将研究与教学相结合，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确定和招收K-12学生，这些学生是他们家庭中第一代上大学的学生，在生物医学工程领域的代表性不足。由于这些学生将从俄亥俄州东北部经济不景气的地区招收，暑期辅导计划包括有偿研究职位，旨在提高学生被大学录取的前景，并帮助他们竞争奖学金。此外，还将招聘高中教师进行研究。理想情况下，这些教师将与他们自己的学生配对，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，并将有机会在K-12和本科生的指导下学习。该提案还力求通过分发教育材料和开发专门为高中教师设计的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从未将生物医学工程视为可行职业道路的学生的兴趣。

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.