Once 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.
