This interdisciplinary program aims to bring new developments in self-assembled materials to bear on frontier problems in bioactive nucleic acid (NA) delivery and gene expression. Materials comprising nucleic acids and lipids, assembled based on a balance of electrostatic, hydrophobic and hydration forces, form stable, layered films, alternating nucleic acid and lipid layers. Recently published work from our group has shown that this structure can be manipulated in various ways, for example, by changing the temperature or state of hydration, or by varying the molecular weights of the nucleic acids included. This provides a versatile platform of potentially enabling technology to advance and develop new capabilities in gene delivery and the regulation of gene expression. The first aim of the proposed work is to optimize these constructs for the stated delivery applications. Preliminary work included in the proposal demonstrates that nucleic acid delivery, leading to transfection of stem cells, is possible with these constructs. Maximization of this capability will be explored by varying lipid choices (to have the best possible disassembly characteristics and to minimize any toxicity), by including multiple nucleic acids to have the desired structural features, and more importantly, to be able to have simultaneous, multiple transfection ability. The structures and disassembly profiles of all of these constructs will be thoroughly characterized. A second aim of this work, which will be conducted in parallel with the first, is to use these constructs to examine transfection efficiency for mouse embryonic stem cells in culture, using expression of green fluorescent protein as an indicator of efficiency. Stem cells are notably difficult to transfect; our aim is to exploit the high concentration of nucleic acids in our constructs, and the direct physical contact of the cells with the nucleic acid-lipid layers to increase transfection efficiency. A third aim of the work will be to apply our new material delivery vehicles to the delivery of microRNAs. MicroRNAs (miRNAs) are a novel class of small, regulatory non-coding RNA, serving as potent regulators for gene expression at posttranscriptional level. Contact-mediated delivery will be explored to accomplish this goal. The further goal of the third phase of this work is to integrate plasmid DNA and miRNA delivery to reprogram adult cells into induced, pluripotent stem cells. iPS cells have numerous profound scientific and biomedical implications in personalized therapies and platforms for high-throughput screening of pharmaceuticals. The technical challenge with microRNA delivery for reprogramming is not delivery efficiency, per se, but rather sustained delivery to achieve sustained expression over a span of approximately ten days.
Interdisciplinary Nature of the Proposed Research:
The team assembled spans several different disciplines from chemical engineering and materials science, to stem cell and tissue engineering, to the molecular and cellular biology of gene regulation. The proposed work will take a discovery in materials science quite far toward new enabling technology in engineering nucleic acid delivery and gene expression. The connection this team embodies, among chemical and materials engineering, biology and biological engineering, is essential to realize the potential of this new delivery system, both to have the insight into how to optimize the materials involved, and to assure that the biological engineering objectives are achieved in a meaningful way.
Broader Impacts:
An exciting, interdisciplinary development project such as this is an ideal opportunity to bring undergraduate engineering students to the forefront of an important research area. The very nature of this project, spanning three laboratories in chemical, materials and biological engineering, as well as molecular and cellular biology, gives capacity to bring undergraduates with varied interests into participation in this work. The specific plan is to engage two undergraduate students per year, in the summer (six total over the project lifetime), from underrepresented groups as participants in this research. These students will be admitted to the Amgen Scholars Summer Research Program at UC Berkeley. The Amgen Scholars Program is a national program attracting approximately 25 participants each year. Joining this group of 25, the two undergraduate participants will benefit significantly in numerous ways as members of the summer research cohort. They will participate in all program activities including weekly meetings and the poster session and oral presentations at the end of the summer. As a result of these collaborative activities, the undergraduate participants in this project will be fully involved in a broad and comprehensive summer experience.
