IDR: Nucleic Acid-Lipid Films - Programmable Structural Transitions for Drug Delivery and Regulating Gene Expression

1015026 tirrelll智力优势：该跨学科项目旨在将自组装材料的新发展带到生物活性核酸（NA）传递和基因表达的前沿问题上。由核酸和脂质组成的材料，在静电、疏水和水合力的平衡基础上组装，形成稳定的层状膜，交替的核酸和脂质层。我们小组最近发表的研究表明，这种结构可以通过各种方式进行操纵，例如，通过改变温度或水合状态，或通过改变所含核酸的分子量。这为潜在的技术提供了一个通用的平台，以推进和开发基因传递和基因表达调控的新能力。提出的工作的第一个目标是为所述的交付应用程序优化这些结构。提案中包括的初步工作表明，核酸传递导致干细胞转染，是可能的与这些结构。将通过不同的脂质选择（尽可能具有最佳的拆卸特性并最小化任何毒性），通过包含具有所需结构特征的多种核酸，更重要的是，能够同时具有多次转染能力，来探索这种能力的最大化。所有这些构造的结构和拆卸概况将被彻底地描述。这项工作的第二个目标，将与第一个目标并行进行，是使用这些结构来检查培养中的小鼠胚胎干细胞的转染效率，使用绿色荧光蛋白的表达作为效率的指标。干细胞明显难以转染；我们的目标是利用我们构建的核酸的高浓度，以及细胞与核酸-脂质层的直接物理接触来提高转染效率。这项工作的第三个目标是将我们的新材料递送载体应用于microrna的递送。MicroRNAs （miRNAs）是一类新的小的、可调节的非编码RNA，在转录后水平上作为基因表达的有效调节剂。为实现这一目标，将探索以接触为媒介的分娩。这项工作第三阶段的进一步目标是整合质粒DNA和miRNA递送，将成年细胞重编程为诱导的多能干细胞。iPS细胞在个性化治疗和高通量药物筛选平台方面具有许多深刻的科学和生物医学意义。microRNA传递重编程的技术挑战不是传递效率本身，而是在大约十天的时间内持续传递以实现持续表达。提议研究的跨学科性质：该团队汇集了几个不同的学科，从化学工程和材料科学到干细胞和组织工程，再到基因调控的分子和细胞生物学。这项工作将使材料科学的发现在工程核酸传递和基因表达方面取得相当大的进展。该团队体现了化学和材料工程、生物学和生物工程之间的联系，这对于实现这种新的输送系统的潜力至关重要，既可以深入了解如何优化所涉及的材料，又可以确保以有意义的方式实现生物工程目标。更广泛的影响：一个令人兴奋的，跨学科的发展项目，如这是一个理想的机会，把本科工程专业的学生到一个重要的研究领域的前沿。该项目横跨化学、材料和生物工程以及分子和细胞生物学三个实验室，其本质赋予了本科生不同兴趣的参与能力。具体计划是每年夏天从代表性不足的群体中招募两名本科生（在项目生命周期中总共有六名）参与这项研究。这些学生将被加州大学伯克利分校的安进学者暑期研究项目录取。安进学者计划是一个国家计划，每年吸引大约25名参与者。加入这个由25人组成的小组，这两名本科生将作为暑期研究小组的成员在许多方面受益良多。他们将参加所有的项目活动，包括每周的会议和海报会议，并在夏季结束时进行口头报告。作为这些合作活动的结果，这个项目的本科生参与者将充分参与到一个广泛而全面的暑期体验中。

1015026TirrellIntellectual Merit: 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.