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

1015026Tirrell智能优点：这一跨学科计划旨在带来自组装材料的新发展，以解决生物活性核酸(NA)传递和基因表达的前沿问题。由核酸和类脂组成的材料，根据静电、疏水和水合力的平衡进行组装，形成稳定的层状膜，核酸和类脂层交替。我们小组最近发表的工作表明，这种结构可以通过各种方式来操纵，例如，通过改变温度或水合状态，或者通过改变所包括的核酸的分子质量。这提供了一个多用途的平台，潜在地使技术能够在基因传递和基因表达调控方面推进和开发新的能力。拟议工作的第一个目标是为所述的交付应用优化这些结构。该提案中包括的初步工作表明，利用这些构建物可以进行核酸传递，从而导致干细胞的转基因。这种能力的最大化将通过不同的脂质选择来探索(具有最好的可能的分解特性并将任何毒性降至最低)，通过包括具有所需结构特征的多个核酸，以及更重要的是，能够同时具有多个转染性。所有这些结构的结构和拆卸轮廓将被彻底表征。这项工作的第二个目的，将与第一个平行进行，是使用这些构建物来检测小鼠胚胎干细胞在培养中的转染率，使用绿色荧光蛋白的表达作为效率的指标。干细胞很难转化；我们的目标是利用我们构建的结构中高浓度的核酸，以及细胞与核酸-脂层的直接物理接触来提高转化效率。这项工作的第三个目标将是将我们的新材料输送工具应用于microRNA的输送。MicroRNAs(MiRNAs)是一类新的小分子、调节性非编码RNA，在转录后水平上对基因表达起着强有力的调节作用。为了实现这一目标，将探索以接触为媒介的交付。这项工作的第三阶段的进一步目标是整合质粒DNA和miRNA传递，以将成体细胞重新编程为诱导的多能干细胞。IPS细胞在个人化治疗和药物高通量筛选平台方面具有许多深刻的科学和生物医学意义。MicroRNA递送用于重新编程的技术挑战不是递送效率本身，而是持续递送以在大约10天的跨度内实现持续表达。拟议研究的跨学科性质：该团队组成的团队跨越了几个不同的学科，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。这项拟议的工作将把材料科学的一项发现推向工程核酸输送和基因表达的新使能技术。这个团队体现了化学和材料工程、生物和生物工程之间的联系，对于实现这一新交付系统的潜力至关重要，既要洞察如何优化所涉及的材料，又要确保以有意义的方式实现生物工程目标。更广泛的影响：像这样一个令人兴奋的跨学科发展项目是将本科生带到一个重要研究领域的前沿的理想机会。这个项目跨越化学、材料和生物工程以及分子和细胞生物学三个实验室，其性质使具有不同兴趣的本科生能够参与这项工作。具体的计划是在夏季每年从代表性不足的群体中招募两名本科生(在整个项目生命周期内总共六名)作为这项研究的参与者。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。安进学者方案是一项国家方案，每年吸引大约25名参与者。加入这个由25人组成的小组，作为夏季研究队列的成员，这两名本科生参与者将在许多方面受益匪浅。他们将参加所有计划活动，包括每周会议、海报会议和夏季结束时的口头陈述。作为这些合作活动的结果，这个项目的本科生参与者将完全参与到广泛和全面的暑期体验中。

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.