Collaborative Research: Designing Multivalent Ligands for Plasmid DNA Purification

本合作提案的总体研究目标是质粒DNA纯化的多价配体的分子工程和基本机制的理解。基因治疗已经成为一种有吸引力的治疗策略，以遗传畸变为特征的疾病。目前，超过四分之一的基因治疗临床试验正在使用质粒DNA （pDNA）。对pDNA日益增长的需求将需要有效的纯化方法，以成功地将治疗性pDNA从实验室工作台转移到患者床边。目前使用的质粒DNA纯化策略很大程度上依赖于最初为蛋白质开发的配体和色谱方法，但主要由于pDNA独特的物理化学性质而具有低容量、低选择性和低产量的特点。具有高容量和/或pDNA选择性的新配体的发现将导致高效和经济的分离过程。假设在分子支架上结合多拷贝的伪亲和DNA结合分子将导致多价配体（MLs）与pDNA具有高亲和性。分子动力学（MD）模拟将用于消除弱结合的MLs，并研究配体- dna结合的基本物理化学现象。在MD模拟的指导下，将合成一个小的ml库。将以平行方式评估ML结合质粒DNA的能力，结合数据的平衡热力学建模将有助于阐明ML与pDNA结合时多价性和协同性的作用。高亲和力的MLs将被结合到有机和无机膜上，以便从细胞培养液中纯化质粒DNA。这种膜层析方法是基于大孔（400nm）膜的高结合能力和低运输阻力。因此，拟议的研究将导致发现用于pDNA纯化的新型多价配体，对这些配体结合活性的基本见解，以及用于pDNA纯化的膜层析的使用。这项研究将对未来的生物加工产生重大影响，从而降低基因治疗和DNA疫苗的质粒DNA成本，这对社会有巨大的好处。所提出的工作将最终在对分子相互作用，多价性和配体发现的合作作用的基本理解中达到高潮，这对于涉及生物分子纯化的其他应用是有用的。参与该项目的两名研究生将在PI Rege和PI Jayaraman的指导下，通过a)每月通过Skype进行项目进展会议，b)在项目过程中进行一次为期6个月的学生交换，在模拟和实验技术方面接受全面的培训。此外，两位PI都致力于将亚利桑那州立大学和科罗拉多大学的本科生的研究、教育和培训相结合。虽然PI Rege是亚利桑那州立大学富尔顿本科生研究计划（FURI）的积极参与者，但PI Jayaraman通过科罗拉多大学（CU）的本科生研究机会计划（UROP）为本科生提供支持。作为拟议外展活动的一部分，PI Rege最近与梅萨的梅萨公立学校生物技术学院建立了合作关系。PI Jayaraman是科罗拉多大学工程学院多样性行动委员会的成员，他将继续参与他们的外展活动。这两个项目都将整合研究生和本科生在高中学生拓展活动期间的实验室演示参与。参加外展活动的研究生和本科生将受益于外展计划中提出的教学和指导活动。预计外展计划将对凤凰城大都会和丹佛地区的高中教育做出重大贡献，并将激励女学生和那些来自代表性不足的人口的学生继续接受科学和工程方面的教育

1067840/1066998Rege/JayaramanThe overall research objective of this collaborative proposal is the molecular engineering and fundamental mechanistic understanding of multivalent ligands for plasmid DNA purification. Gene therapy has emerged as an attractive therapeutic strategy for diseases characterized by genetic aberrations. Over one-quarter of gene therapy clinical trials currently underway employ plasmid DNA (pDNA). Increasing demand for pDNA will require efficient purification methods to successfully transition therapeutic pDNA from the laboratory bench to the patient bedside. Currently used plasmid DNA purification strategies largely rely on ligands and chromatographic methods originally developed for proteins but are characterized by low capacities, selectivities, and yields mainly due to the unique physicochemical properties of pDNA. The discovery of new ligands with high capacities and/or selectivites for pDNA will lead to efficient and cost-effective separation processes. It is hypothesized that incorporation of multiple copies of pseudo-affinity DNA binding molecules on molecular scaffolds will lead multivalent ligands (MLs) with high binding affinities to pDNA. Molecular dynamics (MD) simulations will be employed to eliminate weakly binding MLs and investigate fundamental physicochemical phenomena underlying ligand-DNA binding. A small library of MLs, guided by MD simulations, will be synthesized. MLs will be evaluated for binding plasmid DNA in a parallel fashion, and equilibrium thermodynamic modeling of binding data will lead to elucidation of the roles of multivalency and co-operativity in ML binding to pDNA. High-affinity MLs will be conjugated to organic and inorganic membranes in order to purify plasmid DNA from cell culture broths. This membrane chromatography approach is based on the combination of high binding capacities due to MLs and low transport resistances in large pores ( 400 nm) of the membranes. The proposed research will therefore lead to the discovery of novel multivalent ligands for pDNA purification, fundamental insights into the binding activity of these ligands, and the use of membrane chromatography for pDNA purification. The proposed research will significantly impact future bioprocessing, leading to lowering the cost of plasmid DNA for gene therapy and DNA vaccines, which is of tremendous benefit to society. The proposed work will culminate in a fundamental understanding of the role of molecular interactions, multivalency and co-operativity for ligand discovery which can be useful for other applications involving purification of biomolecules. The two graduate students working on this proposed project will receive a well-rounded training both in simulation and experimental techniques under the guidance of PI Rege and PI Jayaraman through a) monthly project progress meetings through Skype and b) through a 6-month student exchange once during the course of the project, Additionally both PIs are committed to integrating research, education, and training of undergraduate students at ASU and Colorado, respectively. While PI Rege is an active participant in the Fulton Undergraduate Research Initiative (FURI) program at ASU, PI Jayaraman has supported undergraduates through the Undergraduate Research Opportunities Program (UROP) at University of Colorado (CU). As part of proposed outreach activity, PI Rege has recently established a collaboration with the Mesa Pubic Schools Biotechnology Academy in Mesa. PI Jayaraman is a member of the Diversity Action Committee in the College of Engineering at University of Colorado and will continue to participate in their outreach activities. Both PIs will integrate the participation of graduate and undergraduate students in laboratory demonstrations during outreach activities to the high-school students. Graduate and undergraduate in the outreach activity will benefit from the teaching and mentoring activity proposed in the outreach program. It is anticipated that the outreach program will contribute significantly to high school education in the Phoenix Metropolitan and Denver areas and will motivate women students and those from underrepresented populations to pursue further education in sciences and engineering