Collaborative Research: Designing Multivalent Ligands for Plasmid DNA Purification

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

1067840/1066998 Rege/Jayaraman The 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