Quinine polymer beacons for probing encapsulation and cellular delivery of CRISPR-Cas9

Non-Technical Abstract:

With support from the Biomaterials Program in the Division of Materials Research, Professors Reineke and Frontiera are developing a series of new polymeric materials that bind, compact, and transport nucleic acids and gene editing systems into living cells. The development of new delivery vehicles is crucial to reducing the cost and toxicity of gene editing therapies. The goal of gene editing is to modulate the expression of gene products to treat diseases by delivering therapeutic nucleic acids and/or ribonucleoprotein complexes. Traditionally, gene editing systems, have been delivered by modified viruses, which are extremely expensive and can lead to detrimental immune responses. Polymeric vehicles are promising candidate systems for delivery as they bind nucleic acids and proteins into compact nanoscale vehicles, while simultaneously reducing side effects from traditionally used viral vehicles. Here, the PIs propose using a tunable series of polymers containing quinine, a natural product long used to treat malaria, to promote delivery and unpackaging of gene therapy components in living cells. The PIs will synthesize and characterize a family of polymers rationally designed for efficient gene editing construct delivery, and comprehensively characterize their structure and packaging efficiency. These polymers will be screened for genome editing efficiency and lack of toxicity. The most promising candidate formulations will be examined with advanced spectroscopies in order to determine the mechanism of cellular delivery. Additionally, the PIs will establish an interactive mentoring and learning environment by attracting students to the McNair Scholars program and hosting them for summer research and mentoring experiences.

Technical Abstract:

Development of new materials that bind, compact, and transport nucleic acids and gene editing systems into living cells in a manner that is efficient, nontoxic, and does not interfere with normal cell function is essential for the success of many facets of medical research. Polymeric nucleic acid vehicles show great promise in circumventing the problems associated with viral vectors (immunogenicity, difficulty in scalability, and high cost) because their structures can be modified synthetically to endow them with properties for nontoxic and highly efficient transport. The objective of this project is to develop a novel family of seven delivery vehicle 'beacons' containing quinine that form complexes with nucleic acids and Cas9 protein, are low in toxicity, and can be readily tracked for payload packaging and intracellular trafficking. The project will entail synthesis of a family of tunable quinine-containing polymers, with quantitative characterization by comprehensive optical and vibrational spectroscopies. This suite of polymers will be assessed for cell viability and genome editing efficiency, with statistical identification of the most promising formulations. These systems will be examined in detail, with discovery of the mechanisms of intracellular delivery, kinetics of unpackaging, and editing payload location. The exploration of new delivery systems with unique properties to facilitate multicomponent complex formation, spectroscopic probing of payload packaging, cellular delivery, and imaging could offer transformative potential to the field of non-viral based gene editing system development.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.