Metabolic labelling approach for the incorporation of functional groups in therapeutic DNA enabling advanced payload delivery strategies in peptide-based gene delivery

Project description

Project Summary

Non-viral gene delivery strategies have the potential to provide advanced regenerative solutions for cost-effective approaches to improve treatment and restore function of tissues for patient benefit. Effective delivery of therapeutic nucleic acids in a safe and targeted manner using biomaterials in conjunction with defined peptide agents for assembly and delivery could potentially accelerate the translation of non-viral gene therapeutics. This would address significant clinical challenges in dental and musculoskeletal research aiming at the restoration of tissue defects. Research aimed at generating pilot data for such technologies have potential to generate new intellectual property, facilitate interdisciplinary collaboration, lead to the generation of high impact publications and provide valuable data for subsequent larger grant applications.

Aims and Objectives

The current project proposes the incorporation of functional groups into therapeutic plasmid DNA via a novel metabolic labelling strategy during amplification in E. coli using thiophosphate as a phosphate analogue. As the DNA is amplified in E. coli, thiophosphate is incorporated in the plasmid DNA, introducing accessible functional thiol-groups that enable the conjugation of the produced DNA payload to biomaterial surfaces and delivery agents for enhanced delivery. The advantage of using a thiol-based system lies in the compatibility of said system with currently available novel biomaterial systems (as part of a collaboration with Dr. Tronci using collagen and gelatine biomaterial systems developed at Leeds) and advanced peptide systems for gene delivery (provided as part of an ongoing collaboration with Imperial College, London) thus facilitating interdisciplinary collaboration within the University of Leeds and beyond. Furthermore, it has been shown that incorporation of thiophosphate into DNA improves its resistance to nuclease attack, further improving its stability in vivo and the compatibility of the thiol-group approach for attachment opens further avenues for combination of gene therapy approaches with currently investigated self-assembling peptide technologies at the department. Disulphide linkage of payload and delivery agent/material finally allows automatic payload release upon cellular uptake as disulphide groups are reduced in the cytoplasm.

Initial experiments demonstrated the feasibility of the metabolic labelling strategy to introduce thiol-groups into expression capable therapeutic DNA. Furthermore, there is a plethora of synthetic pathways that would facilitate cross linkage between the introduced thiophosphate to the biomaterial surfaces and delivery agents, including (but not limited to) thiol Michael-type reactions or by simply controlling the local redox environment. All synthetic strategies considered will be mild and involve non-toxic catalysts. This project aims at providing the pilot data for application of the principle within biomaterial and peptide-based delivery systems for enhance non-viral DNA delivery in vitro. This pilot data will feed into further grant applications, aimed at translating the most promising delivery strategy in in vivo models of musculoskeletal tissue regeneration via follow-up projects.

Entry requirements

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How to apply (phone)

+44 (0)113 343 7497