Investigating Current Delivery Vehicles for Efficient and Targeted Delivery of Therapeutic RNA and Future Perspectives Part A - Investigating current delivery vehicles for efficient and targeted delivery of therapeutic RNA and future perspectives/ Part B - The development of novel polymeric nanoparticles as efficient non-viral RNA delivery vectors

Sachan, Isha (2023) Investigating Current Delivery Vehicles for Efficient and Targeted Delivery of Therapeutic RNA and Future Perspectives Part A - Investigating current delivery vehicles for efficient and targeted delivery of therapeutic RNA and future perspectives/ Part B - The development of novel polymeric nanoparticles as efficient non-viral RNA delivery vectors. MRes thesis, University of Nottingham.

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Abstract

Part A:

The development of efficient nucleic acid delivery systems is a critical aspect of modern biomedical research aimed at harnessing the potential of gene therapy. Whilst nucleic acid-based therapies have shown success in the biomedical field, they face several key limitations that restrict their effectiveness as therapeutics. A major challenge is the efficient delivery of nucleic cargo to target tissues and cells. Due to nucleic acids being large, negatively charged molecules, they face barriers such as poor cellular uptake, degradation and endosomal escape. Overcoming these challenges to achieve efficient delivery is vital for therapeutics success. Prominent delivery systems such as lipid nanoparticles, polymer nanoparticles, and polyplexes show promise in surpassing these limitations, alongside the therapeutic potential of messenger-RNA and antisense oligonucleotides compounded with primarily lipid nanoparticles. Further research into overcoming the limitations is displayed through hypotheses of the use of polymer micelles and the proton sponge effect. Overall, this paper aims to provide a comprehensive overview of current successful delivery systems, recent advancements, and further novel methods to overcome the delivery limitations. Understanding of the strengths and limitations of these systems alongside the emerging approaches can be optimised by researchers to further their design and foster the translation of nucleic acid-based therapies into clinical practise, ultimately providing a route for the development of treatments for a plethora of diseases.

Part B:

In recent years, the development of mRNA-based therapeutics has emerged as a promising strategy for addressing a wide range of diseases, including genetic disorders, infectious diseases, and cancer. However, the efficient delivery of nucleic acids to the cytoplasm of cells remains a formidable challenge. Polymeric nanoparticles (PNPs) have gained significant traction as versatile and effective carriers for mRNA delivery due to their tuneable properties, biocompatibility, and ability to protect nucleic acid cargo from degradation. However, their successful application hinges on their ability to efficiently internalise through the cell membrane to the endosome, and to subsequently escape the endosome to release their nucleic cargo. The composition of the PNPs influence their ability to overcome these challenges, and two novel polymers named TEPA-3L2 and TETA-3L2 with different nitrate-phosphate (N/P) ratios are explored in this review. Investigations using the HEK293T cell line were conducted into the endosome-disrupting compound chloroquine combined with the N/P derivates of the PNPs to observe if transfection could be improved. The Cy-5 eGFP mRNA cell reporter system was used to visualise the internalisation and transfection capabilities of the PNPs. Furthermore, investigations into inhibition of endocytosis pathways were performed to understand the uptake mechanism of TEPA-3L2 and TETA-3L2. In transfection and cytotoxicity assays, the N/P derivatives displayed high transfection and low toxicity, however when combined with chloroquine the transfection efficacy significantly reduced, suggesting endosomal escape is not the bottleneck in the success of the PNPs. On the other hand, the endocytosis pathway inhibitor studies displayed that the N/P ratios of TEPA-3L2 were able to successfully reduce transfection and prevent co-localisation of mRNA. Initial uncertainty into whether decreased transfection was due to cytotoxic effects were eventually overcome and the role of the clathrin-dependent endocytosis pathway in the uptake of TEPA-3L2 became evident. The experimental findings shed light on the current issues of effective mRNA transfection of TEPA-3L2 and TETA-3L2, which can guide their development towards achieving higher efficacy and lower cytotoxicity, resulting in optimal transfection.

Item Type: Thesis (University of Nottingham only) (MRes)
Supervisors: Alexander, Cameron
Keywords: nucleic acid delivery systems, therapeutics, RNA
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
R Medicine > RM Therapeutics. Pharmacology
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 76031
Depositing User: HARDING, Prof Stephen
Date Deposited: 12 Dec 2023 04:40
Last Modified: 08 Jul 2024 08:48
URI: https://eprints.nottingham.ac.uk/id/eprint/76031

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