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Smith, Lauren (2022) Establishing and Utilising In Vitro Lung Models to Demonstrate Peptide Mediated Non-Viral Delivery. PhD thesis, University of Nottingham.

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Abstract

Despite their success as gene therapy delivery systems, viral vectors have some unresolved issues; pathogenicity, immunogenicity, and off target effects can be detrimental and sometimes even oncogenic. Commercialised vectors can also still come with a higher price point.

An alternative method that might aid in reducing both the negative biological effects and could prove more cost effective is non-viral gene therapy delivery systems. Many are utilised for a variety of diseases currently in research, with varying levels of success. The main focus of this project was to investigate the efficiency of a peptide-based delivery system mediated by the process of Glycosaminoglycan enhanced transduction (GET). This cell penetrating peptide (cell penetrating peptide) contains enhanced peptide domains that allow for interaction with heparan sulphate cell surface sugars, aiding in cell entry. The overall molecule charge also allows binding with double stranded DNA (dsDNA), compacting into a nanoparticle (NP) which can enter the nucleus and allow for transient expression of chosen genes on modified plasmids.

This PhD project combines the novel Glycosaminoglycan enhanced transduction peptide non-viral delivery system (GET system) with two in vitro lung based models, in an attempt to test non-viral delivery without excessive animal use, and to determine this particular non-viral delivery methods efficiency in vitro. The lung models used were murine precision cut lung slices (PCLS), which utilises ex vivo tissue; and human cell Air liquid interface (ALI) cultures, which utilise lung cell lines in vitro. The lung was focussed on due to the potential for non-viral delivery to this organ, particularly in the context of genetic lung disorders such as Cystic Fibrosis (CF).

Within this project, peptide mediated delivery of reporter and enhanced luciferase plasmids to an in vitro lung model was demonstrated using the GET non-viral system; air liquid Interface cultures were also attempted to further demonstrate peptide mediated non-viral delivery in a human relevant in vitro model.

The demonstration of GET non-viral delivery to an in vitro lung model could potentially provide an alternative non-viral methods in the future, for correction of mutations such as the ∆F508 mutation found in 70% of cystic fibrosis patients, or in any other genetic diseases where the mutation is monogenic.

Adding to the body of research on non-viral delivery methods is key to improving non-viral delivery systems; further research can potentially provide more useful information as to the genetic diseases they may be utilised to successfully treat.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Rose, Felicity Dixon, James Tatler, Amanda
Keywords:	viral vectors, non-viral gene therapy delivery, peptides
Subjects:	Q Science > QP Physiology > QP501 Animal biochemistry Q Science > QR Microbiology > QR355 Virology
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	71579
Depositing User:	Smith, Lauren
Date Deposited:	01 Nov 2023 13:19
Last Modified:	14 Dec 2024 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/71579

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