Hanson, Miriam;
(2025)
Nanoparticle Delivery for the
Genetic Therapy of Cystic Fibrosis.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Cystic fibrosis (CF) is a common autosomal recessive disease. The dysfunctional CFTR protein leads to thickened mucus, bacterial infections, and reduced lung function. Modulators are effective treatments for many CFTR genotypes, but ~10% of patients remain untreatable. Gene editing is an attractive therapy to correct CFTR mutations, but lung delivery poses a significant challenge. We aimed to optimise a receptor-targeted nanocomplex (RTN) formulation to deliver plasmid DNA (pDNA) to airway epithelial cells, identify barriers to transfection and explore the inflammatory response to transfection. Lastly, a prime editing approach was explored using a lipid-based delivery system for the Q220X nonsense mutation. RTNs, comprising liposome, peptide and pDNA/mRNA, were successfully optimised in airway epithelial cells using luciferase assays and flow cytometry. They were further characterised for size, charge, encapsulation efficiency and copy number. Two methods of liposome preparation were compared, showing similar performance. RTN cellular uptake in submerged airway cultures exceeded 90%. Higher mRNA GFP transgene expression was observed compared to pDNA in CFBE cells. Microscopy and flow cytometry identified endosomal escape and/or translation as major barriers to transfection for submerged epithelial cells, with additional limiting factors for primary cells. Differentiated airway epithelial cells showed negligible transfection efficiency with RTN delivery. Mucus was a significant barrier, reducing the cilia beat frequency which was partially removed by N-acetylcysteine. There was evidence of RTN-induced inflammation from increased IL-6 and IL-8 secretions in submerged cultures. This short-term effect (< 72 hours) could be reduced by dexamethasone. Finally, prime editing components were delivered by a lipid-based transfection to correct the Q220X mutation. Exogenous CFTR cDNA encoding Q220X in HEK293T cells was corrected to 8.3-20.0%. RTNs remain an effective and relatively safe delivery method for CF. Further optimisation stages will allow these RTNs to deliver gene editing components, paving the way for curative treatments of the disease.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Nanoparticle Delivery for the Genetic Therapy of Cystic Fibrosis |
| Language: | English |
| Additional information: | Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10203879 |
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