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Hardie, Derek T. (2024) Investigating the Differential Regulation of Cell Surface GAG in Cancer: Using NGPD to Isolate Cancer Cell Specific Peptides. PhD thesis, University of Nottingham.

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Abstract

Glioblastoma is the most prevalent primary malignant brain tumour in adults. The standard treatment consists of maximal surgical resection, chemotherapy using the alkylating agent temozolomide (TMZ), and radiotherapy. Despite current treatments, long term survival remains low, and there are high recurrence rates. Gene therapy, which aims to generate a therapeutic effect via the delivery of exogenous nucleic acids, could offer a new approach. However, to be successful gene therapy requires delivery vehicles which are able to facilitate uptake into target cells whilst protecting genetic cargo. The glycosaminoglycan (GAG)-binding Enhanced Transduction (GET)-peptide is able to electrostatically form complexes with nucleic acids and facilitate enhanced transfection in a non-specific manner via a synergistic effect between a GAG-binding domain and cell penetrating peptide (CPP). In order to modify the peptide to specifically deliver to glioblastoma cells Next Generation Phage Display (NGPD) was employed to screen for GET-variants specific to KNS42 cells, resulting in the peptide termed KR being identified. After screening, multiple modified versions of the peptide were made to include LK15 domains, which are present in the GET peptide.

This project first sought to ensure that the modified phage-isolated peptides had potential as a gene delivery vector by investigating their ability to complex pDNA. Utilisation of a YO-PRO-1 assay confirmed their complexation capacity and agarose gels post DNase I treatment confirmed the phage-isolated peptides ability to protect genetic cargo. The physiochemical properties also confirmed the complexes were positively charged nanoparticles. The phage-isolated peptide/pDNA complexes were proven to be able to transfect reporter genes into mammalian cells in vitro. Furthermore, the phage-isolated peptides showed preference for targeting KNS42 cells over NIH3t3 cells. The particles were further optimised on both pDNA dosage and charge ratio which resulted in particles which retained specificity when delivering to KNS42 cells compared with BJ, iHMSC, NIH3t3, and even against another glioblastoma cell line U87. The original phage-isolated peptide without modifications (KR) was also investigated in a mouse biodistribution study. Through comparison between a control GET-peptide (PR) and Alexa Fluor 680 only, which mimicked “free” drug, the peptide again showed improved specificity particularly 1 h post injection. It was also seen that the phage-isolated peptide was quickly excreted from the body, more so than PR. Due to the positive preliminary results new vectors were designed for future phage display libraries. Through the use of a novel phagemid with a eukaryotic expression cassette, peptides can be directly screened for their transfection capabilities. By combining these vectors with modified helper phage (a phage required for superinfection to provide all other required phage proteins for production of an intact virion), it will provide a toolbox to isolate candidate GET-peptides for a variety of delivery challenges including for the treatment of glioblastoma.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Dixon, James E. Gough, Kevin C. Allegrucci, Cinzia
Keywords:	Nanoparticles, Gene therapy, Glioblastoma, nucleic acids
Subjects:	R Medicine > RC Internal medicine > RC 254 Neoplasms. Tumors. Oncology (including Cancer) R Medicine > RM Therapeutics. Pharmacology
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	77413
Depositing User:	Hardie, Derek
Date Deposited:	24 Jul 2024 04:41
Last Modified:	24 Jul 2024 04:41
URI:	https://eprints.nottingham.ac.uk/id/eprint/77413

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