Engineering a Mastoparan Peptide Concatemer Prodrug From CircRNA for Cancer Therapy

Grewcock, Declan (2023) Engineering a Mastoparan Peptide Concatemer Prodrug From CircRNA for Cancer Therapy. PhD thesis, University of Nottingham.

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CircRNAs are covalently closed loops of RNA formed as products of RNA backsplicing in mammalian cells. Engineered circRNAs containing a desired coding sequence have been produced using self-splicing introns. Translatable circRNAs require an internal ribosomal entry site or m6A methylation site for translation initiation. CircRNAs with a nucleotide length a multiple of three, a start codon, and no stop codon in the same frame have an infinite open reading frame. This project aimed to produce a mastoparan peptide concatemer prodrug from circRNA for treatment in cancer therapeutics. Anabaena group I self-splicing introns were used to circularise a mastoparan prodrug containing a metalloproteinase cleavage site for activation (construct named Anabaena Mastoparan). RNA circularisation was achieved in vitro but not in mammalian cells, indicating that group I Anabaena introns do not have the catalytic ability to splice in mammalian cells. Mastoparan peptides were detected in vitro and in vivo after adding a Flag tag to the Anabaena Mastoparan construct. However, only peptides produced from unspliced RNA translation were detected. Mastoparan peptides extracted from Anabaena Mastoparan transfected cells caused cytotoxicity when added to the culture medium of MDA-MB-231 and MCF-7 cells. Anabaena Mastoparan transfection did not directly lead to cytotoxicity, demonstrating the effectiveness of mastoparan as a prodrug, only being activated by metalloproteinase cleavage in the extracellular environment.

This project aimed to identify endogenous circRNAs that have the coding potential to produce a peptide with a different biological function to their parent gene. Using a Bioinformatics approach, circRNAs containing an ORF through the circular junction were identified. Their ORF through junction peptides were investigated for differences in predicted function to their parent gene using InterProScan and Protein Homology/analogY Recognition (Phyre2). Using this approach, four candidate circRNAs were identified that encode a predicted peptide with a different biological function to their parent gene. The four candidate circRNAs contain either a predicted m6A or an internal ribosomal entry site for translation initiation, and have a codon adaption index score (CAI) between 0.781 and 0.821, comparable to the 75th percentile of ORFs through the circular junction (079), and the mean CAI score of coding sequence mRNA. This project demonstrates that the circular junction of circRNAs can provide the coding potential to produce unique peptides with a different function to their parent gene.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Spriggs, Keith
Collins, Hilary
Keywords: CircRNA, Breast Cancer, Mastoparan, cancer therapeutics, prodrugs
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
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
Related URLs:
Item ID: 73525
Depositing User: Grewcock, Declan
Date Deposited: 22 Jul 2023 04:40
Last Modified: 22 Jul 2023 04:40

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