Multiple strategies to enhance efficacy of oligonucleotide therapeutics

Thesis

Nucleic acids (NAs) such as plasmid DNA, messenger RNA (mRNA), and small interfering RNA (siRNA) are a group of gene therapy agents that hold enormous clinical potential in cancer medicine. Despite their undoubted potential, clinical translation of these molecules, however, has been largely held back by their limited bioavailability in target tissues/cells. To overcome this, many drug delivery systems and strategies have been devised.

This research aims to introduce a recently developed cell-penetrating peptide (CPP)-based delivery platform to enhance the internalisation of nucleic acid therapeutics (NATs) into tumour cells. It also aims to explore strategies to trigger the escape of NAs from endosomes to facilitate intracytoplasmic target engagement and controlled release, thereby improving the efficacy and precision of NA-based cancer therapies.

The recently developed CPP, tri-cTatB, combines the advantages of multimerisation and cyclisation of the archetypal TAT peptide and shows highly efficient tumour cell internalisation capability. In this study, a self-assembling stable complex composed of tri-cTatB and NAs was developed. The complex showed efficient and evident cellular uptake within 1 hour at a low carrier concentration of 1 μM. In a study to identify the mechanism of internalisation, it was found that the siRNA/tri-cTatB complexes enter cells via clathrin-mediated endocytosis and that the NA cargoes become trapped in endosomes and lysosomes.

To trigger NA release from intracellular vesicles, various endosome escape strategies were investigated. It was found that photochemical internalisation (PCI) was an effective additional component to the tri-cTatB-mediated NA complex delivery platform. PCI-controlled delivery of GAPDH siRNA caused marked GAPDH enzyme activity downregulation by 91, 51 and 44% in HeLa, MDA-MB-231 and HEK293T cells compared to untreated groups, respectively. In another application, significant polo-kinase-1 (PLK1) knockdown in cancer cell lines was demonstrated following their exposure to siPLK/tri-cTatB complexes. This PCI-controlled tri-cTatB delivery platform was also applied to other types of nucleic acids, including mRNA and plasmid DNA with efficient delivery demonstrated in both cases. In summary, this research describes the use of a tricyclic Tat construct that enables the intracellular delivery of nucleic acids at effective concentrations as low as 1 μM, and is the first report of the combination of a Tat-derived peptide and PCI to achieve highly efficient and controlled release of nucleic acids.

Authors: Li, S

• DOI: 10.5287/ora-zbbjeyz6n
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: drug delivery; cancer therapeutics delivery; nucleic acid therapeutics
• Subjects: Drug delivery systems; Nucleic acids; Oncology