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dc.contributor.authorChatterjee, Nilesh
dc.date.accessioned2022-07-13T17:22:18Z
dc.date.available2022-07-13T17:22:18Z
dc.date.submitted2022-04-28
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/339054
dc.description.abstractTelomerase hTERT RNA is overexpressed in around 90% of all cancers but targeting it has been unsuccessful to date due to the inability of this approach to kill telomerase-expressing cells leading to the evolution of telomerase-independent cells. The approach proposed in the thesis (NASPER), aims to target cells overexpressing hTERT RNA and cause their apoptosis, preventing this evolution and debulking the tumour mass. NASPER involves bringing two fusion proteins, each of which comprises a custom-designed PUF (Pumilio and FBF) RNA-binding protein and a protease, onto the hTERT RNA into close proximity to activate the protease which should lead to cell death. The proteases tested in this study are the HIV protease (HIVPR) and the split-TEV protease, which require dimerisation/fragment complementation for catalytic activity. In Chapter 3, the two designed PUF proteins were first purified and tested in vitro using fluorescence polarisation experiments to assess RNA binding. The results indicate that the PUF proteins bind specifically to their cognate sequences both independently and in combination. In Chapter 4, expression in E. coli and purification of HIVPR was optimised, and its activity was confirmed in vitro. In cells, the individual PUF-HIVPR fusion proteins appeared to auto-activate without their dimerisation partner despite the use of mutations to reduce auto-activity, and it was concluded that a split system is required such that the protease can only undergo activation when both proteins are present. In Chapter 6, a split-GFP system (fusing two GFP fragments to each of the two PUF proteins) most notably show that, as intended, the fusion proteins bind better when cognate RNA is used versus scrambled RNA, and that engineering the fusion proteins such that the split-GFP are domains oriented towards each other results in improved re-constitution of GFP. Subsequently, a NASPER system designed with the split-TEV protease was tested, in which two TEV protease fragments are fused to each of the two PUF domains. The results were consistent with the split-GFP findings, and NASPER was also able to target overexpressed hTERT mRNA in HeLa cells. No cleavage of a procaspase-3 construct could be detected in apoptosis assays in Chapter 8, and further work is required to elucidate the cause. Chapter 9 describes further experiments to understand the binding characteristics of designed PUF domains using crosslinked RNA-seq methods. Overall, the results presented in the thesis provide new insights into the behaviour of designed PUF proteins in the cell and lay the groundwork for a new therapeutic approach based on targeted protease-induced cell death, as discussed in Chapter 10.
dc.description.sponsorshipHarding Distinguished Postgraduate Scholarship
dc.rightsAll Rights Reserved
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.subjectCancer
dc.subjectPUF Protein
dc.subjectsplit-TEV
dc.subjectNASPER
dc.subjectMultispecific Therapeutics
dc.subjectGene Therapy
dc.subjectRNA
dc.subjectApoptosis
dc.subjectPUF split-TEV Fusion Protein
dc.subjectNucleic Acid Scaffold
dc.subjectDesigner RNA binding protein
dc.subjectBroad Spectrum Cancer Targeting
dc.subjectCancer Targeting
dc.titleNucleic Acid Scaffold-dependent Proximity-mediated Enzyme Response (NASPER) - A Proof of Concept Study
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.date.updated2022-07-07T10:21:11Z
dc.identifier.doi10.17863/CAM.86464
rioxxterms.licenseref.urihttps://www.rioxx.net/licenses/all-rights-reserved/
rioxxterms.typeThesis
cam.supervisorItzhaki, Laura
cam.depositDate2022-07-07
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement


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