Redirecting the cellular information flow with programmable dCas9-based chimeric receptors

Thesis

Signal integration and transduction by cell-surface receptors is a complex, multi-layered process resulting in tight regulation of downstream mediators, which in turn elicit pre-defined native cellular responses. The modular architecture of transmembrane receptors provides a unique opportunity for engineering de novo sensor/effector circuits, enabling the development of custom cellular functions for research and therapeutic applications.

The signal transduction module of most existing chimeric receptors consists of either native intracellular domains or effectors domains fused to non-programmable DNA binding proteins. Therefore, these receptors can only engage in natural signalling pathways or drive the expression of artificial, pre-integrated transgenes. By harnessing the programmability of a nuclease deficient CRISPR/Cas9 (dCas9) signal transduction module and leveraging the evolutionarily optimised ligand-sensing capacity of native receptors, I have created a novel class of dCas9-based synthetic receptors (dCas9-synR).

I demonstrate that an optimised split dCas9-based core architecture and custom protease-based signal release mechanism can be standardised across multiple classes of extracellular domains to engineer receptor tyrosine kinase (RTK)-based and G-protein-coupled receptor (GPCR)-based chimeric receptors. dCas9-synRTK and dCas9-synGPCR integrate a broad variety of input signals (peptides, proteins, lipids, sugars) with highly specific and robust activation of any custom output transcriptional programme in an agonist dose-dependent manner. Finally, to showcase the therapeutic potential of dCas9-synRs, I used them to convert a pro-angiogenic signal into an anti-angiogenic response, deploy a chemokine/cytokine programme in response to tumour-enriched biomolecules, and induce insulin expression following glucose stimulation. The performance of dCas9-synRs and their unique versatility in redirecting the information flow makes them ideally suited to engineer designer cells capable of sensing specific disease markers and in turn drive various therapeutic programmes.

Authors: Baeumler, TA

• DOI: 10.5287/ora-rjbx82j6n
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: SYNTHETIC RECEPTORS; GENOME ENGINEERING; SYNTHETIC BIOLOGY; CRISPR