Engineering aynthetic Circuits for research and therapeutic applications using CRISPR-based sensors and extracellular vesicles

Lead Research Organisation: University of Oxford
Department Name: Synthetic Biology DTC

Abstract

1. Development of CRISPR-based sensors for cellular reprogramming

1.1 Brief introduction
Tracking RNA expression in living cells is essential in understanding development, disease and cellular responses to environmental stimuli. The goal of this study is to develop a new platform for live RNA detection and actuation in mammalian cells using Cas9-based transcriptional-activators coupled with inducible spacer-blocking hairpins single guide RNAs (iSBH-sgRNA) developed by Ferry et al.1
1.2 Specific aims
a. Providing proof-of-concept that iSBH-sgRNA can be used for detection of exogenously expressed target RNAs
b. Understanding base-pairing requirements between synthetic RNA triggers and iSBH-sgRNA
c. Extending length of synthetic triggers and determining the tolerated range of trigger lengths
d. Understanding how RNA secondary structures impact trigger activity
e. Extending the original iSBH-sgRNA design and identifying the optimal configurations for detection of long RNA triggers
f. Development of a computational pipeline for screening sub-sequence domains in endogenous targets that could act as triggers. Starting from endogenous RNA targets, this pipeline would output an optimal sequence for iSBH-sgRNAs
g. Test and optimise computational pipeline using bacterial systems. Link detection of target mRNAs with transgene expression
h. Test and optimise computational pipeline in mammalian cells
i. Develop disease-oriented applications by linking detection of endogenous target RNAs with expression of therapeutic transgenes or endogenous genes

2. Development of strategies for loading and delivering CRISPR components using extracellular vesicles

2.1 Brief introduction
Due to their non-immunogenic properties and their ability to cross the blood-brain barrier, extracellular vesicles (EVs) have become valuable tools for drug delivery as well as for engineering communication between mammalian cells3. Although recent studies have attempted to deliver Cas9 to target cells using EVs, limited success has been achieved so far 4,5. This project aims to establish more efficient methods for Cas9 delivery using EVs and to developed entailing EV-based delivery of programable transcriptional activators of clinically relevant genes.
2.2 Specific aims
a. Providing proof-of-concept evidence that EVs could be loaded with Cas9 transcriptional activators and guide RNAs
b. Developing reporter cell lines that trigger expression of fluorescent markers in the presence of active Cas9 transcriptional activators
c. Providing proof-of-concept evidence that EVs loaded with Cas9 transcriptional activators could trigger detectable responses in target cells
d. Performing a mouse biodistribution study for understanding which cells might represent good targets for the modified EVs
e. Developing this system towards upregulation of clinically-relevant gene targets

3. Engineering behaviour of target cellular populations using CRISPR-based sensors and extracellular vesicles

This third project will aim to combine the technologies developed in Part 1 and Part 2
3.1 Project conditions
- Successful implementation of inducible gRNAs capable of linking detection of disease-associated RNAs with the expression of therapeutically relevant genes
- Successful delivery of Cas9-transcriptional activators to target cells using EVs

3.2 Specific aims
a. Proof-of-principle evidence that inducible gRNAs could restore disease phenotype in vitro
b. Loading Cas9-transcriptional activators and iSBH-sgRNA into EVs
c. Optimising the amount of Cas9 and iSBH-sgRNA to be delivered to target cells for obtaining a relevant cellular response
d. Testing strategy in mouse models

This project falls within the EPSRC Synthetic Biology research area and will include EvOX Therapeutics as an industrial partner.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/R512333/1 01/10/2017 30/09/2021
1935000 Studentship EP/R512333/1 01/10/2017 30/09/2021 Oana Pelea
 
Description My DPhil aims to develop a proof-of-concept therapeutic strategy based on RNA-inducible CRISPR sensors and EVs. In order to achieve this, development of engineered sgRNAs for sensing endogenous eukaryotic transcripts as well as optimising CRISPRa delivery using extracellular vesicles is a prerequisite. After accomplishing these tasks, attempts will be made to generate a therapeutic strategy that integrates transcriptional information before executing a CRISPR-mediated therapeutic function.
So far, significant progress has been made towards designing RNA-inducible CRISPR sensors.
Exploitation Route I believe that this project could have implications in both basic research as well as development of novel therapeutic strategies. RNA-inducible CRISPR systems could enable real-time monitoring of RNA transcription. This could be of a particular interest for answering basic research questions regarding RNA functions as well as it would enable further development of RNA-based synthetic circuits. Coupling RNA-sensing with CRISPR-Cas9 technology would also be useful for development of novel therapeutic applications, especially when coupled with novel CRISPR-delivery vehicles such as extracellular vesicles.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description EvOX Therapeutics- Industrial Collaboration 
Organisation EvOx Therapeutics
Country United Kingdom 
Sector Private 
PI Contribution As part of my second year DPhil, I started to work on a project that could be of a potential interest for EvOX as well as I will carry out a placement at this company over the next summer. This research project represents a work in progress and, so far, I initiated several meetings with EvOX in order to make sure to update them regularly on my research plans.
Collaborator Contribution EvOX has kindly sponsored my DPhil and it provides me with an industrial supervisor.
Impact So far, no important outcomes have resulted. The research project I do in collaboration with EvOX is a work in progress and hopefully good outcomes would result soon.
Start Year 2018