Prof Sophie Hambleton, Dr Karin Engelhardt
Applications accepted all year round
Funded PhD Project (Students Worldwide)
About the Project
Gene variants that impair the development or function of T cells cause combined immunodeficiency, leaving infants and young children vulnerable to life-threatening infection. Whereas some of the affected genes do jobs confined to the immune system (e.g. in immune cell signalling), others have broader roles in cell biology, yet immune problems predominate in the associated deficiency state. Defects in RNA biology are increasingly recognized to cause immune dysfunction, reduced humoral immunity…
and viral susceptibility. Understanding how RNA transcription, processing and translation regulate immune homeostasis will provide new insights into disease mechanisms and identify potential therapeutic targets.
In this PhD, you will work on families with combined immunodeficiency and novel homozygous variants in different genes involved in the production and maturation of non-coding RNAs. You will: model the mutations in relevant cell types by gene editing and validate and explore the functional consequences; analyse how well these pathways, involving RNA polymerase III, its transcription factors and other enzymes, function in mutant cells compared to control cells; and examine the abundance, type and maturation of tRNAs, which have essential functions in protein synthesis. Having delineated the associated cellular phenotype in immune cells, you will undertake and validate genomic correction by CRISPR editing.
Project Objectives
- Introduce disease-causing variants by CRISPR gene editing into T- and B-cell lines.
- Assay the function of edited/non-edited cells: T-cell proliferation, activation markers, apoptosis, cell cycle analysis, differentiation of B cells into antibody-producing cells.
- Quantify tRNA abundance and maturation in resting and stimulated cells.
- Measure translational output and compare to mRNA abundance.
Correct patient cells by CRISPR editing to restore cellular function. Validate gene editing efficiency/off-target effects.
Research Methodologies
- CRISPR/Cas9 gene editing.
- qPCR to detect levels of relevant RNA species. Western blot for protein detection. Northern blot for detection of differences in RNA processing.
- (Spectral) flow cytometry for activation markers, apoptosis, proliferation assay, cell cycle analysis by EdU/BrdU double labelling, immunophenotyping
- In vitro immunological assays eg T-cell stimulation with antibody-coated beads. B cell differentiation to plasma cells.
- SUnSET (or other puromycin incorporation assay) or Ribo-seq (ribosome profiling) for assessment of translation efficiency.
- Pre-tRNA, bulk RNA and mRNA sequencing to assess abundance of different RNA species.
- Bioinformatic analysis of the above datasets in R.
Potential Project Impact
This project will validate the pathogenic effect and mechanism of novel forms of immunodeficiency. A molecular diagnosis makes a huge difference for affected children and their families, by enabling genetic counselling and informing clinical decision-making. Addition to the virtual panel of disease genes will enable screening among patients undergoing diagnostic genome sequencing for suspected immunodeficiency. This project will also furnish functional assays to help ascertain the significance of any new variants detected in the same genes. Understanding molecular pathogenesis opens the door to potential precision therapies including the gene correction strategy this project will begin to explore.
Proposed Project Timelines
- Years 1-2: CRISPR-editing of variants into cell lines, functional assays, RNA sequencing, Translation analysis
- Years 3-4: CRISPR-gene correction in patient cells, functional assays of corrected cells
Potential Internship/Exceptional Training Opportunities
Learning CRISPR-gene editing technologies at the University of Oxford. Learning pre-tRNA sequencing technology at the University of Cambridge.
Opportunities for student participation in PPIE
Yearly “Genetic Matters” public engagement event in Newcastle. Interaction with local charity for inborn errors of immunity, the Bubble Foundation. Participation in MRC CoRE-wide PPIE training and activities.
MRC CoRE-TG Scholarships
Applicants to MRC CoRE-TG projects may be nominated for a CoRE-TG DPhil scholarship, which involves a second interview following the departmental interview. Due to UKRI limits on international student recruitment, only UK national students are eligible for these MRC CoRE-TG DPhil scholarships.
The MRC CoRE-TG DPhil scholarship provides funding for:
- Course fees for the duration of fee liability.
- A living stipend at the UKRI rate, paid for four years.
- A £20,000 Research Training Support Grant (RTSG) to support research and training costs.
- A £1,200 travel allowance to support research-related travel and development opportunities.
The stipend rate is reviewed annually by UKRI/MRC.
Ready to join us? Applications are competitive and will be assessed according to the admissions requirements of the relevant host department or university.
For questions about the MRC CoRE in Therapeutic Genomics programme, please contact mrccoretg@paediatrics.ox.ac.uk.