Kennedy Trust Prize Studentships
Genome editing of stem cells for cartilage repair and regeneration
Genome editing is revolutionising biomedical research. It enables the introduction of targeted genomic sequence changes in isolated cells or whole organisms, and as such is an extremely powerful research tool with huge therapeutic potential. Cas9 is a nuclease guided by small RNAs (sgRNAs) to complementary sites in the genome. It induces double stranded breaks (DSBs) which repair through non-homologous end-joining (NHEJ), or in the presence of a suitable DNA template, by homology-directed repair (HDR). The CRISPR-Cas9 system thus enables highly specific gene editing giving it great potential for correction of genetic (particularly monogenic) diseases or for revolutionising current cell therapies, such as autologous chondrocyte implantation, where the cells could be edited in vitro before re-implantation back in the body. In the present proposal we will apply genome editing techniques recently developed with our collaborators (1) with the aim of creating modified populations of human chondrocytes with enhanced capacity for cartilage repair and regeneration.
We have previously found that hypoxic signalling (predominantly via HIF transcription factors) promotes cartilage synthesis (2) and prevents its breakdown (3). We will now use CRISPR-Cas9 to manipulate this pathway in both primary human chondrocytes and human chondrogenic stem cells, specifically induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs). One of our key approaches will be to target upstream regulators of the HIF transcription factors, previously identified in our lab (4), in an effort to enhance chondrogenic differentiation of stem cells and cartilage matrix production of chondrocytes. Genome edited cell populations will be tested in ex vivo human cartilage repair assays. Such edited cells will form the basis for the generation of new cell-based regenerative medicine strategies aimed at repair and maintenance of the joints.
The Kennedy Institute is a world-renowned research centre and is housed in a state-of-the-art research facility. Ideally situated on the Old Road Campus, it sits next to the Big Data Institute, the Ludwig Insitute for Cancer Research, The Target Discovery Institute and the Wellcome Trust Centre for Human Genetics. Training will be provided across the range of cutting-edge cell, molecular, and bioinformatics techniques. A core curriculum of 20 lectures will be taken in the first term of year 1 to provide a solid foundation in various aspects of biomedical research and data analysis. Students will attend weekly departmental meetings, journal clubs, and will be expected to attend seminars within the department and those relevant in the wider University. Subject-specific training will be received through our group's weekly lab meetings. Students will also attend external scientific conferences where they will be expected to present their research findings.
- Bassett, A., et al. Understanding functional miRNA-target interactions in vivo by site-specific genome engineering. Nature Communications. 2014; 5:4640.
- Lafont, J.E., Talma, S., Hopfgarten, C. & Murphy, C.L. Hypoxia promotes the differentiated human articular chondrocyte phenotype through SOX9-dependent and -independent pathways. Journal of Biological Chemistry. 2008; 283, 4778-4786.
- Thoms, B.L., Dudek, K.A., Lafont, J.E. & Murphy, C.L. Hypoxia promotes the production and inhibits the destruction of human articular cartilage. Arthritis Rheum. 2013; 65, 1302-1312.
- Thoms, B.L. & Murphy, C.L. Inhibition of hypoxia-inducible factor-targeting prolyl hydroxylase domain-containing protein 2 (PHD2) enhances matrix synthesis by human chondrocytes. Journal of Biological Chemistry. 2010; 285(27):20472-80.
- Genes, Genetics, Epigenetics and Genomics
- Developmental Biology and Stem Cells; Ageing
- Regenerative Medicine
- Musculoskeletal Science
- Molecular, Cell and Systems Biology
- Translational Medicine and Medical Technology
Prof. Chris Murphy, Head of Cartilage Biology & Repair and Director of Graduate Studies, Kennedy Institute