Defining the post-transcriptional regulatory mechanisms controlling the SOX9 gene and their potential for promoting cartilage regeneration

Articular cartilage is a tissue in our joints that allows easy articulation and provides "shock absorbing" properties. Following injury or disease it heals very poorly. The tissue is typically described as having one type of cell in it, called a chondrocyte and it is very important that this cell type retains its ability to effectively produce the correct type of connective tissue and allow cartilage to perform its function properly. A very important gene, called SOX9, is used by chondrocytes to orchestrate the correct production of cartilage tissue and it is absolutely essential to the formation of healthy cartilage. Regulation of SOX9 has been implicated in age-related diseases such as osteoarthritis and its use in gene therapy approaches is able to promote the engineering of cartilage tissue. The ability to promote the activity of the SOX9 gene in chondrocytes is thus desirable but its regulation by these cells is still not fully understood. The applicant has been able to demonstrate that a regulatory process that controls the decay of an important gene intermediate, called mRNA, can affect the activity of the SOX9 gene. This project takes the work forward by examining the molecular mechanisms behind this process and use the findings to improve the quality of engineered cartilage.

This project will firstly build upon preliminary data, which shows that a group of specialised regulatory proteins, which can interact with mRNA and alter its decay rate, inhibit the activity of the SOX9 gene. Using a cell culture system, these proteins will be inhibited and the effect this has on SOX9 gene function will be measured. The effect of inhibiting these proteins will then be combined with the manipulation of other DNA-like molecules called microRNAs that exist in chondrocytes. Overall, the first objective will define combination of proteins and microRNAs, which potently affect the activity of the SOX9 gene.

The second objective of the project is to determine what parts of the SOX9 gene make it a target for mRNA decay. By introducing a molecular "reporter" into chondrocytes, the contribution of different regions of the gene to this decay will be assessed. This work will seek to determine whether certain regulatory parts of the SOX9 gene are responsible for the general instability of the mRNA whilst others allow changes to be made to the rate of decay in response to environmental cues. Further experiments will then be conducted to examine how these parts of the SOX9 gene interact with the specialised mRNA interacting proteins examined during the first objective.

The first two objectives of the project examine the basic biological mechanisms, which control an important aspect of SOX9 gene regulation. These are valuable experiments, which will provide an understanding of the control of an essential cartilage gene and of how its altered activity may contribute to the development of musculoskeletal diseases in the aged. The final aim of the project is to take these findings and use them to increase SOX9 gene activity, by reducing mRNA decay through the manipulation of relevant mRNA interacting proteins and microRNAs in human adult stem cells. The effect of these interventions on the quality and quantity of cartilage tissue produced by the stem cells will then be assessed.

To conclude, the outcomes of the project will be the identification of factors which regulate the SOX9 gene, the definition of the characteristics of the gene that make it susceptible to this regulation and the evaluation of the potential of these findings to improve our ability to engineer cartilage tissue. In delivering these outcomes the project aims to better understand the fundamental regulation of a critical cartilage gene, which will impact on how we understand age-related tissue pathology and improve tissue regeneration techniques.

The transcription factor SOX9 is a critical regulator of the chondrocyte phenotype and is involved in the regulation of a large number of cartilage extracellular matrix genes. Reduced expression of SOX9 is a hallmark of diseases such as osteoarthritis, which are strongly associated with old age. In addition, it has been demonstrated that elevated levels of SOX9 are able to strongly promote the formation of appropriate extracellular matrix during cartilage tissue engineering. Therefore, it is sensible that promotion of SOX9 expression forms a part of cartilage regeneration approaches. This project builds on the applicant's published and preliminary data to examine the molecular mechanisms that control this important cartilage gene at the post-transcriptional level. Using siRNA knockdown experiments, the RNA binding proteins responsible for promoting SOX9 mRNA decay will be identified and the relationship between their regulation and that of microRNAs that target SOX9 will be determined. AU rich elements within the SOX9 3'UTR that specifically induce destabilisation will be identified using a cell reporter assay and the RNA binding proteins that interact with these elements will be identified using electromobility shift assays. Finally, the findings will be translated into a tissue engineering approach by examining how knockdown of RNA destabilising proteins, identified in the first set of experiments, or expression of 3`UTR decoy molecules, derived from sequences identified in the latter experiments, can promote the chondrocytic differentiation and subsequent cartilage matrix synthesis of adult human stem cells grown in three-dimensional culture systems. Overall, the project promises to deliver in two major ways: (1) characterise a key step in the regulation of a critical cartilage gene, which has great relevance to degenerative diseases affecting the elderly and (2) translate the findings into a technique that will promote the regeneration of cartilage tissue.

Degeneration and damage to articular cartilage results in pain and disability for millions of people around the world. In the UK alone, it is estimated that treating diseases such as arthritis combined with loss of working hours costs the economy around £18 billion. As the age of the population continues to increase, it is important that we address the degeneration of the musculoskeletal system so that people can lead their longer lives in a happier and more productive manner. This work aims to better understand basic aspects of cartilage biology and apply this knowledge to generating improved protocols for the generation of cartilage in the laboratory. This will benefit other scientists in the fields of arthritis research, connective tissue biology and ageing. Furthermore, tissue engineers will be able to use and build on the improvements to cartilage culture conditions that the project will define. The gene examined in the project also performs essential roles in other tissues, including the brain, heart, gut and reproductive system and so the project's finding will be of interest to researchers in these diverse areas. These impacts will occur over a period of months or years as the work is disseminated at conferences and in scientific literature.
The project will impact upon society. Improved understanding of cartilage biology will be the basis upon which treatments for individuals suffering from degenerative joint diseases will be based. This applies particularly to older people as well as to very active people e.g. sportsmen and women. Such treatments will relieve pain and immobility for a great many people, vastly improving their quality of life. A further consequence of better treatments for cartilage disease and injury include lower treatment costs and a reduced care burden within the National Health Service. In addition, the UK economy would benefit through savings in disability and mobility benefit payments as would employers in the public and private sector as a result of reduced sickness pay and lost working hours. These are long-term societal impacts (years or decades), in the shorter term the project will benefit orthopaedic charities such as Arthritis Research UK and Orthopaedic Research UK due to the additional funds invested in their area of interest, further achieving their aims and raising their profile. Furthermore, public engagement activities such as hosting work experience placements and taking part in outreach activities in local schools will help to increase awareness of the science conducted.
In understanding how a critical cartilage gene is regulated, this work will be of interest to companies that are keen to develop products that can prevent or treat degenerative joint diseases such as osteoarthritis. The project will also develop novel cartilage tissue-engineering approaches. The findings have the potential to be commercially exploitable leading to the production of new spin out companies and to partnership with or expansion of existing enterprise. The project will employ a post-doctoral research associate for three years, training them in relevant molecular and cell biological techniques, as well as the use of stem cells in research. In addition, they will gain transferable skills such as science writing, data presentation, project management, and commercialisation through their work on the project and additional impact related activities. This will result in an individual who possesses a skillset that would benefit employers in the UK public or private sector.
Timescales for these impacts could be as soon as a few months from the end of the project for public engagement and academic beneficiaries. Applying the findings to clinical treatments and commercialisation to a level that may benefit the general public could take 10 years or more and the further development of these treatments to a scale where they are able to affect welfare on a national scale may be measured in decades.