Title: Enhancing chondrogenesis using CRISPR-cas9
Funder: Dunhill Medical Trust

Abstract: OA is a significant burden on health resources globally. Novel approaches are needed to tackle the increased morbidity caused by this disease and provide alternative options to the mechanical solutions currently available for terminal disease; such as joint replacement surgery. Tissue engineering approaches are needed to provide biological solutions for the treatment of OA without the drawbacks observed using inorganic materials. There have been significant advances in the ability to generate cartilage tissue in the laboratory from mesenchymal derived stem cells (MSCs) by controlling their differentiation into hyaline cartilage tissue. MSCs are readily isolated from various tissues, including adipose and peripheral blood, but most work has utilised bone marrow-derived MSCs (BM-MSCs). BM-MSCs have been used with moderate success for the treatment of cartilage defects in animal models in their multipotent state or after differentiation into chondrocytes. As a cellular therapy MSC-derived chondrocytes are believed to offer the most potential. However, during current approaches to MSC-chondrogenesis the cells essentially undergo an endochondral ossification programme, in that cells can enter hypertrophy (characterised by their expression of type X collagen) and then further differentiate to osteoblasts or undergo apoptosis.(Zhong et al., 2015; Mwale et al., 2006) Mechanisms to adapt the progression of MSC-chondrogenesis, as occurs for articular chondrocytes, are likely to be of benefit for tissue engineering approaches to cartilage repair. The recent advance of CRISPR-Cas9 technology allows the manipulation of the human genome not just through genome editing - addition or deletion, but also through the activation or repression of the expression of specific genes; in this case those involved in chondrocyte differentiation. (Chavez et al., 2015; Gilbert et al., 2013) Gene expression during embryological chondrogenesis is well understood, and the limitations of MSC-chondrogenesis in vitro are also well documented. Our strategy is to apply this understanding to modulate gene expression in MSC-chondrogenesis. Our aim is to genetically engineer MSC-chondrogenesis to advance articular chondrocyte phenotype and enhance cartilage extracellular matrix expression and analyse the physiological, structural and mechanical properties of the cartilage produced. Finally our optimised engineered cells will be tested within a ceramic-polymer composite to mimic the endogenous tissue and enhance survival and phenotype. (Vukajlovic et al., 2019; Liu et al., 2017) The purpose of this research is to aid and enhance the biological treatment options of patients with cartilage defects and osteoarthritis. This may lead to translational research testing the optimum engineered MSC-chondrocytes in an in vivo rat cartilage-defect model.

Lay abstract: Joint damage known as OsteoArthritis is caused by the age-related erosion of the joint surfaces where two bones meet and move. It is a leading cause of suffering and loss of working hours in the UK. Approximately 8.5 million people in the UK suffer from moderate to severe joint damage. Treatment for this condition often requires joint replacement using materials not intended to be placed in the body such as metal, ceramic or plastic. These materials often wear out, become loose, react with the human body or harbour infection. There is a need to develop treatments that match the normal joint surface tissue to treat or delay the need for joint replacement. Body tissues are formed from groups of cells that work together. Stem cells are cells that can be turned into any cell in the body and offer a new treatment avenue for creating new joint surface tissue. The process of forming tissue from stem cells can be controlled, using laboratory techniques, into different types of specialist cells by manipulating their genetic code. We have designed a protocol for forming new joint surface tissue by turning off certain genes; that cause stem cells to die or turn to bone, and turn on certain genes; that cause the stem cells to turn to functioning joint surface tissue cells. The ability to create new joint surface tissue in the laboratory has the potential to offer a repair option for joint damage without the problems of current joint replacement surgery options. To date, joint surface tissue created in a lab is of insufficient quality when compared to native joint surface tissue that we are not able to use it as a treatment with reliable long term outcomes. We aim to create a joint surface tissue that is of similar properties to normal human tissue. This has the potential to revolutionise the treatment of this disease. This study is the first of its kind to use a modification of a new gene editing technology to finely control gene function in stem cells to create artificial joint surface tissue in a laboratory. This research has the potential to lead to the production of joint surface tissue, of sufficient quality, to be placed into damaged joints in humans, treat suffering, and delaying or even preventing the need for joint replacement surgery.

Research activity codes: 5.3 MEDICAL DEVICES;5.2 CELLULAR AND GENE THERAPIES
Health categories: MUSCULOSKELETAL