Osteoarthritis (OA) is a debilitating joint disease that severely affects elderly populations. At present there are no effective treatments for OA and mechanisms of disease progression are poorly understood. Previous work has identified that neuronal-Interleukin-16 (nIL-16) was significantly up-regulated in cartilage during the later stages of OA. Preliminary investigations identified co-localisation of nIL-16 with the Transient Receptor Potential cation ion-channel sub-Family-V-member-4 (TRPV4) in the primary cilium and the pericellular matrix of human OA chondrocytes. Perturbation of both TRPV4 and cilia are strongly associated with OA. We hypothesised that nIL-16 and TRPV4 work in tandem in a pathway that leads to chondrocyte hypertrophy and calcification that is seen in late OA and contributes to the loss of joint integrity. This makes it a promising target for development of a gene therapy to combat the disease. With the aim of elucidating the mechanism involved, nIl-16 knock-out cell lines generated using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system will be used to knock out nIl-16 PDZ domains to investigate whether this is the mechanism in which nIL-16 functions to anchor TRPV4 to the membrane of chondrocytes at the primary cilium. This work will be carried out using an immortalized hTERT mesenchymal stromal cell (MSC) cell line and effects on terminal MSC chondrogenesis, where hypertrophy mimics the process of calcification seen in OA, will be used to define functional effects of the knockout. Cell lines will be made using the RALA peptide (Phion Therapeutics), a bioinspired nanoparticle, for delivery the CRISPR/Cas9 system

Low back pain (LBP), caused by intervertebral disc (IVD) degeneration represents one of the most significant socioeconomic conditions facing Western economies. Novel regenerative therapies, however, have the potential to restore function and relieve pain. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation to nucleus pulposus (NP) cells of the IVD, offering a potential treatment for LBP. The aims of this study were to i) elucidate GDF6 cell surface receptor profile and signalling pathways to better understand mechanism of action; and (ii) develop a microparticle (MP) delivery system for GDF6 stimulation of ASCs. GDF6 receptor expression by ASCs (N=6) was profiled through western blot, immunofluorescence (IF) and flow cytometry. Signal transduction through Smad1/5/9 and non-Smad pathways following GDF6 (100ng/ml) stimulation was assessed using western blotting and confirmed using pathway specific blockers and type II receptor sub-unit knockdown using CRISPR. Release kinetics of GDF6 from MPs was calculated (BCA assay, ELISAs) and ASC differentiation to NP cells was assessed. BMPR profiling revealed high BMPR2 expression on ASCs. GDF6 stimulation of ASCs resulted in significant increases in Smad1/5/9 and Erk phosphorylation, but not p38 signalling. Blocking GDF6 signalling confirmed differentiation to NP cells required Smad phosphorylation, but not Erk. GDF6 release from MPs was controlled over 14days in vitro and demonstrated comparable NP-like differentiation to exogenous GDF6 delivery. This study elucidates the signalling mechanisms responsible for GDF6-induced ASC differentiation to NP cells and also demonstrates an effective and controllable release vehicle for GDF6