Design and application of TRAFTACs to improve the osteoarthritis chondrocyte phenotype

Osteoarthritis (OA), the most common musculoskeletal synovial joint disorder, has turned out to be a global threat crisis due to high prevalence in human population and absence of effective Disease Modifying Osteo-Arthritis Drugs (DMOADs). Targeted degradation of OA-causing catabolic Transcription Factors (TFs), prove to be promising approach of treating OA and designing effective DMOAD. Unfortunately, TFs are usually undruggable due to absence of ligand binding sites. In 2021, TRAnscription Factor TArgeting Chimeras (TRAFTAC) were reported, which were able to efficiently target and degrade two transcription factors: NFκβ and brachyury. First generation TRAFTACs comprise of a HaloTag fused to a catalytically dead CRISPR-associated protein (HT7-dCas9), in complex with a chimeric oligonucleotide consisting of trans-activating RNA (tracrRNA) covalently attached the double-stranded (ds) DNA binding sequence of the TF of interest (TOI). Halo-PROTACs are utilized to recruit E3-ubiquitin ligase, whilst the desired transcription factors bind to the DNA consensus motif. Consequently, this leads to close proximity between E3-ubiquitin ligase and the targeted transcription factor, enabling ubiquitination of the transcription factor, followed by its proteasomal degradation. Second generation TRAFTAC comprises of dsDNA having binding motif sequence of TOI, covalently attached to E3 ubiquitin ligase ligand. Therefore, requirement for ectopic expression of fusion protein and haloPROTAC is bypassed. In this project, we aim to use bespoke TRAFTACs engineered to degrade specific catabolic transcription factors, involved in osteoarthritis pathogenesis and evaluate the therapeutic efficiency of TRAFTACs in treating disease.