Silencing of inflammatory pathways through crispr cas9 knockout of tak1 in human chondrocytes

Purpose: Osteoarthritis (OA) is a debilitating disease with few effective therapeutic options. Chronic inflammation is a risk factor for poorer outcome, and it remains unclear how the presence of proinflammatory cytokines can affect cartilage repair. To address this question, we investigated the role of the main effector of the inflammatory pathways, Tak1, a MAP kinase associated with OA progression. Tak1 was knocked out in chondrocytes using CRISPR/Cas9 and the ability of edited chondrocytes to undergo chondrogenesis in the presence of IL-1β, lipopolysaccharide (LPS) and TNFα was studied. Chondrocytes from polydactyly patients (< 1 year old) were used as these are a promising allogenic cell source due to their ability to support chondrogenesis up until passage 5 as well as their immune-privileged properties. Methods: Chondrocytes were edited with the CRISPR/Cas9 ribonucleoprotein approach. Guide RNAs were screened for their ability to produce an effective knockout (KO) of the Tak1 gene as determined by Sanger sequencing. Sequencing Results were evaluated with the TIDE online tool for KO prediction. The most efficient combination was used for the other experiments, using three different donors to account for donor variability. Residual protein levels were investigated through Western blotting of Tak1. Cells were treated with 10 ng/ml of IL-1β, LPS or TNFα and expression of inflammation-associated genes were studied by qPCR. NF-κB translocation into the nucleus was assessed by counting the NF-κB positive nuclei with immunofluorescence (IF) on cells treated with IL-1β for 48 hours. Chondrogenesis was evaluated by a pellet assay, with or without 10 ng/ml of IL-1β, LPS and TNFα during the last 7 days. The Tak1-inhibitor 5Z-7-Oxozeaenol was used as a positive control. To test the ability of KO cells to repair cartilage, fluorescent tracker-labelled cells were adhered ex vivo to bovine cartilage discs and imaged at day 1, 3 and 7. Results: The most efficient KO showed retention of the editing for at least two passages (Fig.1A) and a residual protein expression of 12.4%±3.35 compared to WT (Fig.1B, C). qPCR results demonstrated that the Tak1 KO decreased the expression of inflammatory cytokines IL1-β and IL-6, as well as matrix metalloproteinase 13 and NF-κB, compared to WT (Fig 2A). Furthermore, NF-κB translocation into the nucleus due to IL-1β treatment was elevated in the WT (13,83%) and Oxozeaenol treated cells (37,01%) but was almost completely absent in the edited cells (0,3%; Fig.2B). In the presence of LPS, pellets made of Tak1-KO cells showed a better retention of extracellular matrix. Both edited and WT human chondrocytes showed a strong ability to adhere to the surface of bovine cartilage explants, and to proliferate until confluency. Conclusions: We report here on the generation of a highly efficient and stable population of edited human chondrocytes with low expression of Tak1 protein, that are less sensitive to inflammatory stimuli normally present in an OA environment. These cells retain the ability to differentiate into mature cartilage and show reduced expression of inflammatory interleukins and MMPs. We also report a lower activation of the NF-κB pathway by qPCR and IF, confirming the central role of Tak1 in the transmission of inflammation. These edited chondrocytes have potential to promote regeneration of OA-afflicted cartilage.View Large Image Figure ViewerDownload Hi-res image Download (PPT)