CRISPR-Cas9 Targeting of the MMP13 Gene Locus Towards Treating Osteoarthritis

Abstract

Osteoarthritis (OA) is a degenerative joint disease that causes the continuous breakdown of cartilage that results in pain, reduced joint function, and disability. There is a need for a treatment for OA that not only aids in symptom management, but permanently stops OA progression. PTOA caused by injury, the focus of this work, is characterized by an imbalance between Matrix Metalloproteinase 13 (MMP13), which degrades type II collagen, and its endogenous inhibitor, the Tissue Inhibitor of Metalloproteinases 3 (TIMP3). We aim to develop an MMP13-targeting CRISPR-Cas9 ribonucleoprotein (RNP) and TIMP3 donor DNA template for the targeted insertion of TIMP3 into the MMP13 gene locus via homology directed repair (HDR), i.e., targeted knock-in (KI). This will create a gene circuit that produces TIMP3 under the control of the endogenous MMP13 promoter. The hypothesis is that this gene circuit will generate TIMP3 “on-demand” whenever the MMP13 promoter is activated by mechanical stimuli and other OA mediators. The results show that NHEJ-mediated MMP13 KO via CRISPR-Cas9 RNP delivery in vitro resulted in up to 95% KO that was maintained after months of subculture. The high KO efficiency was also maintained in a 3D ATDC5 aggregate model; A TIMP3 dox-inducible circuit that was stably transfected into KO (95% indel) and WT ATDC5s showed promising results of TIMP3-mediated MMP13 inhibition. An additional control using recombinant TIMP3 protein directly added to the aggregate treatment medium also showed MMP13 inhibition. Optimization of the TIMP3 DNA template (homology arm length) and in vitro transfection of both the Cas9 RNP and DNA template resulted in successful TIMP3 integration into the MMP13 genome. A mutant TIMP3 (mTIMP3) with an increased half-life in tissues was also developed and integrated into MMP13. However, neither the TIMP3 nor mTIMP3 KI cell lines showed significant MMP13 inhibition in the 3D ATDC5 aggregate model. Overall, TIMP3 was validated as a viable target to inhibit MMP13 activity and subsequent cartilage degradation in the context of PTOA, but further optimizations need to be made in the proposed gene circuit. This data demonstrates a proof-of-concept of the therapeutic potential of on-demand TIMP3 production upon MMP13 activity stimulation to permanently block PTOA progression.