Utilizing CRISPRa engineered stem cells for regeneration of the temporomandibular joint - Patients with Temporomandibular joint osteoarthritis (TMJ-OA) present with articular mandibular surface degeneration, that when severe, leaves patients with no regenerative treatment options available and total joint arthroplasty as the only surgical option when they can no longer function. Towards regeneration of the surface of the TMJ condyle, we developed the goat as a large animal model to test tissue engineering strategies for the TMJ. Our previous approaches have shown robust matrix deposition; however, bone was not observed to regenerate and cartilage-bone interface was irregular and not organized. Thus, we have decided to explore cell-based approaches to better control the architecture of the cartilage and bone layers. To this end, we will employ synthetic biology approaches to guide progenitor cell differentiation into both chondrocytes and osteoblasts, and then create bi-layer constructs with both cell types. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) epigenome modifications facilitate targeted, multiplex, long-term regulation of endogenous gene expression without altering the base pairs of the target genes. CRISPR based upregulation of collagen2 (COL2A1) combined with aggrecan (ACAN), and CRISPRi based downregulation of noggin (NOG) in human adipose stem cells (hASCs) has been demonstrated to promote chondrogenic or osteogenic phenotypes, respectively. In addition, Sox9 and Runx2 are known regulators of chondrogenic and osteogenic differentiation. We hypothesize that we can combine these technologies into a bilayer hydrogel comprised of 1) a top layer consisting of gelatin hydrogel seeded with Sox9/COL2A1/ACAN epigenome edited BMSCs and 2) a bottom layer consisting of a gelatin hydrogel seeded with Runx2/NOG epigenome edited BMSCs that will better regenerate the cartilage-bone interface of the mandibular condyle compared to hydrogel seeded with non-target edited BMSCs. To test this hypothesis, we propose the following specific aims. In Aim1, we will demonstrate the ability of Sox9/COL2A1/ACAN epigenome edited BMSC seeded hydrogels to form cartilage tissue in vitro. In Aim2, we will establish the ability of Runx2/NOG epigenome edited BMSC seeded hydrogels to form mineralized tissue in vitro. In Aim3, we will develop a bilayer hydrogel, composed of a Sox9/COL2A1/ACAN edited BMSC layer and a Runx2/NOG edited BMSC layer, to form a cartilage-bone tissue interface in vitro. Finally, we will test the ability of the Sox9/COL2A1/ACAN and Runx2/NOG edited BMSC seeded bilayer hydrogels to regenerate the surface of the TMJ condyle in our goat large animal model of TMJ-OA. Overall, these results will demonstrate the ability of CRISPR epigenome editing Sox9/COL2A1/ACAN and Runx2/NOG expression in BMSCs to drive chondrogenic and osteogenic differentiation, respectively, and establish the use of CRISPR epigenome edited BMSC based therapeutics as a potential treatment strategy for regeneration of the surface of the TMJ condyle.
