Project Summary Cartilage defects in patients with knee osteoarthritis are often filled with fibrocartilage. Inflammation in joints renders cartilage defects a refractory disease. Mesenchymal stromal/stem cells (MSCs), a promising cell source for the treatment of cartilage defects, exhibit a site-dependent differentiation preference: bone marrow- derived MSCs (BMSCs) exhibit chondrogenic differentiation but end with endochondral ossification; adipose- derived MSCs (ADSCs) are readily available but have limited chondrogenic potential and tend toward fibrocartilage instead of articular cartilage; and synovium-derived MSCs (SDSCs) are a tissue-specific stem cell for chondrogenic differentiation but have limited accessibility. Moreover, a greater number of MSCs is needed for tissue engineering and regeneration; however, MSCs are prone to become senescent during ex vivo expansion leading to a loss of their proliferation and differentiation potentials. Given that MSCs and fibrocartilage have extensive expression of collagen I (COL1A1), in this proposal, we hypothesize that integration of OStrio (both OCT4 and SOX trio) at the COL1A1 locus allows for MSC rejuvenation and articular differentiation while suppressing fibrocartilage by a negative feedback loop. To achieve this hypothesis, we plan to use cutting-edge CRISPR-Cas9 Genome Editing technology for precise integration of OStrio at the COL1A1 locus of MSCs (Aim 1), followed by fine-tuning of proliferation and chondrogenic differentiation of OStrio-expressing MSCs via WNT signals (Aim 2); lastly, genetically engineered MSCs will be assessed in vivo through subcutaneous implantation (with a sufficient vascular supply) of premature tissue constructs from human OStrio-expressing MSCs in an immunodeficient mouse model for future clinical translation (Aim 3.1) and intra-articular implantation of premature tissue constructs from rabbit OStrio-expressing MSCs in an osteochondral defect rabbit model (Aim 3.2). Our expectation is that OStrio-expressing MSCs have superior proliferation and chondrogenic differentiation capacity in an in vitro study as well as superior ability in cartilage resurfacing and resistance to deterioration from inflammation and oxidation as well as angiogenesis in animal studies. Our objective is to engineer “ideal” MSCs which can be prodigiously expanded and specifically differentiated toward articular cartilage with a robust capacity to resist the harsh osteoarthritic environment by preventing hypertrophic cartilage and fibrocartilage formation. To translate this finding into the clinical setting, we will use cutting-edge CRISPR-Cas9 Genome Editing technology to introduce these factors by targeted integration instead of using lentiviral vectors to improve the safety profile of this new therapy. This project has the potential to bypass a critical bottleneck in articular cartilage regeneration by safely providing large quantities of patient-specific functional chondroprogenitors that c...