Brunger, J. | bioRxiv Science

bioRxiv2019

Long non-coding RNA GRASLND enhances chondrogenesis via suppression of interferon type II signaling pathway

Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression and cellular processes; however, their roles in musculoskeletal development, disease, and regeneration remain poorly understood. Here, we identified a novel lncRNA, Glycosaminoglycan Regulatory ASsociated Long Non-coDing RNA (GRASLND) as a regulator of mesenchymal stem cell (MSC) chondrogenesis, and we investigated its basic molecular mechanism and its potential application towards regenerative medicine. GRASLND, a primate-specific lncRNA, is upregulated during MSC chondrogenesis and appears to act directly downstream of SRY-Box 9 (SOX9), but not Transforming Growth Factor Beta 3 (TGF-{beta}3). Utilizing the established model of pellet formation for MSC chondrogenesis, we showed that the silencing of GRASLND resulted in lower accumulation of cartilage-like extracellular matrix, while GRASLND overexpression, either via transgene ectopic expression or by endogenous activation via CRISPR, significantly enhanced cartilage matrix production. GRASLND acts to inhibit interferon gamma (IFN-{gamma}) by binding to Eukaryotic Initiation Factor-2 Kinase EIF2AK2. We further demonstrated that GRASLND exhibits a protective effect in engineered cartilage against interferon type II across different sources of chondroprogenitor cells. Our results indicate an important role of GRASLND in regulating stem cell chondrogenesis, as well as its therapeutic potential in the treatment of cartilage-related diseases, such as osteoarthritis.\n\nSignificanceLong non-coding RNAs (lncRNAs) play critical roles in gene regulation and cellular physiology; however, the role of lncRNAs in controlling stem cell chondrogenesis remains to be determined. Here, we utilized next generation sequencing of adult stem cell chondrogenesis to identify a set of potential lncRNA candidates involved in this process. We identified lncRNA Glycosaminoglycan Regulatory ASsociated Long Non-coDing RNA (GRASLND) and characterized its molecular mechanism of action. We described a novel role of GRASLND in positive regulation of chondrogenesis via its inhibition of type II interferon. Importantly, we showed that overexpression of GRASLND augments stem cell chondrogenesis, providing a promising approach to enhancing stem cell chondrogenesis and cartilage regeneration.

cell biology↗

bioRxiv2019

A Genome-engineered Bioartificial Implant for Autoregulated Anti-Cytokine Drug Delivery

Biologic drug therapies are effective treatments for autoimmune diseases such as rheumatoid arthritis (RA) but may cause significant adverse effects, as they are administered continuously at high doses that can suppress the immune system. Using CRISPR-Cas9 genome editing, we engineered stem cells containing a synthetic gene circuit expressing biologic drugs to antagonize interleukin-1 (IL-1) or tumor necrosis factor (TNF) in an autoregulated, feedback-controlled manner in response to activation of the endogenous chemokine (C-C) motif ligand 2 (Ccl2) promoter. To test this approach in vivo, cells were tissue-engineered into a stable cartilaginous construct and implanted subcutaneously in mice with inflammatory arthritis. Bioengineered anti-cytokine implants mitigated arthritis severity as measured by joint pain, structural damage, and systemic and local inflammation. The coupling of synthetic biology with tissue engineering promises a range of potential applications for treating chronic diseases using custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗