Development of Self-regulatory Gene Circuits for Cartilage Tissue Engineering
Wu, Biming
2018
Abstract
Articular (hyaline) cartilage protects the subchondral bone from the high mechanical load during joint movement. This mechanical function of articular cartilage largely relies on the specialized composition and organization of the extracellular matrix deposited by chondrocytes, specifically, aggrecan, type II collagen, and sulfated glycosaminoglycans. The avascular nature of articular cartilage prevents access to progenitor cells and factors that mediate the endogenous healing response inherent in many other tissues. Thus, focal defects that result from traumatic injuries in articular cartilage do not heal and the intense biomechanical loading environment of the tissue leads to the painful and debilitating joint disease osteoarthritis. Efforts to develop mesenchymal stem cell (MSC)-based functional cartilage regeneration are hindered by the unstable phenotype that chondrocytes derived from these cells adopt using common cartilage tissue engineering strategies. Typically, MSC-derived chondrocytes (MdChs) express a transient articular phenotype before further differentiating through the stages of endochondral ossification, leading to the hypertrophic phenotype. Hypertrophic chondrocytes, driven by the transcription factor RUNX2 (Runt-related transcription factor 2), stop producing and start degrading the structural matrix macromolecules aggrecan and type II collagen, compromising the mechanical integrity of the overall tissue. Therefore, biological interventions that suppress chondrocyte maturation and encourage matrix retention can improve the functional outcome of MSC-derived cartilage tissues. This thesis focuses on developing a self-regulatory RUNX2 silencing gene circuit to improve accrual of articular cartilage-specific matrix by MSC-derived chondrocytes via tunable negative-feedback regulation of RUNX2 activity. Specifically, we engineered a synthetic cis promoter to initiate RNA interference of Runx2 exclusively during chondrocyte hypertrophy. To induce chondrocyte-specific RUNX2 silencing, synthetic cis promoters were engineered with a single or multiple copies of cis-enhancers upstream of the Col10a1 basal promoter. We showed that these promoters can direct transcription exclusively in pre-hypertrophic and hypertrophic chondrocytes with minimum activity in undifferentiated progenitor cells. Integrating the cis promoter and RNAi of Runx2 into a gene circuit, we further demonstrated that the cis-RUNX2 silencing circuit can: 1) induce loss of RUNX2 function specifically during chondrocyte hypertrophy, 2) resist elevation of intracellular RUNX2 activity via negative-feedback regulation, and 3) provide adjustable levels of RUNX2 suppression. With these gene circuits, we observed improved matrix accumulation and downregulation of hypertrophy markers during chondrogenesis of a murine chondrogenic cell line and primary MSCs. The successful engineering of this gene circuit highlights the potential to introduce artificial regulatory machinery into mammalian cells to modulate their behavior and optimize tissue engineering outcomes.Subjects
Cartilage Tissue Engineering Gene Therapy Synthetic Biology
Types
Thesis
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