Development of a siRNA-activated scaffold enabling targeted knockdown of pro-inflammatory signalling pathways for enhanced cartilage tissue regeneration
Creators: name: Kelly, Domhnall
Publisher: name: Royal College of Surgeons in Ireland
Year: 2023
Subjects: classificationCode: 320606
subject: Regenerative medicine (incl. stem cells)

description: Due to its limited intrinsic healing capacity, articular cartilage remains one of the most challengingtissues to repair,and current treatment strategies fail to provide long-lasting functional tissue regeneration. Biomaterial scaffolds and cell-based approaches have demonstrated some promise for repairingsmall cartilage defects (&lt;4 cm2). However, the development of strategies for treatinglarge chondral defects or cartilage damage resulting from degenerative diseases, such as osteoarthritis (OA), remains a significantchallenge due to the highly inflamed microenvironmentformed in the diseasedor injured joint.The chronic expression of pro-inflammatory cytokines inhibitsmesenchymal stem cell (MSC) chondrogenesis upon implantation. Therefore, the development of approaches promotingMSC chondrogenesis whilst overcoming the effects of pro-inflammatory cytokines may facilitateenhanced cartilage tissue repair. The primary goal of this thesis was to develop a gene-activated scaffold for the controlled delivery of small interfering (si)RNA for targeted knockdown of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathway in MSCs. Following optimisation, non-viral nanoparticles (NPs) were incorporated into atype Icollagen-hyaluronic acid (CHyA)scaffold. The ability of this siRNA-activatedscaffold to prevent the aberrant activation of the NF-κB signalling pathway through transient knockdown of the p65 subunit was then assessed as an advancedapproach to the treatment of OA and cartilage repair. Chapter 2 focused on the comparative assessment of a series of non-viral vectors determining their specific ability to deliver siRNA to MSCs safely and efficiently. The development of a robust formulation and screening methodology enabled the optimisation of a range of non-viral vectors for siRNA delivery. A comparative assessment of these vectors resulted in identifying the glycosaminogly can-binding enhanced transduction (GET) peptideas a promising non-viral vector for siRNA deliveryto MSCs. Further optimisation of this vector enabled the formation ofNPs with favourable physicochemical properties (&lt;200nm diameter, cationic zeta-potential, high degree of encapsulation and no aggregation), contributing to efficient cellular uptake. In Chapter 3, GET-siRNA NPs wereinvestigated fortargeted knockdown ofthe p65 subunit of the NF-κB inMSCs. Transfection with GET-siRNA NPs successfully prevented IL-1β-induced activation of the NF-κB signalling pathway in MSCs. Transient knockdown of the p65 subunit attenuated the upregulation of downstream catabolic mediators, offering a promising strategy for overcoming the inhibitory effects of the inflamed environment. These siRNA-loaded NPs presentan attractive method of preventing further degradation of articular cartilage via intra-articular (IA) delivery of the NPsorfacilitatingenhanced MSC chondrogenesis via ex vivotransfection. However,challenges with the delivery of such therapeutics prevent a prolonged therapeutic timeframe. The final chapter of this thesis set out to develop asiRNA-activated scaffold capable of functional tissue engineering whilst overcoming the negative effects of pro-inflammatory cytokines. In Chapter 4, the incorporation of GET-siRNA NPsinto a chondrogenic CHyAscaffold designed specifically for cartilagerepair supported MSC attachment, proliferation, and viability.These platforms demonstrate the potential to repair cartilage tissue by directing MSC chondrogenesis whilst resistingthe inhibitory effects of pro-inflammatory cytokines. Controlled, localized delivery of siRNA demonstrated the ability to restore balance to the anabolic-catabolic axis, resulting in the attenuation ofcatabolic MMP expression, recapitulation of SOX9expression and a reduction in geneexpressionassociated with MSC hypertrophy (RUNX2). Collectively, this thesis has led to the development of a collagen-based scaffold for the controlled and localized delivery of siRNA to MSCs,offering an advanced approach for the treatment of OA and cartilage tissue regeneration. This thesis demonstrates that combining advanced biomaterialsand gene therapy can facilitate the generation of functional cartilage tissue through MSC chondrogenesis. Moreover, this approach can overcomethe negative effects of pathophysiological concentrationsof pro-inflammatory cytokines, ultimately offering enhanced tissue formation and long-term success for cartilage repair strategies.