Scaffold-Based Delivery of Gene Therapeutics for the Repair of Large Volume Defects in Both Healthy and Osteoporotic Bone
Creators: name: Power, Rachael
Publisher: name: Royal College of Surgeons in Ireland
Year: 2024
Subjects: classificationCode: 300199
subject: Agricultural biotechnology not elsewhere classified
classificationCode: 320699
subject: Medical biotechnology not elsewhere classified
classificationCode: 400311
subject: Tissue engineering
classificationCode: 320606
subject: Regenerative medicine (incl. stem cells)

description: Non-union bone fractures caused by significant trauma do not heal without interventions and can be further complicated by co-morbidities such as osteoporosis. Gene-activated scaffolds provide an opportunity to enhance bone repair by upregulating expressionof osteogenic genes or inhibiting expression of aberrantly expressed genes that impair fracture repair. The goal of this thesis was to develop a versatile gene-activated scaffold platform capable of delivering plasmid (pDNA) and microRNA therapeutics to direct enhanced bone repair in both healthy and osteoporotic fractures. 
Chapter 2 focussed on identifying the optimal vector for pDNA delivery to mesenchymal stem cells (MSCs) by comparinga broad range of vectors with known transfection potential. The glycosaminoglycan enhanced transduction (GET)peptide was selected as it was the most effective vector with respect to MSC transfection in this study, and increased efficacy in serum will likely prove advantageous under physiological conditions. Furthermore, the multi-domain design of the GET peptide provides potential opportunities for modification and potentially more effective formulations. 
The research presented in Chapter 3 described the development of an advanced gene-activated scaffold platform for pDNA delivery to MSCs, incorporating a modified version of the GET peptide (FLR), designed to enhance plasmid pDNA delivery by substitution of arginine residues with histidine (FLH). GET* N/P 8 nanoparticles incorporated into collagen–nanohydroxyapatite (coll–nHA) scaffolds at a 1:1 ratio of collagen:nanohydroxyapatite was cytocompatible with MSCs and attained significantly higher transfection efficiency compared with other gene-activated scaffolds, hence this formulation was selected as the optimal gene-activated scaffold platform.   
Chapter 4 focussed on utilising the optimised GET* coll–nHA platform as a delivery system for therapeutic pDNA encoding stromal-derived factor 1α (pSDF-1α), an angiogenic chemokine which plays a role in BMP mediated differentiation of MSCs. In vitro, a combinatorial approach of pSDF-1α delivery and co-culture of MSCs with endothelial progenitor cells (EPCs) significantly enhanced angiogenesis. The chorioallantoic (CAM) model was utilised to demonstrate the highly pro-angiogenic effect of pSDF-1α activated scaffolds in vivo,successfully targeting host cells in the absence of any pre-seeded cells.
The research presented in Chapter 5 utilised the GET* coll–nHA platform as a gene-activated scaffold for repair of osteoporotic fractures by targeting and inhibiting miRNA-100, a miRNA which is overexpressed in bone tissue and serum of patients suffering from osteoporosis. GET*-antimiR-100 N/P 8 was selected as the lead formulation based on characterisation in osteoporotic MSCs, and significantly downregulated miR-100 expression, by approximately 15-20-fold when incorporated into coll-nHA scaffolds. 
In Chapter 6, gene-activated scaffolds developed in previous chapters were tested in a weight bearing rat segmental femoral defect in vivo. pSDF-1α activated scaffolds significantly increased vasculature and formation of new bone within the defect, compared with gene-free controls. The antimiR-100 activated scaffolds were tested in ovariectomised (OVX) rats, as a model of osteoporosis, and demonstrated enhanced reparative capacity compared with gene-free controls, particularly at early timepoints. 
Overall, the results from this thesis developed an advanced gene-activated scaffold platform capable of delivering indication specific cargos and regenerating bone in a weight bearing defect in vivo. More specifically, pSDF-1α activated scaffolds were successfully developed to repair bone defects using a pro-angiogenic approach, and antimiR-100 activated scaffolds were shown to enhance repair of osteoporotic defects. In summary, the positive outcomes of both gene-activated scaffolds highlighted the versatility and potential applicability of the GET* coll-nHA gene-activated scaffolds for a variety of tissue engineering applications