Date of Award
PhD (Doctor of Philosophy)
Professor Sally-Ann Cryan
Professor Fergal O'Brien
Professor Andreas Heise
Science Foundation Ireland
Star-Polymer, Polypeptide, Gene Therapy, Mesenchymal Stem Cells, Tissue Engineering
The field of tissue engineering (TE) is increasingly using biomaterial scaffolds which are augmented with therapeutics to facilitate enhanced tissue regeneration. The formation of a “gene activated” scaffold, an advanced construct containing gene therapeutics within a 3D scaffold is recognised as a safe method to provide improved spatiotemporal control of growth factor release at a defect site via the in-situ transfection of host cells. However, a versatile and biocompatible gene delivery vector which is capable of functionalising 3D scaffolds for the efficient in vivo delivery of nucleic acids is currently lacking. The primary objective of this thesis was to create a next generation gene activated scaffold which could be applied to multiple TE applications via the incorporation of innovative, bio-inspired gene delivery vectors in the form of star-shaped poly(L-lysine) polypeptides (star-PLLs).
Herein, we systematically evaluate three star-PLL vectors for plasmid DNA (pDNA) delivery to Mesenchymal Stem Cells (MSCs) which encompassed structural variations to the dendrimer core (G3, G4 or G5), the number of poly(L-lysine) arms (16 arms, 32 arms or 64 arms) and the associated number of L-lysine subunits per arm (5 subunits or 40 subunits). These included 1: G3(16)PLL40 (16-star-PLL) 2: G4(32)PLL40 (32-star-PLL) & 3: G5(64)PLL5 (64-star-PLL). Numerous physicochemical and biophysical techniques were employed throughout this thesis to identify star-PLL-pDNA formulations which were non-toxic, non-immunogenic and of suitable size and charge to efficiently deliver pDNA to MSCs both in 2D monolayer culture and in 3D on collagen based scaffolds. The safety and functionality of lead star-PLL-pDNA gene activated scaffolds were then evaluated in vivo using a rodent subcutaneous implant model and a cranial bone defect model.
Results from this thesis demonstrate for the first time that all three star-PLL vectors can self-assemble with pDNA to form stable, non-toxic, non-immunogenic, nano-sized complexes which can efficiently transfect MSCs, a difficult to transfect primary cell type. We highlight the 64-star-PLL structure as a particularly efficient vector, which can facilitate comparable transgene expression in MSCs to commonly used vectors such as polyethyleneimine (PEI) at a lower pDNA dose. Following delivery of the therapeutic transgenes bone morphogenetic protein-2 (pBMP-2) and vascular endothelial growth factor (pVEGF) by the 64-star-PLL vector, we demonstrate enhanced MSC mediated osteogenesis compared to using the PEI vector in both 2D culture and 3D on collagen based scaffolds. Using a rodent subcutaneous implant model, we demonstrate that star-PLL-pDNA gene activated scaffolds are biocompatible in vivo and can facilitate autologous host cell transfection. Finally, using a rodent cranial bone defect model we show that star-PLL-pDNA gene activated scaffolds containing a pBMP-2 and pVEGF (pDual) cargo can facilitate superior bone tissue regeneration compared to a gene free scaffold at just four weeks post implantation.
Collectively, this thesis describes the development of a first in class, biocompatible star-PLL-pDNA gene activated scaffold platform with proven functionality in vivo for TE applications. We have identified a lead star-PLL vector which is non-toxic, non-immunogenic and can efficiently deliver a therapeutic pDNA cargo to cells in 2D monolayer culture, in 3D culture on collagen scaffolds and in vivo. This cell-free gene activated scaffold confers enhanced spatiotemporal control of growth factor release at a tissue defect leading to a transient protein expression profile which is highly desirable for TE applications. We highlight the translational reality of these star-PLL-pDNA gene activated scaffolds via demonstrating the significant regeneration of bone tissue in a rodent calvarial defect model at just four weeks post implantation compared to a gene free scaffold.
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Walsh D. The Evaluation of Star-Shaped Polypeptides for Gene Delivery in Tissue Engineering [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2018.
Available for download on Saturday, May 30, 2020