SBIR-STTR Award

Tissue engineering, grafting procedures, regeneration, and tissue remodeling are developing therapies with great potential medical value. None of these therapeutic modalities are yet fully effective or predictable. Glycosaminoglycan-decorated proteoglycans such as perlecan, once thought to primarily serve as structural components of extracellular matrix, are now being focused on for their role in tissue and cell regulation, particularly angiogenesis and wound healing. Perlecan's domain 1 becomes glycosylated with heparan sulfate in vivo so has the potential to effect several aspects of wound healing and is an excellent candidate for induction in an effort to modulate wound healing. This project is premised on the hypothesis that delivery of heparan sulfate via the perlecan domain 1 will improve wound healing. Agenta Biotechnologies owns IP related to nucleic acid vector presentation of perlecan in wound healing and regeneration. In phase 1 of the project we will complete two aims related to osseous regeneration. To establish replicable in vitro parameters, we will measure delivery of replication-defective adenovirus expressing perlecan domain 1 to endothelial cells and osteoblasts. To establish proof of principle in vivo, we will assess delivery of the adenovirus particles, perlecan domain 1 and heparan sulfate expression, and wound healing parameters relating to vasculogenesis and new bone formation in a mouse osseous defect model. In each aim, particle delivery, via a common bone graft substitute, hydroxyapatite crystals, will be assessed. Outcomes of this phase 1 project will be applicable to many wound healing disciplines but will be targeted first to improvement of osseous regeneration in phase 2 of this project where alternative bone graft substitutes, barrier membranes, and surgical applications will be investigated, and a more extensive biochemical analysis of loading, delivery, and expression will be performed. Molecular heparan sulfate delivery has the potential to improve osseous grafting and the usefulness of bone graft substitutes, which is the ultimate goal of this technology platform and proposed project

The role of proteoglycans and carbohydrate polymers such as heparan sulfate in biological processes are becoming better understood, but this new frontier has only begun to be explored. Tissue regeneration is also an emerging frontier. This proposed research, when completed, will make an important contribution to both the proteoglycan and tissue regenerative fields, with a specific, clinical impact on periodontal regeneration. The objectives of this project are both scientific and developmental in nature. Scientific advancements in the structural and functional understanding of proteoglycans, such as Perlecan, are proposed, along with innovative studies investigating their role as therapeutics in bone healing. Site-directed mutagenesis to the Perlecan core will establish a valuable precedent. One of the major innovations of this technology is the ability to deliver DNA, not protein, to generate natural, endogenous Perlecan expression in the wounds or surgical sites. With this patented technology, a host-glycosylated Perlecan core will be generated in vivo, intended to promote vascularity, proliferation, and differentiation for enhanced healing. In addition to being highly cost-effective, this technology offers a low-cost alternative to the costly recombinant biologic adjuncts already in the marketplace. The development of a safe, effective, and useful product for periodontal bone regeneration is another important objective. Phase I support for this project enabled the creation of a prototype Perlecan expression construct for delivery of sequences encoding domain 1 (D1) of the core protein. In situ heparan sulfate glycosylation of the expressed core was validated, and new bone formation appeared to be enhanced by delivery of the Perlecan D1 expression construct in vivo. This Phase II project proposes to develop important modifications to the prototype Perlecan D1 expression construct, to identify other effective bone graft materials for co-delivery with the Perlecan D1 expression construct, and to test the Perlecan D1 expression construct in guided tissue regeneration. Structural modeling has allowed rational design for site-directed mutagenesis of the Perlecan D1 core sequence to increase glycosylation and enhance growth factor binding, while addition of a COOH-terminal tag on the prototype transgene will enhance development and characterization. Effective bone graft materials for co-delivery with the replication-defective adenovirus system, or with plasmid DNA in liposomes, will be identified from common bone graft materials or by copolymerization with resorbable poly D-lactide microparticles through an industry partnership. Periodontal regeneration in vivo will be assessed by radiographic and volumetric magnetic resonance imaging in a university collaboration, by histomorphometric analysis of new bone, new periodontal ligament, new cementum, and immunohistochemical measurement of osteocalcin at a 3 week and 12 week post-surgical time point. Expression of the Perlecan D1 transgene will be assessed locally and systemically while safety and toxicity of molecular Perlecan and heparan sulfate delivery is investigated