The overall goal of this project is to develop a pro-angiogenic wound-healing implant. New tissue that replaces a wound should be vascularized through a process known as angiogenesis. Vascular endothelial growth factor (VEGF) is the major pro-angiogenic factor and its presence at the wound site stimulates healing. Systemic delivery of a highly labile VEGF is difficult and can lead to undesirable side effects. Unfortunately, there is no effective technology for local delivery of VEGF to the wound sites. Current synthetic biodegradable matrices are not easily adaptable for formulation and controlled release of protein therapeutics. On the other hand, natural collagen-based dermal regeneration implants, such as Apligraft TM (Organogenesis), do not hold protein therapeutics because of the required porosity of these materials. Thus, the challenge is to create a biocompatible porous scaffold capable of controlled release of VEGF. Significantly, a similar problem exists for other protein therapeutics that require slow and sustained release. In this project we propose to test feasibility of a novel approach to this problem. We propose to immobilize VEGF inside of a porous matrix in non-covalent complexes with a standardized """"""""adapter"""""""" protein integrated into the matrix. The complexes are formed as a result of interactions between adapter protein and a standardized """"""""docking tag"""""""" genetically fused to VEGF. Thus, instead of controlling release of VEGF via degradation of a matrix, we propose to achieve a sustained release via slow dissociation of VEGF from these complexes. An innovative humanized adapter/docking tag system suitable for slow release has been recently developed in our company and tested in vitro and in vivo. This system includes a 107-aa adapter protein that can be immobilized on a desired scaffold and a 15-aa """"""""docking"""""""" tag fused to a targeting protein. Both docking tag and adapter are parts of human ribonuclease I, and therefore are not expected to be significantly immunogenic. Furthermore, the current version of adapter is engineered to contain a unique cysteine residue for covalent linking to the scaffold. In Phase I of this project, we will test feasibility of this approach with two collagen-based scaffolds: a traditional collagen gel and a recently described electrospun collagen mat. We will establish if adapter immobilized in matrices retains affinity to tagged VEGF, determine the kinetics of VEGF uptake and release, and select the best candidate for pre-clinical development in Phase II. Importantly, technology developed in this project may be used as a platform for other protein therapeutics in need of slow and sustained release.
