Pro-angiogenic Collagen Implants

描述(由申请人提供)： 该项目的总体目标是开发一种有利于血管生成的伤口愈合植入物。取代伤口的新组织应该通过一种被称为血管生成的过程来血管化。血管内皮生长因子(VEGF)是主要的促血管生成因子，其存在于创面可促进愈合。 全身注射高度不稳定的血管内皮生长因子是困难的，并可能导致不良的副作用。遗憾的是，目前还没有有效的技术将血管内皮生长因子局部输送到伤口部位。目前人工合成的生物可降解基质不容易适应蛋白质治疗药物的制剂和控释。另一方面，天然胶原基皮肤再生植入物，如Apligraft TM(器官发生)，由于这些材料所需的多孔性，不含蛋白质疗法。因此，挑战在于创造一种能够控制释放血管内皮生长因子的生物相容的多孔支架。值得注意的是，其他需要缓慢和持续释放的蛋白质疗法也存在类似的问题。 在这个项目中，我们建议测试解决这个问题的新方法的可行性。我们建议将血管内皮生长因子固定在多孔基质中，形成非共价复合体，并将标准化的“接头”蛋白整合到基质中。这些复合体是适配器蛋白和标准化的“对接标签”之间相互作用的结果，该“对接标签”在基因上与血管内皮生长因子融合在一起。因此，我们不是通过降解基质来控制血管内皮生长因子的释放，而是通过缓慢地将血管内皮生长因子从这些复合体中解离出来来实现缓释。 我们公司最近开发了一种适用于缓释的创新的人源化适配器/对接标签系统，并在体外和体内进行了测试。该系统包括一个可以固定在所需支架上的107-AA适配蛋白和一个与靶向蛋白融合的15-AA“对接”标签。对接标签和接头都是人类核糖核酸酶I的一部分，因此预计不会有明显的免疫原性。此外，当前版本的适配器被设计为含有用于共价连接到支架上的独特的半胱氨酸残基。 在这个项目的第一阶段，我们将用两种胶原基支架来测试这种方法的可行性：一种是传统的胶原凝胶，另一种是最近描述的电纺胶原垫。我们将确定固定在基质中的适配器是否与标记的血管内皮生长因子保持亲和力，确定血管内皮生长因子摄取和释放的动力学，并在第二阶段选择最适合临床前开发的候选药物。重要的是，本项目开发的技术可以作为其他需要缓慢和持续释放的蛋白质治疗药物的平台。

DESCRIPTION (provided by applicant): 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.