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促血管生成伤口愈合植入物。新的组织，取代伤口应血管化的过程称为血管生成。血管内皮生长因子（VEGF）是主要的促血管生成因子，其在伤口部位的存在刺激愈合。 高度不稳定的VEGF的全身递送是困难的，并且可能导致不期望的副作用。不幸的是，没有有效的技术用于将VEGF局部递送到伤口部位。目前合成的生物可降解基质不容易适用于蛋白质治疗剂的配制和控释。另一方面，基于天然胶原蛋白的真皮再生植入物，如Apligraft TM（Organogenesis），由于这些材料所需的多孔性，不能容纳蛋白质治疗剂。因此，挑战在于创建能够控制VEGF释放的生物相容性多孔支架。值得注意的是，对于需要缓慢和持续释放的其他蛋白质治疗剂存在类似的问题。 在这个项目中，我们提出了一种新的方法来测试这个问题的可行性。我们建议将VEGF以非共价复合物的形式固定在多孔基质内，并将标准化的“接头”蛋白整合到基质中。复合物是由于衔接蛋白和与VEGF基因融合的标准化“对接标签”之间的相互作用而形成的。因此，我们提出通过VEGF从这些复合物中缓慢解离来实现持续释放，而不是通过基质的降解来控制VEGF的释放。 我们公司最近开发了一种适用于缓慢释放的创新性人源化适配器/对接标签系统，并进行了体外和体内测试。该系统包括可以固定在所需支架上的107-aa衔接蛋白和与靶向蛋白融合的15-aa“对接”标签。对接标签和衔接子都是人核糖核酸酶I的一部分，因此预期不会具有显著的免疫原性。此外，目前版本的衔接子被工程化以含有用于共价连接至支架的独特半胱氨酸残基。 在该项目的第一阶段，我们将测试这种方法的可行性与两个胶原蛋白为基础的支架：传统的胶原蛋白凝胶和最近描述的静电纺丝胶原蛋白垫。我们将确定固定在基质中的衔接子是否保留对标记的VEGF的亲和力，确定VEGF摄取和释放的动力学，并选择用于II期临床前开发的最佳候选物。重要的是，该项目中开发的技术可用作需要缓慢和持续释放的其他蛋白质治疗剂的平台。

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.