Biomaterials for Cell Triggered Gene Transfer

描述（由申请人提供）：可以在组织形成期间的不同时间递送生物活性信号的治疗策略对于复杂组织（如成熟脉管系统）的再生至关重要。在正常的伤口愈合过程中，导致成熟血管形成的事件是由一系列严格调节的事件引起的，这些事件由于环境变化而顺序发生。该提案的重点是设计，合成和体外测试的非病毒基因传递策略，可以提供DNA在时间上控制的方式随着环境的变化。在我们的方法中，阳离子聚合物凝聚的DNA（聚合物）共价固定到生物材料通过基质金属蛋白酶（MMP）敏感肽，可以降解后，MMP添加。将设计用于介导多聚复合物固定的肽，使得它们被特异性MMP切割。因此，编码不同蛋白质的DNA聚合复合物可以通过可以被不同MMP降解的肽固定到生物材料上，并且它们的释放、摄取和表达可以通过在不同时间添加不同MMP来暂时控制。在体内，MMP表达谱在整个伤口愈合过程中受到严格调控，在组织形态发生期间不同的MMP在不同的时间表达。拟议研究的长期目标是利用伤口愈合期间的MMP表达谱，在不同时间递送不同的促血管生成蛋白，以促进成熟脉管系统的形成，从而提高伤口愈合速率。这项建议分为两个目标。目的一是合成并表征由A、B和C三个不同嵌段组成的三嵌段共聚物，其可通过特异性基质金属蛋白酶介导DNA缩合成复合物、DNA复合物的固定和DNA复合物的释放。A嵌段将由MMP不稳定肽组成，其可以通过末端半胱氨酸基团介导固定并通过MMP降解释放。另外两个嵌段B和C将由聚（乙二醇）（PEG）和聚（乙烯亚胺）（PEI）组成，其将负责介导DNA多聚复合物稳定化和DNA缩合。肽合成和胺/羧酸化学将用于合成所提出的ABC三嵌段共聚物。目的2是通过将粘附的细胞接种在具有共价固定在其表面上的DNA聚合物的生物材料上来诱导细胞触发的基因转移。被特异性MMP降解的肽将被用于使多聚复合物稳定，并且预期仅当特异性MMP作为重组蛋白加入或由稳定转染的细胞释放时才导致基因转移。此外，时间控制将通过固定多聚复合物来实现，所述多聚复合物经由被不同MMP降解的MMP不稳定肽编码不同报告基因。因此，可以通过在不同时间加入特定MMP来控制特定复合物的释放。血管生成，即新血管的形成，代表了治疗缺血性伤口的迫切临床需求，并且是组织工程构建物转化的主要障碍。成熟血管生成的一个主要限制是不能在必要的时间递送治疗分子。该提案旨在设计一种基因递送策略，其可以通过使用生物调节分子在伤口愈合期间的特定时间诱导释放，在血管生成所需的时间递送DNA（治疗剂）。

DESCRIPTION (provided by applicant): Therapeutic strategies that can deliver bioactive signals at different times during tissue formation are essential for the regeneration of complex tissues such as a mature vasculature. During normal wound healing, the events that lead to mature blood vessel formation results from a series of tightly regulated events, which occur sequentially as a result of environmental changes. This proposal focuses on the design, synthesis and in vitro testing of a non-viral gene delivery strategy that can deliver DNA in a temporally controlled fashion following environmental changes. In our approach, cationic polymer condensed DNA (polyplex) are covalently immobilized to biomaterials through matrix metalloproteinase (MMP) sensitive peptides that can be degraded following MMP addition. The peptides utilized to mediate polyplex immobilization will be designed so that they are cleaved by specific MMPs. Thus, DNA polyplexes encoding for different proteins can be immobilized to the biomaterial through peptides that can be degraded by different MMPs and their release, uptake and expression can be temporally controlled by the addition of different MMPs at different times. In vivo, the MMP expression profile is tightly regulated throughout the wound healing process with different MMPs being expressed at different times during tissue morphogenesis. The long term goal of the proposed research is to take advantage of this MMP expression profile during wound healing to deliver different pro-angiogenic proteins at different times to promote the formation of a mature vasculature and thus enhance the rate of wound healing. This proposal is divided into two aims. Aim 1 is the synthesis and characterization of triblock copolymers composed of three distinct blocks A, B and C, which can mediate DNA condensation into polyplexes, DNA polyplex immobilization and DNA polyplex release through specific MMPs. The A block will be composed of a MMP labile peptide, which can mediate immobilization through a terminal cysteine group and release through MMP degradation. The other two blocks, B and C, will be composed of poly(ethylene glycol) (PEG) and poly(ethylene imine) (PEI), which will be responsible for mediating DNA polyplex stabilization and DNA condensation. Peptide synthesis and amine/carboxylic acid chemistry will be used to synthesize the proposed ABC triblock copolymers. Aim 2 is to Induce cell triggered gene transfer by plating adhered cells on biomaterials that have DNA polyplexes covalently immobilized on their surface. Peptides that are degraded by specific MMPs will be used to immobilize the polyplexes and are expected to result in gene transfer only when the specific MMP is either added as a recombinant protein or released by stably transfected cells. Further, temporal control will be achieved by immobilizing polyplexes, encoding for different reporter genes via MMP labile peptides that are degraded by different MMPs. Thus, release of specific polyplexes can be controlled by adding specific MMPs at different times. PUBLIC HEALTH REVELANCE Angiogenesis, the formation of new blood vessels, represents a pressing clinical need for the treatment of ischemic wounds and is a major obstacle in the translation of tissue engineered constructs. One major limitation in the generation of mature blood vessels is the inability to deliver therapeutic molecules at the necessary times. This proposal aims to design a gene delivery strategy that can deliver DNA (the therapeutic) at the required times for angiogenesis to take places by using biologically regulated molecules to induce release at specific times during wound healing.