Biomaterials for Cell Triggered Gene Transfer

用于细胞触发基因转移的生物材料

• 批准号: 7471590
• 负责人: Tatiana Segura
• 金额: $ 17.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-13 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7471590
• 关键词: Amines; Angiogenic Proteins; Avidin; Binding; Biocompatible Materials; Biotin; Blood Vessels; Carboxylic Acids; Cell Differentiation process; Cells; Cellular biology; Chemistry; Cleaved cell; Clinical; Complex; Cysteine; DNA; DNA delivery; Data; Development; Endopeptidases; Environment; Ethylene; Ethylene Glycols; Ethylenes; Event; Funding; Gene Delivery; Gene Transfer; Generations; Genes; Genome; Goals; Grant; Hydrogels; Hydrolysis; Imines; Immobilization; In Vitro; Lead; Link; Matrix Metalloproteinases; Mediating; Molecular Profiling; Morphogenesis; Natural regeneration; Patients; Peptide Hydrolases; Peptide Synthesis; Peptides; Physical condensation; Polymers; Process; Proteins; Public Health; Rate; Recombinant Proteins; Reporter Genes; Research; Rewards; Series; Signal Transduction; Stem cells; Surface; Technology; Testing; Therapeutic; Time; Tissue Engineering; Tissues; Transcriptional Activation; Translations; Up-Regulation; Viral; Wound Healing; angiogenesis; base; copolymer; covalent bond; design; desire; environmental change; ethylene glycol; extracellular; in vivo; innovation; non-viral gene delivery; protein aminoacid sequence; research study; response; scaffold; tissue regeneration; tool; transgene expression; uptake

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.

描述（由申请人提供）：能够在组织形成过程中不同时间传递生物活性信号的治疗策略对于复杂组织（如成熟的脉管系统）的再生至关重要。在正常伤口愈合过程中，导致成熟血管形成的事件是由一系列严格调控的事件引起的，这些事件是由于环境变化而依次发生的。本研究的重点是设计、合成和体外测试一种非病毒基因传递策略，该策略可以在环境变化后以暂时可控的方式传递DNA。在我们的方法中，阳离子聚合物凝聚DNA （polyplex）通过基质金属蛋白酶（MMP）敏感肽共价固定在生物材料上，这些肽可以在添加MMP后降解。用于介导多聚体固定的肽将被设计成可以被特定的MMPs切割。因此，编码不同蛋白质的DNA多聚体可以通过多肽固定在生物材料上，多肽可以被不同的MMPs降解，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的MMPs来暂时控制。在体内，MMP的表达谱在整个伤口愈合过程中受到严格调控，在组织形态发生的不同时间表达不同的MMPs。本研究的长期目标是利用MMP在伤口愈合过程中的表达谱，在不同时间传递不同的促血管生成蛋白，促进成熟脉管系统的形成，从而提高伤口愈合的速度。这个建议分为两个目的。目的1是由A、B和C三个不同嵌段组成的三嵌段共聚物的合成和表征，该共聚物可以介导DNA缩聚成多聚体、DNA多聚体固定和DNA多聚体通过特异性MMPs释放。A区将由一个MMP不稳定肽组成，它可以通过末端半胱氨酸基团介导固定化，并通过MMP降解释放。另外两个区块，B和C，将由聚乙二醇（PEG）和聚乙烯亚胺（PEI）组成，它们将负责介导DNA多聚体稳定和DNA缩合。多肽合成和胺/羧酸化学将用于合成ABC三嵌段共聚物。目的2是通过将粘附细胞电镀在表面共价固定有DNA多聚体的生物材料上，诱导细胞触发基因转移。被特异性MMP降解的肽将用于固定多聚体，并且只有当特异性MMP作为重组蛋白添加或被稳定转染的细胞释放时，才能导致基因转移。此外，时间控制将通过固定多聚体来实现，多聚体通过被不同的MMP降解的MMP不稳定肽编码不同的报告基因。因此，可以通过在不同时间添加特定的MMPs来控制特定多路复合物的释放。血管生成，即新血管的形成，是治疗缺血性伤口的迫切临床需求，也是组织工程构建转化的主要障碍。成熟血管生成的一个主要限制是无法在必要的时间输送治疗分子。该提案旨在设计一种基因传递策略，通过使用生物调节分子在伤口愈合的特定时间诱导释放，在血管生成所需的时间递送DNA（治疗药物）。