Biomaterials for Cell Triggered Gene Transfer

用于细胞触发基因转移的生物材料

• 批准号: 7568793
• 负责人: Tatiana Segura
• 金额: $ 21.49万
• 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 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7568793
• 关键词: Amines; Angiogenic Proteins; Avidin; Binding; Biocompatible Materials; Biotin; Blood Vessels; Carboxylic Acids; Cells; Chemistry; Cleaved cell; Clinical; Complex; Cysteine; DNA; DNA delivery; Data; Development; Environment; 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; Recombinant Proteins; Reporter Genes; Research; Series; Signal Transduction; Surface; Technology; Therapeutic; Time; Tissue Engineering; Tissues; Translations; Up-Regulation; Viral Genes; Wound Healing; angiogenesis; base; copolymer; covalent bond; design; environmental change; ethylene glycol; extracellular; high reward; in vitro testing; in vivo; innovation; non-viral gene delivery; protein aminoacid sequence; research study; response; scaffold; stem cell biology; stem cell differentiation; 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(申请人提供)：能够在组织形成的不同时间传递生物活性信号的治疗策略对于复杂组织的再生至关重要，例如成熟的血管系统。在正常的伤口愈合过程中，导致成熟血管形成的事件是一系列严格调控的事件的结果，这些事件是由于环境变化而顺序发生的。这项建议的重点是设计、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时间受控的方式传递DNA。在我们的方法中，阳离子聚合物缩合DNA(Polyplex)通过基质金属蛋白酶(MMPs)敏感的多肽被共价固定到生物材料上，这些多肽可以在加入MMP后被降解。用于介导多链固定化的多肽将被设计成使它们被特定的MMPs切割。因此，编码不同蛋白质的DNA多聚体可以通过可被不同MMPs降解的多肽固定在生物材料上，并可以通过在不同时间加入不同MMPs来控制其释放、摄取和表达。在体内，基质金属蛋白酶的表达谱在伤口愈合过程中受到严密的调控，不同的基质金属蛋白酶在组织形态发生过程中的不同时间表达。这项研究的长期目标是利用伤口愈合过程中这种基质金属蛋白酶的表达谱，在不同的时间输送不同的促血管生成蛋白，促进成熟血管的形成，从而提高伤口愈合的速度。这项提议分为两个目标。目的1合成由A、B、C三个不同嵌段组成的三嵌段共聚物，通过特定的MMPs介导DNA缩合成多链、DNA多链固定化和DNA多链释放。A块将由一种不稳定的基质金属蛋白酶多肽组成，它可以通过末端的半胱氨酸基介导固定化，并通过基质金属蛋白酶的降解来释放。另外两个区段，B和C，将由聚乙二醇(PEG)和聚乙二醇亚胺(PEI)组成，它们将负责介导DNA多链稳定和DNA缩合。多肽合成和胺/羧酸化学将用于合成所建议的ABC三嵌段共聚物。目的2是通过将贴壁细胞接种在表面共价固定了DNA多聚体的生物材料上来诱导细胞触发的基因转移。被特定MMPs降解的多肽将被用来固定多聚体，并且只有当特定的MMPs以重组蛋白的形式加入或由稳定转染的细胞释放时，才有望导致基因转移。此外，时间控制将通过固定复合体来实现，通过被不同MMPs降解的不稳定的多肽来编码不同的报告基因。因此，可以通过在不同的时间添加特定的MMP来控制特定多聚体的释放。新血管的形成代表了临床治疗缺血性创伤的迫切需要，也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无法在必要的时间输送治疗分子。该建议旨在设计一种基因传递策略，通过在伤口愈合过程中使用生物调节分子在特定时间诱导释放，在血管生成所需的时间内传递DNA(治疗性DNA)。

项目成果

DNA delivery from matrix metalloproteinase degradable poly(ethylene glycol) hydrogels to mouse cloned mesenchymal stem cells.

• DOI: 10.1016/j.biomaterials.2008.09.027
• 发表时间: 2009-01
• 期刊: BIOMATERIALS
• 影响因子: 14
• 作者: Lei, Yuguo;Segura, Tatiana
• 通讯作者: Segura, Tatiana

VEGF internalization is not required for VEGFR-2 phosphorylation in bioengineered surfaces with covalently linked VEGF.

• DOI: 10.1039/c1ib00037c
• 发表时间: 2011-09
• 期刊: Integrative biology : quantitative biosciences from nano to macro
• 作者: Anderson SM;Shergill B;Barry ZT;Manousiouthakis E;Chen TT;Botvinick E;Platt MO;Iruela-Arispe ML;Segura T
• 通讯作者: Segura T

Hyaluronic acid and fibrin hydrogels with concentrated DNA/PEI polyplexes for local gene delivery.

• DOI: 10.1016/j.jconrel.2011.01.028
• 发表时间: 2011-08-10
• 期刊: Journal of controlled release : official journal of the Controlled Release Society
• 作者: Lei Y;Rahim M;Ng Q;Segura T
• 通讯作者: Segura T

Two and three-dimensional gene transfer from enzymatically degradable hydrogel scaffolds.

• DOI: 10.1002/jemt.20840
• 发表时间: 2010-09
• 期刊: MICROSCOPY RESEARCH AND TECHNIQUE
• 影响因子: 2.5
• 作者: Lei, Yuguo;Ng, Quinn K. T.;Segura, Tatiana
• 通讯作者: Segura, Tatiana

Incorporation of active DNA/cationic polymer polyplexes into hydrogel scaffolds.

• DOI: 10.1016/j.biomaterials.2010.08.016
• 发表时间: 2010-12
• 期刊: BIOMATERIALS
• 影响因子: 14
• 作者: Lei, Yuguo;Huang, Suxian;Sharif-Kashani, Pooria;Chen, Yong;Kavehpour, Pirouz;Segura, Tatiana
• 通讯作者: Segura, Tatiana