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（由申请人提供）：在组织形成期间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。在正常的伤口愈合期间，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，这些事件依次发生，这是由于环境变化而导致的。该建议着重于非病毒基因输送策略的设计，合成和体外测试，该策略可以在环境变化后以时间控制的方式传递DNA。在我们的方法中，通过基质金属蛋白酶（MMP）敏感肽将阳离子聚合物凝结的DNA（多流线）共价固定在生物材料中，这些肽可在MMP添加后降解。将设计用于介导双流线固定化的肽，以使它们被特定的MMP裂解。因此，编码不同蛋白质的DNA多体可以通过肽固定在生物材料上，这些肽可以通过不同的MMP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MMP在时间上控制。在体内，在整个伤口愈合过程中，MMP表达谱受到严格调节，在组织形态发生过程中，不同时间表达了不同的MMP。拟议的研究的长期目标是利用伤口愈合过程中这种MMP表达谱，在不同时间提供不同的促血管生成蛋白，以促进成熟的脉管系统的形成，从而提高伤口愈合的速度。该提议分为两个目标。 AIM 1是由三个不同的块A，B和C组成的三嵌段共聚物的合成和表征，它们可以通过特定的MMP介导DNA缩合到多聚膜，DNA多聚体固定和DNA多路线释放。 A块将由MMP不稳定肽组成，该肽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。其他两个块B和C将由聚（乙二醇）（PEG）和聚（乙烯亚胺）（PEI）组成，这些块将负责介导DNA多聚体稳定和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镀在具有DNA多聚体的生物材料上，诱导细胞触发的基因转移，这些生物材料在其表面共价固定在其表面上。被特定MMP降解的肽将用于固定多链体，并有望在添加特定MMP作为重组蛋白或通过稳定转染的细胞释放时才导致基因转移。此外，将通过固定多流线来实现时间控制，通过通过不同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MMP来控制特定多流线的释放。公共卫生启示血管生成是新血管的形成，代表了对缺血性伤口治疗的紧迫临床需求，并且是组织工程结构翻译的主要障碍。成熟血管产生的主要局限性是无法在必要时间提供治疗分子。该提案旨在设计一种基因输送策略，该策略可以在血管生成的必要时间下通过使用生物学调节的分子在伤口愈合过程中诱导特定时间释放，以便进行血管生成。

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

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

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

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

Protein-polymer nanoparticles for nonviral gene delivery.

• DOI: 10.1021/bm101354a
• 发表时间: 2011-02
• 期刊: Biomacromolecules
• 影响因子: 6.2
• 作者: Jianjun Zhang;Y. Lei;Anandika Dhaliwal;Quinn K. T. Ng;Juanjuan Du;Ming Yan;Yunfeng Lu;T. Segura