RAMP Mediated Gene Delivery

RAMP介导的基因传递

• 批准号: 8116557
• 负责人: KEVIN G RICE
• 金额: $ 30.87万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8116557
• 关键词: Amino Acids; Animals; Asialoglycoprotein Receptor; Binding; Biodistribution; Bioluminescence; Cells; Coagulation Process; Cysteine; Cytolysis; Cytosol; DNA; DNA delivery; Defect; Development; Dose; Endosomes; Environment; Factor VIII; Gene Delivery; Gene Expression; Gene Transfer; Glutathione; Glycopeptides; Glycoproteins; Hemophilia A; Hepatocyte; Hepatotoxicity; Image; In Vitro; Ligands; Liver; Location; Luciferases; Lytic; Mediating; Membrane; Mus; N-terminal; Penicillamine; Peptides; Plasmids; Polyethylene Glycols; Polylysine; Polymers; Polysaccharides; Preparation; Relative (related person); Research; S Phase; Safety; Solid; System; Tail; Testing; Therapeutic; Toxic effect; Veins; analog; base; disulfide bond; disulfide bond reduction; gene delivery system; improved; in vivo; innovation; late endosome; non-viral gene delivery; novel; physical property; polymerization; polypeptide; public health relevance; success

DESCRIPTION (provided by applicant): This proposal aims to develop and test the gene transfer efficiency of chemically defined Reductively Activated Melittin PEGylated glycoproteins (RAMPs). RAMPs will be prepared on solid support by joining a melittin peptide, PEG-peptide and glycopeptides by disulfide bond formation. The resulting RAMPs will bind to DNA to form polyplexes that target hepatocytes in vivo and undergo a glutathione triggered release of melittin in endosomes. This proposal is innovative in its development of a means to systematically vary the composition, sequence and reductive stability of homogeneous RAMPs to control the intracellular location and release rate of melittin to improve the level of gene expression. The proposed solid phase synthesis of RAMPS is based on our previous success in developing reductively activated peptides containing terminal Cys residues that mediate a triggering release of DNA in the intracellular reducing environment, dramatically increasing gene transfer efficiency. Since this initial discovery, we have developed Cys-terminated polyethylene glycol (PEG) - peptides, and glycopeptides containing a natural triantennary N-glycan asialoglycoprotein receptor ligand. We recently developed Cys-terminated melittin peptides as potent in vitro gene transfer peptides. These advances resulted in the development of Reductively Activated Melittin PEGylated glycoproteins (RAMPs) by a random co-polymerizing of Cys terminated PEG-peptide, glycopeptides and melittin. Randomly co-polymerized RAMP polyplexes mediated specific targeting to heptocytes in mice without observable toxicity and demonstrated significant luciferase expression from a 5 ¿g DNA dose delivered via the tail vein. However, the gene transfer efficiency of randomly co-polymerized RAMP polyplexes can still be significantly improved by precisely controlling the sequence, composition and the intracellular release location of melittin. To enhance the gene transfer efficiency of RAMPs, we propose to develop homogenous RAMPs of precise sequence and composition using a novel solid synthesis. These will be used to test the hypothesis that delaying the release of melittin until the late endosomes by incorporating penicillamine disulfide bonds will enhance gene transfer efficiency. We propose to test RAMP polyplexes of defined sequence, composition and stability for in vitro gene transfer activity in primary mouse hepatocytes. Potent RAMP polyplexes will be used to deliver luciferase expressing plasmids to hepatocytes in vivo. Quantitative bioluminescence imaging (BLI) will be used to determine the efficiency of RAMP polyplexes relative to an equivalent hydrodynamic dose of DNA. We propose to evaluate the liver toxicity of RAMP polyplexes compared to hydrodynamic dosing. RAMP polyplexes will be used to treat Factor VIII deficiency in hemophilic mice. The correction of this coagulation defect in hemophilic mice will be used to establish the therapeutic potential of RAMP polyplexes. The results of this study aim to develop the first homogenous reductively activated nonviral gene delivery carrier to achieve comparable efficiency as hydrodynamic dosing, using much smaller dosing volumes and resulting in less liver toxicity. PUBLIC HEALTH RELEVANCE: This proposal aims to develop and test homogeneous Reductively Activated Melittin PEGylated glycoproteins (RAMPs) as a new class of gene delivery agents that improve the delivery and expression of DNA in animals.

描述（由申请人提供）：该提案旨在开发和测试化学定义的还原激活蜂毒肽聚乙二醇化糖蛋白（RAMP）的基因转移效率。 RAMP 将通过形成二硫键连接蜂毒肽、PEG-肽和糖肽而在固体支持物上制备。由此产生的 RAMP 将与 DNA 结合，形成复合物，在体内靶向肝细胞，并在内体中经历谷胱甘肽触发的蜂毒肽释放。该提案的创新在于开发了一种方法，系统地改变同质RAMP的组成、序列和还原稳定性，以控制蜂毒肽的细胞内定位和释放速率，从而提高基因表达水平。所提出的 RAMPS 固相合成基于我们之前成功开发出含有末端 Cys 残基的还原激活肽，该肽可介导细胞内还原环境中 DNA 的触发释放，从而显着提高基因转移效率。自这一初步发现以来，我们开发了半胱氨酸封端的聚乙二醇 (PEG) 肽和含有天然三触角 N-聚糖脱唾液酸糖蛋白受体配体的糖肽。我们最近开发了半胱氨酸终止的蜂毒肽作为有效的体外基因转移肽。这些进展导致通过半胱氨酸封端的 PEG 肽、糖肽和蜂毒肽的随机共聚开发了还原活化蜂毒肽聚乙二醇化糖蛋白 (RAMP)。随机共聚的 RAMP 复合物介导对小鼠肝细胞的特异性靶向，没有可观察到的毒性，并且通过尾静脉输送的 5 µg DNA 剂量显示出显着的荧光素酶表达。然而，通过精确控制蜂毒肽的序列、组成和细胞内释放位置，随机共聚的RAMP复合物的基因转移效率仍然可以得到显着提高。为了提高 RAMP 的基因转移效率，我们建议使用新型固体合成方法开发具有精确序列和组成的均质 RAMP。这些将用于检验以下假设：通过掺入青霉胺二硫键将蜂毒肽的释放延迟到晚期内涵体将提高基因转移效率。我们建议测试具有确定序列、组成和稳定性的 RAMP 复合物在原代小鼠肝细胞中的体外基因转移活性。有效的 RAMP 复合物将用于将荧光素酶表达质粒递送至体内肝细胞。定量生物发光成像 (BLI) 将用于确定 RAMP 复合物相对于同等流体动力学剂量的 DNA 的效率。我们建议评估 RAMP 复合物与流体动力学给药相比的肝脏毒性。 RAMP 复合物将用于治疗血友病小鼠的因子 VIII 缺乏症。血友病小鼠中这种凝血缺陷的校正将用于确定 RAMP 复合物的治疗潜力。本研究的结果旨在开发第一个同质还原激活的非病毒基因递送载体，以达到与流体动力学给药相当的效率，使用更小的剂量体积并产生更少的肝脏毒性。 公共健康相关性：该提案旨在开发和测试均质还原激活蜂毒肽聚乙二醇化糖蛋白 (RAMP)，作为一类新型基因递送剂，改善动物体内 DNA 的递送和表达。