RAMP Mediated Gene Delivery

描述(申请人提供)：这项建议旨在开发和测试化学定义的还原激活蜂毒素聚乙二醇化糖蛋白(RAMP)的基因转移效率。通过二硫键将蜂毒素多肽、聚乙二醇多肽和糖多肽连接在一起，将在固体载体上制备RAMP。由此产生的RAMPS将与DNA结合，形成以体内肝细胞为靶点的复合体，并经历谷胱甘肽引发的内吞体内蜂毒素的释放。这一建议的创新之处在于，它开发了一种方法，系统地改变均一坡道的组成、序列和还原稳定性，以控制蜂毒素在细胞内的位置和释放速度，从而提高基因表达水平。RAMPS的固相合成是基于我们之前成功开发的含有末端半胱氨酸残基的还原激活肽，它介导了细胞内还原环境中DNA的触发释放，大大提高了基因转移效率。自从这一初步发现以来，我们已经开发了半胱氨酸封端的聚乙二醇肽，以及含有天然三天线N-糖链去唾液酸糖蛋白受体配体的糖肽。我们最近开发了半胱氨酸封端的蜂毒素多肽作为有效的体外基因转移多肽。这些进展导致了通过半胱氨酸封端的聚乙二醇肽、糖肽和蜂毒素的随机共聚合形成还原活性的蜂毒素聚乙二醇化糖蛋白(RAMPS)。随机共聚合的RAMP多聚体在小鼠体内介导了对肝细胞的特异性靶向，没有明显的毒性，并显示出显著的荧光素酶表达，通过尾静脉注射5？g DNA剂量。然而，通过精确控制蜂毒素的序列、组成和胞内释放位置，仍然可以显著提高随机共聚合的RAMP多聚体的基因转移效率。为了提高RAMP的基因转移效率，我们建议使用一种新的固体合成方法来开发具有精确序列和组成的均质RAMP。这些将被用来检验这样的假设，即通过结合青霉胺二硫键将蜂毒素的释放推迟到较晚的内体将提高基因转移效率。我们建议测试确定的序列、组成和稳定性的RAMP多聚体在原代小鼠肝细胞中的基因转移活性。高效的RAMP多聚体将被用来在体内将荧光素酶表达质粒运送到肝细胞。定量生物发光成像(BLI)将被用来确定与同等流体动力剂量的DNA相比，斜坡多聚体的效率。我们建议评估RAMP复合体的肝脏毒性，并将其与流体动力剂量进行比较。斜坡复合体将用于治疗血友病小鼠的因子缺乏症。血友病小鼠凝血缺陷的纠正将用于确定RAMP复合体的治疗潜力。本研究的结果旨在开发第一种均质还原激活的非病毒基因递送载体，以达到与流体动力给药相当的效率，使用更小的剂量体积，并导致更低的肝脏毒性。 公共卫生相关性：这项提案旨在开发和测试均相还原激活蜂毒素聚乙二醇化糖蛋白(RAMPS)，作为一种新型的基因递送剂，可以改善DNA在动物体内的递送和表达。

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.