RAMP Mediated Gene Delivery

描述（由申请人提供）：本提案旨在开发和测试化学定义的还原性活化蜂毒肽聚乙二醇化糖蛋白（RAMP）的基因转移效率。通过二硫键形成连接蜂毒肽、PEG-肽和糖肽，在固体支持物上制备RAMP。所得RAMP将结合DNA以形成体内靶向肝细胞的聚合复合物，并在核内体中经历谷胱甘肽触发的蜂毒肽释放。该提议在其开发的系统地改变均质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的手段方面是创新的。所提出的RAMPS的固相合成是基于我们先前在开发含有末端Cys残基的还原活化肽中的成功，所述末端Cys残基介导在细胞内还原环境中触发DNA释放，从而显著提高基因转移效率。由于这一初步发现，我们已经开发了半胱氨酸封端的聚乙二醇（PEG）肽，和糖肽含有天然三触角N-聚糖脱唾液酸糖蛋白受体配体。我们最近开发了半胱氨酸封端的蜂毒肽作为有效的体外基因转移肽。这些进展导致了通过Cys封端的PEG-肽、糖肽和蜂毒肽的随机共聚合来开发还原活化蜂毒肽PEG化糖蛋白（RAMP）。随机共聚的RAMP聚合复合物介导特异性靶向小鼠肝细胞，没有可观察到的毒性，并显示出通过尾静脉递送的5 μ g DNA剂量的显著荧光素酶表达。然而，通过精确控制蜂毒素的序列、组成和细胞内释放位置，随机共聚RAMP聚合物的基因转移效率仍然可以显著提高。为了提高RAMPs的基因转移效率，我们建议使用一种新的固体合成来开发具有精确序列和组成的同源RAMPs。这些将用于检验以下假设：通过掺入青霉胺二硫键延迟蜂毒肽的释放直到晚期内体将增强基因转移效率。我们建议在原代小鼠肝细胞中测试具有确定序列、组成和稳定性的RAMP复合物的体外基因转移活性。有效的RAMP聚合复合物将用于在体内将荧光素酶表达质粒递送至肝细胞。定量生物发光成像（BLI）将用于确定RAMP聚合复合物相对于DNA的等效流体动力学剂量的效率。我们建议评估RAMP复合物与流体动力学给药相比的肝毒性。RAMP复合物将用于治疗血友病小鼠中的因子VIII缺乏症。血友病小鼠中这种凝血缺陷的校正将用于建立RAMP复合物的治疗潜力。本研究的结果旨在开发第一种均质还原活化非病毒基因递送载体，以实现与流体动力学给药相当的效率，使用更小的给药体积并导致更少的肝毒性。 公共卫生相关性：该提案旨在开发和测试均质还原活化蜂毒肽聚乙二醇化糖蛋白（RAMP）作为一类新的基因递送剂，改善动物中DNA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.