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激活蜂毒聚乙二醇糖蛋白（RAMPs）的基因转移效率。RAMPs将在固体载体上制备，通过二硫键形成连接蜂胶肽、聚乙二醇肽和糖肽。由此产生的RAMPs将与DNA结合形成靶向肝细胞的多聚体，并在体内经历谷胱甘肽触发的内体melittin释放。该建议的创新之处在于，它开发了一种系统地改变均质RAMPs的组成、序列和还原稳定性的方法，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。提出的RAMPS固相合成是基于我们之前成功开发的还原激活肽，其中含有末端Cys残基，在细胞内还原环境中介导DNA的触发释放，显著提高基因转移效率。自这一初步发现以来，我们已经开发了半胱氨酸末端聚乙二醇（PEG）肽，以及含有天然三触角n聚糖asialal糖蛋白受体配体的糖肽类。我们最近开发了cys终止的蜂毒肽作为有效的体外基因转移肽。这些进展导致了还原激活蜂鸟素聚乙二醇糖蛋白（RAMPs）的发展，通过随机共聚合的Cys终止peg肽，糖肽和蜂鸟素。随机共聚合的RAMP多聚物介导小鼠肝细胞特异性靶向，无明显毒性，并通过尾静脉给药5¿g DNA显示显著的荧光素酶表达。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多聚物的基因转移效率。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方法来开发具有精确序列和组成的均质RAMPs。这些将被用来测试这样一个假设，即通过结合青霉胺二硫键将蜂毒素的释放推迟到内体晚期，将提高基因转移效率。我们打算在小鼠原代肝细胞中测试确定序列、组成和稳定性的RAMP多聚体体外基因转移活性。有效的RAMP复合物将被用于在体内将表达荧光素酶的质粒输送到肝细胞。定量生物发光成像（BLI）将用于确定相对于等效流体动力学剂量的DNA的RAMP多聚物的效率。我们建议与水动力给药相比，评估RAMP多聚物的肝毒性。RAMP复合物将用于治疗血友病小鼠的因子VIII缺乏症。对这种血友病小鼠凝血缺陷的纠正将用于确定RAMP复合物的治疗潜力。本研究的结果旨在开发第一个均质还原激活的非病毒基因递送载体，以达到与水动力给药相当的效率，使用更小的给药体积，导致更少的肝毒性。

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.