Polyacridine Peptide Mediated Gene Targeting

多吖啶肽介导的基因靶向

• 批准号: 8306000
• 负责人: KEVIN G RICE
• 金额: $ 28.39万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8306000
• 关键词: Acridines; Affinity; Animals; Binding; Biodistribution; Bioluminescence; Blood Circulation; Cell Count; Cell Line; Cell Nucleus; Cells; Charge; DNA; DNA Binding; Development; Digestion; Dose; Drug Kinetics; Electroporation; Gene Delivery; Gene Expression; Gene Targeting; Gene Transfer; Genes; Half-Life; Hour; In Vitro; Ligands; Limb structure; Liver; Luciferases; Measures; Mediating; Metabolism; Mus; Nude Mice; Octreotide; Pathway interactions; Peptides; Permeability; Property; Receptor Cell; Relative (related person); Reporting; Research; Resistance; Route; S Phase; Serum Proteins; Shapes; Site; Small Interfering RNA; Solid; Somatostatin; Structure; Testing; Time; Tissues; Tracer; Transfection; base; cellular targeting; design; gene delivery system; in vivo; innovation; non-viral gene delivery; novel; plasmid DNA; protein aminoacid sequence; receptor; research study; skeletal; somatostatin receptor 2; targeted delivery; uptake

DESCRIPTION (provided by applicant): This proposal aims to develop long-circulating polyplexes that target cells and mediate gene expression or siRNA knockdown in remote tissues. The approach utilizes a novel DNA binding peptide, composed of six repeats of (Acr-Lys), where Acr is defined as Lys modified with acridine. The peptide binds DNA or siRNA with high affinity by polyintercalation of six acridines. PEGylated-(Acr-Lys) 6 binds DNA with sufficient affinity to form DNAse stable polyplexes that circulate with a half-life of 3 hours and mediate luciferase expression in mice when triggered by hydrodynamic stimulation. Based on these results, we propose to develop long-circulating multi-component PEGylated PolyAcridine Polyplexes (PPAPs) containing a fusogenic peptide and octreotide as a high affinity ligand to target cells expressing the somatostatin subtype-2 receptor (SS2). Solid phase synthesis and bioconjugation will be used to prepare melittin-(Acr-Lys) 6 and octreotide-(Acr-Lys) 6. Precise add- mixtures of PEG-, melittin- and octreotide-(Acr-Lys) 6 will be used to form multi-component DNA and siRNA PPAPs with defined composition, charge, size and shape. Octreotide containing PPAPs will be used to target stably transformed HEK-SS2 and HEK-SS2-Luc cell lines in vitro and in vivo. Multi-component 125I-DNA or 125I- siRNA PPAPs will serve as tracers to measure SS2 receptor mediated uptake in HEK-SS2 and HEK-SS2-Luc cells. In vitro transfection studies using HEK-SS2, SH-SY5Y and G3 cells will establish optimal ratios of PEG, melittin and octreotide to mediate luciferase expression. Multi-component siRNA PPAPs will be optimized by measuring luciferase knockdown following transfection of HEK-SS2-Luc cells. Multi-component 125I-DNA or 125I-siRNA PPAPs will be dosed i.v. in nude mice possessing hind limb xenographs of SS2-cells. Pharmacokinetic and biodistribution experiments will determine the efficiency of octreotide targeted delivery of PPAPs as a function of xenograph size and cell number, multi-component PPAP composition, charge, dose and route of administration. Scrambled octreotide ligand or SS2 antagonist will be substituted into PPAPs to measure the relative contribution of targeted delivery versus the enhanced permeability and retention effect. Optimal multi-component DNA and siRNA PPAPs will be administered i.v. to xenograph mice and measured for luciferase expression or knockdown using quantitative bioluminescence (BLI) as a function of time, xenograph size and multi-component PPAP composition. These experiments aim to test the hypothesis that multi-component combinations of polyacridine peptides possessing PEG, ligand and fusogenic peptide form functionally active PPAPs that target DNA and siRNA to remote tissue sites in animals following i.v. dosing. The proposed studies are innovative in their use of short, high affinity polyacridine peptides to simultaneously bind PEG, melittin and octreotide to DNA and siRNA. The studies are further innovative in the proposed novel use of octreotide as a gene targeting ligand. The successful execution of the proposed studies will establish fundamental parameters to incorporate in the design of i.v. dosed nonviral gene delivery polyplexes targeted to cells outside the liver. These studies are essential to advance nonviral gene delivery toward long-circulating polyplexes that target to remote tissue sites.

描述(申请人提供)：这项提议旨在开发以细胞为靶点的长循环复合体，并在远程组织中介导基因表达或siRNA敲除。该方法利用了一种新的DNA结合肽，由6个重复的(ACR-Lys)组成，其中ACR被定义为用吖啶修饰的Lys。该多肽与DNA或siRNA以高亲和力结合，通过六个吖啶类化合物的多嵌插作用结合。聚乙二醇化的(ACR-Lys)6以足够的亲和力结合DNA，形成半衰期为3小时的DNA酶稳定多聚体，并在流体动力刺激触发下介导小鼠体内荧光素酶的表达。基于这些结果，我们建议开发含有融合肽和奥曲肽的长循环多组分聚乙二醇化吖啶多聚体(PPAPs)，作为表达生长抑素亚型2受体(SS2)的靶细胞的高亲和力配体。采用固相合成和生物偶联的方法制备蜂毒素-(ACR-Lys)6和奥曲肽-(ACR-Lys)6。将聚乙二醇、蜂毒素和奥曲肽-(ACR-Lys)6精确加成，形成具有特定组成、电荷、大小和形状的多组分DNA和siRNA PPAP。含有PPAPs的奥曲肽将在体外和体内靶向稳定转化的HEK-SS2和HEK-SS2-Luc细胞系。多组分的125I-DNA或125I-siRNA PPAP将作为示踪剂来测量SS2受体在HEK-SS2和HEK-SS2-Luc细胞中的摄取。利用HEK-SS2、SH-SY5Y和G3细胞进行的体外转染研究将建立聚乙二醇酯、蜂毒素和奥曲肽的最佳比例来介导荧光素酶的表达。多组分siRNA PPAP将在HEK-SS2-Luc细胞转染后通过测量荧光素酶敲除来优化。多组分的125I-DNA或125I-siRNA PPAP将静脉注射。在有SS2细胞后肢异种移植的裸鼠身上。药代动力学和生物分布实验将确定奥曲肽靶向递送PPAP的效率与异种照相尺寸和细胞数量、多组分PPAP组成、电荷、剂量和给药途径的函数关系。打乱的奥曲肽配体或SS2拮抗剂将被取代到PPAP中，以测量靶向传递与增强的通透性和滞留效应的相对贡献。将静脉注射最佳的多组分DNA和siRNA PPAP。使用定量生物发光(BLI)测量荧光素酶的表达或击倒，作为时间、包埋大小和多组分PPAP组成的函数。这些实验旨在验证这样一种假设，即具有聚乙二醇、配体和融合多肽的多组分组合形成具有功能活性的PPAP，这些PPAP靶向DNA和siRNA到动物体内的远程组织部位。给药。这项研究的创新之处在于，他们使用了短的、高亲和力的聚吖啶多肽来同时将聚乙二醇、蜂毒素和奥曲肽与DNA和siRNA结合。这些研究在提出将奥曲肽作为基因靶向配体的新用途方面具有进一步的创新性。拟议研究的成功执行将确立纳入静脉注射设计的基本参数。剂量的非病毒基因递送复合体靶向肝脏外的细胞。这些研究对于推动非病毒基因向以远程组织为靶点的长循环复合体的传递至关重要。