Polyacridine Peptide Mediated Gene Targeting

多吖啶肽介导的基因靶向

• 批准号: 8447530
• 负责人: KEVIN G RICE
• 金额: $ 27.39万
• 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/8447530
• 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（由申请人提供）：本提案旨在开发靶向细胞并介导远程组织中基因表达或siRNA敲低的长循环多聚物。该方法利用一种新的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。该肽通过6个吖啶的多插层结合DNA或siRNA，具有高亲和力。PEGylated-(Acr-Lys) 6以足够的亲和力与DNA结合，形成DNA酶稳定的多聚体，其半衰期为3小时，在水动力刺激下介导小鼠荧光素酶的表达。基于这些结果，我们建议开发含有促聚变肽和奥曲肽的长循环多组分聚乙二醇化聚吖啶多聚物（PPAPs）作为高亲和力配体靶向表达生长抑素亚型-2受体（SS2）的细胞。采用固相合成和生物偶联法制备蜂毒素-(Acr-Lys) 6和奥曲肽-(Acr-Lys) 6。PEG-，蜂毒素-和奥曲肽-(Acr-Lys) 6的精确添加混合物将用于形成具有确定组成，电荷，大小和形状的多组分DNA和siRNA PPAPs。含PPAPs的奥曲肽将用于体外和体内靶向稳定转化的HEK-SS2和HEK-SS2- luc细胞系。多组分125I- dna或125I- siRNA PPAPs将作为示踪剂，测量HEK-SS2和HEK-SS2- luc细胞中SS2受体介导的摄取。在HEK-SS2、SH-SY5Y和G3细胞的体外转染研究中，将建立PEG、蜂毒素和奥曲肽介导荧光素酶表达的最佳比例。多组分siRNA PPAPs将通过检测转染HEK-SS2-Luc细胞后荧光素酶的敲除来优化。多组分125I-DNA或125I-siRNA PPAPs将在具有ss2细胞后肢异种照相的裸鼠体内静脉注射。药代动力学和生物分布实验将确定奥曲肽靶向递送PPAPs的效率，这与xenograph的大小和细胞数量、多组分PPAP的组成、电荷、剂量和给药途径有关。重组的奥曲肽配体或SS2拮抗剂将被取代到PPAPs中，以测量靶向递送的相对贡献与增强的渗透性和保留效应。将最佳的多组分DNA和siRNA PPAP静脉注射到xenograph小鼠，并使用定量生物发光（BLI）作为时间、xenograph尺寸和多组分PPAP组成的函数来测量荧光素酶的表达或敲低。这些实验旨在验证具有PEG、配体和促聚变肽的聚吖啶肽的多组分组合在动物体内静脉给药后形成功能活性ppap，将DNA和siRNA靶向到远处的组织部位。拟议的研究在使用短的、高亲和力的聚吖啶肽同时将PEG、蜂毒蛋白和奥曲肽与DNA和siRNA结合方面具有创新性。这些研究进一步创新了奥曲肽作为基因靶向配体的新用途。该研究的成功实施将建立基本参数，用于设计针对肝外细胞的静脉注射剂量的非病毒基因递送复合物。这些研究对于推进非病毒基因向靶向远程组织部位的长循环多聚体传递至关重要。