Polyacridine Peptide Mediated Gene Targeting

描述（由申请人提供）：该提案旨在开发靶向细胞并介导远端组织中的基因表达或siRNA敲低的长循环复合物。该方法利用了一种新的DNA结合肽，由六个重复的（Acr-Lys）组成，其中Acr被定义为用吖啶修饰的Lys。该肽通过六个吖啶的多嵌插以高亲和力结合DNA或siRNA。PEG化-（Acr-Lys）6以足够的亲和力结合DNA以形成DNA酶稳定的聚合复合物，其以3小时的半衰期循环，并且当由流体动力学刺激触发时介导小鼠中的荧光素酶表达。基于这些结果，我们建议开发长循环多组分聚乙二醇化聚吖啶复合物（PPAP），其中含有融合肽和奥曲肽作为表达生长抑素亚型2受体（SS 2）的靶细胞的高亲和力配体。将使用固相合成和生物缀合来制备蜂毒肽-（Acr-Lys）6和奥曲肽-（Acr-Lys）6。PEG-、蜂毒肽-和奥曲肽-（Acr-Lys）6的精确添加混合物将用于形成具有确定的组成、电荷、大小和形状的多组分DNA和siRNA PPAP。含有奥曲肽的PPAP将用于体外和体内靶向稳定转化的HEK-SS 2和HEK-SS 2-Luc细胞系。多组分125 I-DNA或125 I-siRNA PPAP将用作示踪剂，以测量HEK-SS 2和HEK-SS 2-Luc细胞中SS 2受体介导的摄取。使用HEK-SS 2、SH-SY 5 Y和G3细胞的体外转染研究将建立介导荧光素酶表达的PEG、蜂毒肽和奥曲肽的最佳比例。将通过在转染HEK-SS 2-Luc细胞后测量荧光素酶敲低来优化多组分siRNA PPAP。多组分125 I-DNA或125 I-siRNA PPAP将在具有SS 2细胞后肢异种移植物的裸鼠中静脉内给药。药代动力学和生物分布实验将确定PPAP的奥曲肽靶向递送的效率作为异种移植物大小和细胞数量、多组分PPAP组成、电荷、剂量和给药途径的函数。将杂乱的奥曲肽配体或SS 2拮抗剂取代到PPAP中以测量靶向递送相对于增强的渗透性和保留效应的相对贡献。将最佳的多组分DNA和siRNA PPAP静脉内给予异种移植小鼠，并使用定量生物发光（BLI）测量荧光素酶表达或敲低，作为时间、异种移植物大小和多组分PPAP组成的函数。这些实验旨在检验以下假设：具有PEG、配体和融合肽的聚吖啶肽的多组分组合形成功能活性PPAP，其在静脉内给药后将DNA和siRNA靶向动物中的远程组织部位。所提出的研究是创新的，在他们使用短，高亲和力聚吖啶肽，同时结合PEG，蜂毒肽和奥曲肽的DNA和siRNA。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。所提出的研究的成功执行将建立基本参数，以纳入靶向肝外细胞的静脉内给药的非病毒基因递送复合物的设计中。这些研究对于推进非病毒基因递送向靶向远程组织部位的长循环复合物是必不可少的。

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.