Core--SiRNA manufacturing and toxicology

核心--SiRNA制造和毒理学

• 批准号: 7679623
• 负责人: MUTHIAH MANOHARAN
• 金额: $ 42.06万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7679623
• 关键词: Biological; Biological Assay; Catalytic RNA; Cell Line; Cells; Chemicals; Cholesterol; Clinical; Drug Formulations; Drug Kinetics; Gene Silencing; Goals; HIV; In Vitro; Infection; Infection prevention; Ligands; Macaca mulatta; Mammalian Cell; Mannose; Modification; Modification Type; Mucous Membrane; Mus; Numbers; Oligonucleotides; Pharmaceutical Preparations; Positioning Attribute; Proteins; RNA; Range; Resistance; Small Interfering RNA; Sugar Phosphates; System; Testing; Therapeutic; Toxicology; Vaginal Route of Drug Administration; Vaginal delivery procedure; Vertebral column; Viral; antigene; chemical stability; design; experience; improved; in vivo; microbicide; nuclease; nucleobase; phosphorothioate; sugar; transmission process; uptake

The goal of this Core is to design, synthesize and evaluate selected chemical modifications to small interfering RNAs (siRNAs) for improved chemical stability and delivery to cells within the cervicovaginal mucosa that are important in HIV transmission. Previous experience with in vivo applications of oligonucleotides (including antigene, antisense, decoy, and ribozyme therapies) has clearly demonstrated the need for chemical modification to improve pharmacokinetics in mammalian systems. Although there are virtually no technical limitations regarding chemical modification of the nucleobase, sugar and phosphate moieties of RNA, interactions of the siRNA with the proteins involved in gene silencing puts constraints on the types of modifications and the number of residues that can be modified without severely impairing the efficacy of the siRNA. Optimization of delivery, stability, and activity of siRNAs for vaginal delivery will require an assessment of a range of chemical modifications for incorporation within and near the termini of siRNAs in combination with cholesterol or CCR5-ligand conjugation for enhancement of uptake into cells susceptible to infection. Finally, the selected siRNAs will be appropriately formulated for clinical intravaginal application. This Core will provide modified and formulated siRNAs to each of the Projects for in vitro and in vivo assays to test the potential of specific modifications for gene silencing and for protection against viral transmission in cell lines, primary cells and cervicovaginal explants (Project by Lieberman), in mice (Project by Palliser) and in rhesus macaques (Project by Veazey). The ultimate commercial product from this study will be a drug that can be used vaginally to prevent infection by HIV. In particular, our aims are to (1) improve biological stability of siRNA duplexes by adding phosphorothioate linkages to the backbone and by modification of the 2' position of the sugar residues; (2) improve cell targeting and permeation by conjugation of cholesterol, CCR5-specific ligands, or mannose to siRNA; and (3) develop an siRNA formulation suitable for clinical intravaginal application.

该核心的目的是设计，合成和评估所选的化学修饰，以进行小型干扰 RNA（siRNA）改善了化学稳定性和向宫颈阴道粘膜内的细胞传递 在HIV传播中很重要。以前的寡核苷酸体内应用经验 （包括抗基因，反义，诱饵和核酶疗法）清楚地表明了需要 化学修饰以改善哺乳动物系统中的药代动力学。虽然几乎没有 有关化学修饰的核碱，糖和磷酸化学修饰的技术限制 RNA，siRNA与涉及基因沉默的蛋白质的相互作用构成了对的类型的限制 修改和可以修改的残留数量，而不会严重损害 sirna。 siRNA的递送，稳定性和阴道递送活动的优化将需要 评估在siRNA末端内和附近的一系列化学修饰的评估 与胆固醇或CCR5配体共轭结合使用，以增强对细胞的摄取 感染。最后，选定的siRNA将适当制定用于临床阴道内应用。 该核心将为体外和体内测定的每个项目提供改良和配制的siRNA 测试特定修饰的基因沉默和防止病毒传播的潜力 细胞系，原代细胞和宫颈阴道外植体（Lieberman的Project），在小鼠（Palliser的项目）和恒河猕猴中 （Veazey的项目）。这项研究的最终商业产品将是一种可以阴道使用的药物 防止艾滋病毒感染。特别是，我们的目的是（1）通过 将磷光酸盐连接添加到主链并通过修饰糖的2'位置 残留物； （2）通过胆固醇，CCR5特异性配体或 Mannose到sirna; （3）开发适合临床阴道内应用的siRNA公式。