SELF-NEUTRALIZING OLIGONUCLEOTIDES WITH ENHANCED CELLULAR UPTAKE

增强细胞吸收的自中和寡核苷酸

• 批准号: 9281767
• 负责人: David R Tabatadze
• 金额: $ 58.79万
• 依托单位: ZATA PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9281767
• 关键词: Achievement; Adverse effects; Animal Model; Animals; Biological; Biophysics; Boston; Breast Cancer cell line; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Charge; Chemicals; Chemistry; Collaborations; Complex; DNA; Data; Development; Disease; Exhibits; Failure; Formulation; Gene Silencing; Gene Targeting; Glioblastoma; Glioma; Goals; Hela Cells; Hospitals; Human; Hybrids; Hydrophobicity; In Vitro; Inhibition of Cancer Cell Growth; Institution; Legal patent; Licensing; Liquid substance; MCF7 cell; Maintenance; Mediating; Methods; MicroRNAs; Modeling; Modification; Oligonucleotides; Oncogenes; Oncogenic; Pathway interactions; Penetration; Pharmacologic Substance; Phase; Plasma; Postdoctoral Fellow; Preparation; Problem Solving; Progress Reports; Property; RNA; Reporting; Research Personnel; Resources; Rivers; Safety; Scheme; Serum; Solubility; Study Subject; Sugar Phosphates; System; Technology; Testing; Therapeutic; Time; Toxic effect; Treatment Efficacy; Validation; Vertebral column; Woman; Work; Xenograft procedure; analog; anti-cancer therapeutic; aqueous; base; cancer cell; cancer genetics; cytotoxicity; drug candidate; experimental study; improved; in vivo; medical schools; microorganism; monomer; mouse model; novel; phase 1 study; phase 2 study; phosphoramidite; public health relevance; scale up; screening; technology development; tertiary amine; tumor; tumor growth; uptake

 DESCRIPTION (provided by applicant): There is enormous potential for oligonucleotides (ON) as therapeutics, but the challenge remains how to effectively deliver ON into cells. Cell membranes resist the cellular uptake of currently used charged ON. The application of various delivery systems has only partially solved the problem and is often associated with therapeutically unacceptable side effects. Low level cellular uptake has been the main reason of the failure of large number of ON targeting cancer, genetic-, and microorganism-mediated diseases. Specific aims for the Phase I were 1) development and validation of two new types of phosphoramidite monomers, 2) their use for the synthesis of ZATA ON with enhanced cellular uptake, and 3) demonstration that ZATA ON possess an optimal combination of properties necessary for high in vivo therapeutic activity, such as enhanced cell penetration, high efficacy toward silencing of target genes, low or lack of toxicity at therapeutic concentrations, maintenance of natural hybridization properties, stability in plasma/biological fluids, solubility n aqueous media and robust method of synthesis allowing scale-up. As demonstrated in the Progress Report section, we fully completed all Phase I specific tasks and, for the first time, have developed ON with new composition of matter that practically satisfies the complex criteria outlined herein. Particularly, novelties implemented in ZATA ON enabled a) 4 times higher cellular uptake vs. similar oligonucleotides without ZATA modifications, b) over 95% inhibition of cancer cell growth in culture with single treatment at a concentration as low as 1 µM, c) high stability in serum, and d) lack of cytotoxicity at a concentration as high as 10 µM. Our achievements can briefly be defined as a novel class of ON synthesized via standard phosphoramidite chemistry which permits facile attachment of Charge Neutralizing Groups (CNG) bearing positive charges at their termini capable of reaching the adjacent negative charges and neutralizing them. Charge-neutralization in combination with added partial hydrophobicity across the backbone of ON dramatically enhance cellular uptake and gene silencing efficacy. Our major goal for the Phase II of this technology development is further validation of ZATA ON by demonstrating their high therapeutic efficacy in vitro and in vivo (mouse) models. The main tasks for Phase II study are: 1) Optimization and scale-up of the synthesis of all four 2′-modified RNA phosphoramidites enabling the incorporation of optimal CNG (i.e. 1,3-Bis(2-(dimethylamino) ethoxy)propan-2-ol) into the backbones of our ON; 2) Synthesis and screening of over two dozen ON targeting oncogenic miR10b and miR21 in human glioblastoma and breast cancer cell lines; 3) Scale-up of the best ON drug candidate(s) and testing in vivo in mouse model using human glioblastoma xenografts as a target. Novelties developed in the Phase I study are subject to ZATA's new PCT patent application.

 描述（由应用提供）：作为治疗的寡核苷酸（ON）具有巨大的潜力，但挑战仍然是如何有效地输送到细胞中的挑战。细胞膜抵抗当前充电的细胞摄取。各种输送系统的应用仅部分解决了该问题，并且通常与治疗上不可接受的副作用有关。低水平的细胞摄取一直是大量靶向癌症，遗传和微生物介导的疾病失败的主要原因。 Specific aims for the Phase I were 1) development and validation of two new types of phototrophoramidite monomers, 2) their use for the synthesis of ZATA ON with enhanced cellular uptake, and 3) demonstration that ZATA ON possesses an optimal combination of properties necessary for high in vivo therapeutic activity, such as enhanced cell penetration, high efficiency towards silencing of target genes, low or lack of toxicity at治疗浓度，自然杂交特性的维持，血浆/生物流体的稳定性，溶解度N水性培养基以及合成的鲁棒方法，可以扩大规模。如“进度报告”部分所示，我们完全完成了I阶段特定任务，并首次以新的物质组成进行开发，这些物质实际上满足了此处概述的复杂标准。特别是，在Zata中实施的新颖性启用a）a）细胞摄取量高4倍，而没有ZATA修饰的类似的寡核苷酸，b）超过95％以上的95％抑制了培养物中癌细胞生长的95％以上，单一治疗的浓度低至1 µm，c）在血清中高稳定性高达1 µm，并且缺乏浓度为10 µM的细胞毒性。我们的成就可以短暂地定义为通过标准磷光岩化学的合成类别的新型类别，该类别允许在其末端带有正电荷的电荷中和基团的容易附着，能够达到相邻的负电荷并中和它们。电荷中和化与跨主体的局部疏水性相结合，可显着增强细胞摄取和基因沉默效率。我们对这一技术开发的第二阶段的主要目标是通过证明其在体外和体内（小鼠）模型的高治疗效率来进一步验证Zata。第二阶段研究的主要任务是：1）所有四个2'修饰的RNA磷光体的合成的优化和扩大，使最佳CNG（即1,3-双（2-（Dimethylamino）乙氧基）丙氧氧基丙氧烷2-醇）掺入我们的主骨; 2）在人胶质母细胞瘤和乳腺癌细胞系中靶向致癌的miR10b和miR21的合成和筛选； 3）使用人胶质母细胞瘤Xenographograptic作为靶标，在小鼠模型中对最佳药物候选者的最佳缩小和在体内进行了测试。在I期研究中发展的新颖性受Zata的新PCT专利应用。