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（由申请人提供）：寡核苷酸（ON）作为治疗剂具有巨大的潜力，但挑战仍然是如何将ON有效地递送到细胞中，以抵抗目前使用的带电ON的细胞摄取。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相关，低水平的细胞摄取是大量针对癌症、遗传和微生物介导的疾病的 ON 失败的主要原因。是1) 开发和验证两种新型亚磷酰胺单体，2) 将其用于合成增强细胞摄取的 ZATA ON，以及 3) 证明 ZATA ON 具有高体内治疗活性所需的最佳特性组合，例如增强的细胞渗透性、对靶基因沉默的高效性、治疗浓度下的低毒性或无毒性、维持自然杂交特性、在血浆/生物液体中的稳定性、在水性介质中的溶解性以及稳健的合成方法使得正如进度报告部分所展示的，我们完全完成了第一阶段的所有具体任务，并且首次开发了具有新物质成分的 ON，该物质实际上满足了此处概述的复杂标准，特别是在 ZATA 中实现的新颖性。 ON 实现了 a) 与未经 ZATA 修饰的类似寡核苷酸相比，细胞摄取量提高了 4 倍，b) 在浓度低至 1 µM 的单次处理下，对培养物中癌细胞生长的抑制超过 95%，c) 血清中的高稳定性，以及d) 在浓度高达 10 µM 时缺乏细胞毒性，我们的成果可以简单地定义为通过标准亚磷酰胺化学合成的一类新型 ON，它允许在其末端轻松连接带有正电荷的电荷中和基团 (CNG)。到达邻近的负电荷并中和它们，再加上ON主链上增加的部分疏水性，可显着增强细胞摄取和基因沉默功效。该技术开发的 II 期是通过在体外和体内（小鼠）模型证明 ZATA ON 的高治疗功效来进一步验证 ZATA ON。 II 期研究的主要任务是： 1) 优化和扩大所有四种 2 的合成。 '-修饰的 RNA 亚磷酰胺能够将最佳 CNG（即 1,3-双（2-（二甲氨基）乙氧基）丙-2-醇）纳入我们的 ON 的主链中；在人胶质母细胞瘤和乳腺癌细胞系中合成和筛选超过两种 ON 靶向致癌 miR10b 和 miR21；3) 扩大最佳 ON 候选药物的规模并使用人胶质母细胞瘤异种移植物作为靶点在小鼠模型中进行体内测试第一阶段研究中开发的新颖性将受到 ZATA 的新 PCT 专利申请的约束。