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，但挑战仍然是如何有效地将ON递送到细胞中。细胞膜抵抗细胞对目前使用的带电ON的摄取。各种递送系统的应用仅部分地解决了该问题，并且通常与治疗上不可接受的副作用相关联。低水平的细胞摄取是大量ON靶向癌症、遗传和微生物介导的疾病失败的主要原因。 I期的具体目标是1）开发和验证两种新型亚磷酰胺单体，2）它们用于合成具有增强的细胞摄取的ZATA ON，和3）证明ZATA ON具有高体内治疗活性所需的性质的最佳组合，例如增强的细胞渗透，对靶基因沉默的高功效，在治疗浓度下毒性低或没有毒性、保持天然杂交性质、在血浆/生物流体中的稳定性、在水性介质中的溶解性和允许放大的稳健的合成方法。如进度报告部分所示，我们完全完成了第一阶段的所有具体任务，并首次开发出了具有新物质成分的ON，该物质成分实际上满足本文概述的复杂标准。特别地，在ZATA ON中实施的新颖性使得a）与没有ZATA修饰的类似寡核苷酸相比，细胞摄取高4倍，B）在低至1 μM的浓度下，用单一处理在培养物中抑制超过95%的癌细胞生长，c）在血清中的高稳定性，以及d）在高至10 μM的浓度下缺乏细胞毒性。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的ON，其允许在其末端携带正电荷的电荷中和基团（CNG）的容易附着，所述电荷中和基团能够到达相邻的负电荷并中和它们。电荷中和与ON主链上增加的部分疏水性组合显著增强细胞摄取和基因沉默功效。 该技术开发的第二阶段的主要目标是通过在体外和体内（小鼠）模型中证明其高疗效来进一步验证ZATA ON。II期研究的主要任务是：1）优化和放大所有四种2′-修饰的RNA亚磷酰胺的合成，使得能够掺入最佳CNG（即1，3-双（2-（二甲氨基）乙氧基）丙-2-醇）插入到我们的ON的主链中; 2）合成和筛选二十多种在人胶质母细胞瘤和乳腺癌细胞系中靶向致癌miR 10 b和miR 21的ON; 3）最佳ON药物候选物的规模扩大和使用人胶质母细胞瘤异种移植物作为靶标在小鼠模型中的体内测试。在第一阶段研究中开发的新产品受到ZATA新的PCT专利申请的限制。