SELF-NEUTRALIZING OLIGONUCLEOTIDES WITH ENHANCED CELLULAR UPTAKE

增强细胞吸收的自中和寡核苷酸

• 批准号: 8775829
• 负责人: David R Tabatadze
• 金额: $ 36.38万
• 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 2015-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8775829
• 关键词: Acids; Address; Adverse effects; Ammonium; Base Pairing; Binding; Biological; Breast Epithelial Cells; Cell Line; Cell Membrane Permeability; Cell membrane; Cells; Cellular Membrane; Charge; Chemicals; Chemistry; Computer Assisted; Custom; DNA; Data; Deoxyribonucleotides; Detection; Development; Disease model; Drug Formulations; Drug Kinetics; Epithelial; Equilibrium; Fibroblasts; Figs - dietary; Fluorescein; Gene Silencing; Goals; HL60; Hereditary Disease; Human; Hydrophobicity; Imagery; Ions; Label; Legal patent; Length; Liposomes; Mammary gland; Manuals; Messenger RNA; Methods; Modeling; Modification; Mus; Nature; Nucleic Acids; Nucleotides; Oligodeoxyribonucleotides; Oligonucleotides; Oligoribonucleotides; Penetration; Peptide Nucleic Acids; Performance; Pharmacologic Substance; Phase; Problem Solving; Procedures; Property; Protocols documentation; RNA; Reporter; Ribonucleotides; Serum; Side; Site; Small Interfering RNA; Sodium Chloride; Solubility; Solutions; Specificity; Staining method; Stains; System; Techniques; Technology; Temperature; Testing; Therapeutic; Therapeutic Effect; Therapeutic Uses; Transfection; Validation; Variant; Vertebral column; Virus; amino group; analog; aqueous; base; chemical group; chemical stability; design; improved; infectious disease treatment; inorganic phosphate; lipophilicity; melting; methylphosphonate; nanoparticle; novel; phosphoramidite; public health relevance; research study; uptake; water solubility

DESCRIPTION (provided by applicant): There is enormous potential of oligonucleotides (ON) as therapeutics, but the challenge remains how to effectively deliver ON into cells. Currently, there are no effective and reliable ways of delivery. Outer cell membranes resist the cellular uptake of charged ON, and charges-eliminating backbone modifications such as those in peptide nucleic acids (PNA) and methylphosphonates reduce but do not solve the problem, because such structural changes compromise their aqueous solubility. Use of delivery vehicles (formulations), such as virus-based delivery systems, liposomes, nanoparticles and transporter chemical groups, have not solved this problem fully and are often associated with significant side effects. Development of optimal oligotherapy for the treatment of infectious and genetic diseases still remains unrealized. ZATA Pharmaceuticals, Inc. is developing a nucleic acid technology platform that will enable the synthesis of self-neutralizing ON with enhanced intracellular penetration capabilities. In ZATA's compounds negative charges will be neutralized (not eliminated!) by formation of intramolecular ammonium/phosphate ion-pairs. The resulting modified ON (MON) should possess sufficient solubility for optimal pharmacokinetic (PK) properties and improved cell penetration. We will first synthesize novel phosphoramidite synthons containing branched amino-terminated linkers (BATLs) with positive charges at their termini, in order to neutralize negative backbone charges of the final ON. The length of each branch will allow the terminal positively charged groups to reach neighboring phosphate groups and neutralize their negative charges. Additionally, the BATLs will introduce partial hydrophobic properties to the ON backbone. Our preliminary data and computer assisted modeling indicate that introduction of those modifications will not disturb the natural Watson-Crick hybridization properties. Second, we will use these modified synthons to prepare 21-mer ribo-, and deoxyribonucleotides bearing different numbers of charge-neutralizing groups, and to test their solubility, chemical and serum stability, Watson-Crick base paring specificity and duplex stability. We will test these MONs for their intracellular uptake and mRNA knockdown experiments in C127 mouse mammary epithelial, HL-60 human lymphoblastoma, and human fibroblasts cells. This set of experiments will satisfy the main goals of Phase I: 1) validate the methods of synthesis and purification of ZATA's MONs, and 2) demonstrate their biological validity. The proposed platform ON technology will apply equally to oligodeoxy- and oligoribonucleotide derivatives. Variation of the number, site, and type of the charge-neutralizing BATLs will allow for optimal balance between hydrophobicity and water solubility of the MONs, thus maximizing intracellular penetration and minimizing non-specific binding and poor PK properties. We anticipate that this new platform may be used without the need of additional vehicles. Upon successful validation of our concept, we will continue in phase II to study and optimize the biological stability, PK properties, gene silencing properties, and therapeutic effect in disease models, alone and in combination with other compatible platforms.

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 传递到细胞中。目前，尚无有效、可靠的交付方式。外细胞膜抵抗细胞对带电 ON 的吸收，而消除电荷的主链修饰（例如肽核酸 (PNA) 和甲基膦酸盐中的修饰）会减少但不能解决问题，因为这种结构变化会损害其水溶性。使用递送载体（制剂），例如基于病毒的递送系统、脂质体、纳米粒子和转运化学基团，并没有完全解决这个问题，并且通常与显着的副作用相关。用于治疗传染病和遗传性疾病的最佳寡疗法的开发仍未实现。 ZATA Pharmaceuticals, Inc. 正在开发一个核酸技术平台，该平台将能够合成具有增强细胞内渗透能力的自中和 ON。在 ZATA 的化合物中，负电荷将通过分子内铵/磷酸根离子对的形成而被中和（而不是消除！）。所得修饰 ON (MON) 应具有足够的溶解度，以实现最佳药代动力学 (PK) 特性并改善细胞渗透。我们将首先合成含有支链氨基末端连接体（BATL）的新型亚磷酰胺合成子，其末端带有正电荷，以中和最终ON的负主链电荷。每个分支的长度将允许末端带正电荷的基团到达邻近的磷酸基团并中和它们的负电荷。此外，BATL 将为 ON 主链引入部分疏水性。我们的初步数据和计算机辅助建模表明，引入这些修饰不会干扰自然的沃森-克里克杂交特性。其次，我们将使用这些修饰的合成子来制备带有不同数量电荷中和基团的21聚核糖核苷酸和脱氧核糖核苷酸，并测试它们的溶解度、化学和血清稳定性、Watson-Crick碱基配对特异性和双链体稳定性。我们将在 C127 小鼠乳腺上皮细胞、HL-60 人淋巴母细胞瘤和人成纤维细胞中测试这些 MON 的细胞内摄取和 mRNA 敲低实验。这组实验将满足第一阶段的主要目标：1）验证ZATA MONs的合成和纯化方法，2）证明其生物学有效性。 拟议的 ON 技术平台将同样适用于寡脱氧和寡核糖核苷酸衍生物。电荷中和的数量、位点和类型的变化 BATL 将实现 MON 的疏水性和水溶性之间的最佳平衡，从而最大限度地提高细胞内渗透性并最大限度地减少非特异性结合和不良 PK 特性。我们预计这个新平台可以在不需要额外车辆的情况下使用。在成功验证我们的概念后，我们将继续在第二阶段研究和优化生物稳定性、PK特性、基因沉默特性和治疗效果 在疾病模型中，单独或与其他兼容平台结合使用。