SELF-NEUTRALIZING OLIGONUCLEOTIDES WITH ENHANCED CELLULAR UPTAKE

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。目前，没有有效和可靠的交付方式。细胞外膜抵抗带电ON的细胞摄取，并且消除电荷的骨架修饰（例如肽核酸（PNA）和甲基膦酸酯中的那些修饰）减少但不解决问题，因为这样的结构变化损害它们的水溶性。使用递送载体（制剂），例如基于病毒的递送系统、脂质体、纳米颗粒和转运化学基团，尚未完全解决该问题，并且通常与显著的副作用相关。用于治疗传染病和遗传病的最佳寡疗法的开发仍然没有实现。 ZATA Pharmaceuticals，Inc.正在开发一种核酸技术平台，该平台将能够合成具有增强的细胞内渗透能力的自中和ON。在ZATA的化合物中，负电荷将被中和（而不是消除！）通过形成分子内铵/磷酸盐离子对。所得的改性ON（MON）应具有足够的溶解度以获得最佳的药代动力学（PK）性质和改善的细胞渗透。我们将首先合成新型的亚磷酰胺配体，其含有末端带正电荷的分支氨基末端连接体（BATLs），以中和最终ON的负骨架电荷。每个分支的长度将允许末端带正电荷的基团到达相邻的磷酸基团并中和它们的负电荷。此外，BATL将向ON主链引入部分疏水性质。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天然的沃森-克里克杂交特性。第二，我们将使用这些修饰的双链体来制备具有不同数量的电荷中和基团的21聚体核糖核苷酸和脱氧核糖核苷酸，并测试它们的溶解性、化学和血清稳定性、沃森-克里克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。 所提出的平台ON技术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、位置和类型的变化 BATL将允许MON的疏水性和水溶性之间的最佳平衡，从而使细胞内渗透最大化并使非特异性结合和不良PK特性最小化。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II期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，单独和与其他兼容平台组合。

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.