SELF-NEUTRALIZING OLIGONUCLEOTIDES WITH ENHANCED CELLULAR UPTAKE

 描述(申请人提供)：寡核苷酸(ON)作为治疗药物有巨大的潜力，但挑战仍然是如何有效地将其传递到细胞中。细胞膜抵抗细胞对当前使用的带电的吸收。各种给药系统的应用只部分解决了这个问题，而且经常伴随着治疗上不可接受的副作用。低水平的细胞摄取是大量ON靶向癌症、遗传和微生物介导性疾病失败的主要原因。第一阶段的具体目标是1)开发和验证两种新型亚磷酰胺单体，2)它们用于合成ZATA ON，增强细胞摄取，3)证明ZATA ON具有体内治疗活性所需的各种特性的最佳组合，例如增强细胞穿透性、高效地沉默靶基因、在治疗浓度下毒性低或无毒性、保持自然杂交性质、在血浆/生物液中的稳定性、在水介质中的溶解以及允许扩大规模的稳健的合成方法。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，并且第一次以新的物质组成发展起来，实际上满足了这里概述的复杂标准。特别是，在ZATA上实施的创新使a)细胞摄取量比没有ZATA修饰的类似寡核苷酸高4倍，b)在低至1微米的单一处理下对癌细胞生长的抑制率超过95%，c)在血清中高度稳定，以及d)在高至10微米的浓度下无细胞毒性。我们的成就可简要定义为通过标准亚磷酰胺化学合成的新型ON，它允许电荷中和基团(CNG)容易地附着在其末端具有正电荷的能够到达相邻负电荷并中和它们的电荷。电荷中和与增加的跨越ON主干的部分疏水性相结合，极大地提高了细胞摄取和基因沉默效率。我们这项技术开发第二阶段的主要目标是通过展示其在体外和体内(小鼠)模型的高疗效来进一步验证ZATA ON。第二阶段研究的主要任务是：1)优化并放大所有四个2‘-修饰的RNA亚胺的合成，从而使优化的CNG(即1，3-双(2-(二甲氨基)乙氧基)丙烷-2-醇)能够整合到我们的ON的骨架中；2)合成并筛选20多个靶向人胶质母细胞瘤和乳腺癌细胞系中致癌的miR10b和miR21的药物；3)放大最佳候选药物(S)，并以人脑胶质母细胞瘤异种移植瘤为靶点进行体内实验。在第一阶段研究中开发的新颖性以Zata新的PCT专利申请为准。

 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.