New Building Blocks for the Synthesis of Modified RNAs

用于合成修饰 RNA 的新构件

• 批准号: 8252504
• 负责人: Na Li
• 金额: $ 18.01万
• 依托单位: AM BIOTECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8252504
• 关键词: Address; Affinity; Area; Binding; Biochemical; Biological; Biological Sciences; Biotechnology; Chemicals; Chemistry; Communities; Cultured Cells; DNA; Drug Kinetics; Equipment; Exhibits; Family suidae; Functional RNA; Gene Expression Regulation; Gene Silencing; Genes; Hereditary Disease; Human; Isomerism; Marketing; MicroRNAs; Modeling; Modification; Pharmaceutical Preparations; Phase; Phosphorus; Positioning Attribute; Production; Property; Proteins; Protocols documentation; Public Health; RNA; Reagent; Research; Resistance; Ribonucleosides; Ribose; S Phase; Sales; Small Business Innovation Research Grant; Small Interfering RNA; Solid; Specificity; System; Testing; Therapeutic; Thrombin; Vertebral column; Virus Diseases; Work; Yang; aptamer; base; biological systems; cancer genetics; gene function; human disease; improved; in vivo; industry partner; meetings; monomer; nervous system disorder; novel; nuclease; phosphorodithioic acid; phosphorothioate; secretase; success; sugar; synthetic construct; tool

DESCRIPTION (provided by applicant): Functional RNA molecules such as aptamers, siRNAs, miRNAs, and related compounds have enormous potential as human therapeutics and as tools for elucidating gene regulation in vivo. To reach this potential, such molecules must be highly potent and highly nuclease resistant. Unmodified RNAs typically do not meet these requirements. A variety of chemical modifications have been explored to overcome these limitations. In particular, some success has been achieved in various systems using 2'-F-ribose and phosphorothioate backbone modifications, alone or in combination. However, further improvements are highly desirable. In addition, phosphorothioate modifications are chiral, resulting in two distinct isomers at each backbone substitution. Thus, there is a need for improved chemical modifications that can be incorporated into functional RNAs. AM Biotechnologies will address these critical issues by developing 2'-F-ribonucleoside thiophosphoramidites (2'-F-thioamidites) to enable synthesis of phosphorodithioate 2'-F-RNA (PS2-2'-F- RNA). We have previously shown that PS2 modifications at selected backbone positions of DNA aptamers enhance binding affinity to target proteins without loss of specificity. In addition, selected PS2 modifications in siRNAs significantly improve gene silencing activities. Thus, selected PS2-2'-F-RNA modifications will significantly increase binding affinity and potency of 2'-F-RNA aptamers, and will offer new avenues for synthesis of highly potent siRNAs. PS2-2'-F-RNAs will also be achiral at phosphorus, eliminating the variable biochemical, biophysical, and biological properties of diastereomeric phosphorothioate substituted RNAs. This Phase I project will: 1) develop the chemistry to produce four 2'-F-thioamidites (ABz, CBz, GIbu and U); 2) optimize the synthesis of PS2-2'-F-RNAs; 3) evaluate the effects of PS2-2'-F modifications on the binding affinity of a model 2'-F-RNA aptamer; and 4) evaluate the effects of PS2-2'-F modifications on the gene silencing activities of siRNAs targeting 2-secretase. In Phase II, AM will (a) scale reagent production up to commercial quantities and purity; (b) optimize a robust protocol for synthesis of PS2-2'-F-RNA; (c) evaluate the effects of PS2-2'-F modifications on aptamers and siRNA activity in vivo; and (d) fully characterize the pharmacokinetic properties of PS2-2'-F-RNA. AM in Phase II may also offer for sale limited quantities of research-grade reagents for market beta testing. Upon successful completion of Phase II, AM will work with its existing industry partners to commercialize the 2'-F-thioamidites and enable the entire life science community to use these unique reagents in developing improved high-potency RNA drugs for a wide variety of human disease applications. PUBLIC HEALTH RELEVANCE: Functional RNA molecules such as aptamers and siRNAs have exciting potential as therapeutics in areas such as viral infections, cancer, genetic disorders, and neurological diseases. However, these potential RNA drugs require chemical modifications to achieve the necessary potency and stability. AM Biotechnologies (AM) will develop 2'-F-ribonucleoside thiophosphoramidite reagents that will allow the life science community to produce high potency, highly stable phosphorodithioate 2'-F-RNA-based drugs. The unique reagents that AM will develop under this project could have a profound impact on public health.

描述（由申请人提供）：功能性 RNA 分子，例如适体、siRNA、miRNA 和相关化合物，作为人类治疗药物和阐明体内基因调控的工具具有巨大的潜力。为了实现这一潜力，此类分子必须具有高效能和高核酸酶抗性。未修饰的 RNA 通常不能满足这些要求。人们已经探索了多种化学修饰来克服这些限制。特别是，单独或组合使用2'-F-核糖和硫代磷酸酯主链修饰的各种系统已取得一些成功。然而，进一步的改进是非常需要的。此外，硫代磷酸酯修饰是手性的，在每个主链取代处产生两种不同的异构体。因此，需要可以掺入功能性RNA中的改进的化学修饰。 AM Biotechnologies 将通过开发 2'-F-核糖核苷硫代亚磷酰胺 (2'-F-thioamidites) 来合成二硫代磷酸 2'-F-RNA (PS2-2'-F-RNA) 来解决这些关键问题。我们之前已经证明，在 DNA 适体的选定主链位置进行 PS2 修饰可增强与靶蛋白的结合亲和力，而不会损失特异性。此外，siRNA 中选定的 PS2 修饰显着提高了基因沉默活性。因此，选定的PS2-2'-F-RNA修饰将显着增加2'-F-RNA适体的结合亲和力和效力，并将为合成高效siRNA提供新途径。 PS2-2'-F-RNA 在磷上也将是非手性的，从而消除了非对映体硫代磷酸酯取代的 RNA 的可变生化、生物物理和生物学特性。 该第一阶段项目将： 1) 开发生产四种 2'-F-硫代酰胺（ABz、CBz、GIbu 和 U）的化学方法； 2) 优化PS2-2'-F-RNA的合成； 3) 评估PS2-2'-F修饰对模型2'-F-RNA适体结合亲和力的影响； 4)评估PS2-2'-F修饰对靶向2-分泌酶的siRNA的基因沉默活性的影响。在第二阶段，AM 将 (a) 将试剂生产规模扩大到商业数量和纯度； (b) 优化 PS2-2'-F-RNA 合成的稳健方案； (c) 评价PS2-2'-F修饰对适体和siRNA体内活性的影响； (d) 全面表征 PS2-2'-F-RNA 的药代动力学特性。第二阶段的 AM 还可能提供有限数量的研究级试剂用于市场 beta 测试。第二阶段成功完成后，AM 将与其现有的行业合作伙伴合作，将 2'-F-thioamidites 商业化，并使整个生命科学界能够使用这些独特的试剂为多种人类开发改进的高效 RNA 药物。疾病应用。 公共健康相关性：核酸适体和 siRNA 等功能性 RNA 分子在病毒感染、癌症、遗传性疾病和神经系统疾病等领域具有令人兴奋的治疗潜力。然而，这些潜在的 RNA 药物需要进行化学修饰才能达到必要的效力和稳定性。 AM Biotechnologies (AM) 将开发 2'-F-核糖核苷硫代亚磷酰胺试剂，使生命科学界能够生产高效、高度稳定的二硫代磷酸酯 2'-F-RNA 药物。 AM 将在该项目下开发的独特试剂可能会对公众健康产生深远的影响。