New Building Blocks for the Synthesis of Modified RNAs

描述（由申请人提供）：功能性RNA分子如适体、siRNA、miRNA和相关化合物具有作为人类治疗剂和作为阐明体内基因调控的工具的巨大潜力。为了达到这种潜力，这些分子必须是高度有效和高度耐核酸酶的。未经修饰的RNA通常不满足这些要求。已经探索了各种化学修饰来克服这些限制。特别地，在单独或组合使用2 '-F-核糖和硫代磷酸酯骨架修饰的各种系统中已经取得了一些成功。然而，进一步的改进是非常期望的。此外，硫代磷酸酯修饰是手性的，在每个骨架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-硫代亚酰胺）来解决这些关键问题，以实现二硫代磷酸2 '-F-RNA（PS2- 2'-F-RNA）的合成。我们先前已经表明，在选定的DNA适体骨架位置的PS2修饰增强了对靶蛋白的结合亲和力，而不丧失特异性。此外，siRNA中选择的PS2修饰显著提高基因沉默活性。因此，选择的PS2- 2 ′-F-RNA修饰将显著增加2 ′-F-RNA适体的结合亲和力和效力，并将为合成高效siRNA提供新的途径。PS2- 2 ′-F-RNA在磷上也是非手性的，消除了非对映体硫代磷酸酯取代的RNA的可变生物化学、生物物理和生物学特性。 第一阶段项目将：1）开发化学以产生四种2 ′-F-硫代酰胺（ABz、CBz、GIbu和U）; 2）优化PS2- 2 ′-F-RNA的合成; 3）评估PS2- 2 ′-F修饰对模型2 ′-F-RNA适体的结合亲和力的影响;和4）评估PS2- 2 ′-F修饰对靶向β 2-分泌酶的siRNA的基因沉默活性的影响。在II期，AM将（a）将试剂生产规模扩大至商业数量和纯度;（B）优化用于合成PS2- 2 '-F-RNA的稳健方案;（c）评估PS2- 2'-F修饰对体内适体和siRNA活性的影响;以及（d）充分表征PS2- 2 '-F-RNA的药代动力学性质。II期AM还可能提供销售有限数量的研究级试剂，用于市场β测试。在第二阶段成功完成后，AM将与现有的行业合作伙伴合作，将2 '-F-硫代酰胺商业化，并使整个生命科学界能够使用这些独特的试剂开发用于各种人类疾病应用的改进的高效RNA药物。 公共卫生相关性：功能性RNA分子如适体和siRNA在病毒感染、癌症、遗传疾病和神经疾病等领域具有令人兴奋的治疗潜力。然而，这些潜在的RNA药物需要化学修饰以实现必要的效力和稳定性。AM生物技术公司（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力、高稳定性的基于2'-F-RNA的二硫代磷酸酯药物。AM将在该项目下开发的独特试剂可能对公共卫生产生深远的影响。

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.