New Building Blocks for the Synthesis of Modified RNAs

描述(申请人提供)：功能性RNA分子，如适体、siRNAs、miRNAs和相关化合物，作为人类治疗和阐明体内基因调控的工具具有巨大的潜力。为了达到这一潜力，这些分子必须是高度有效和高度抗核酸酶的。未经修改的RNA通常不符合这些要求。为了克服这些限制，人们探索了各种化学修饰方法。特别是，在使用2‘-F-核糖和硫代磷酸主链修饰的各种系统中，单独或组合使用已经取得了一些成功。然而，进一步的改进是非常可取的。此外，硫代磷修饰是手性的，导致每个主干取代处有两个不同的异构体。因此，需要改进的化学修饰可以并入到功能RNA中。AM BioTechnologies将通过开发2‘-F-核糖核苷硫代磷酰胺(2’-F-硫代酰胺)来解决这些关键问题，从而能够合成二硫代磷酸2‘-F-RNA(PS2-2’-F-RNA)。我们以前已经证明，在DNA适配子的选定主干位置上的PS2修饰增强了与目标蛋白的结合亲和力，而不会失去特异性。此外，siRNAs中的PS2修饰显著提高了基因沉默的活性。因此，选择性的PS2-2‘-F-RNA修饰将显著提高2’-F-RNA适配子的结合亲和力和效力，并将为合成高效的siRNA提供新的途径。PS2-2‘-F-RNAs在磷原子上也是去手性的，消除了非对映异构体硫代硫代RNAs可变的生化、生物物理和生物学特性。这个第一阶段的项目将：1)发展产生四个2‘-F-硫代酰胺(ABZ，CBZ，Gibu和U)的化学；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；以及4)评估PS2-2‘-F修饰对靶向2-分泌酶的siRNA基因沉默活性的影响。在第二阶段，AM将(A)将试剂生产扩大到商业数量和纯度；(B)优化合成PS2-2‘-F-RNA的可靠方案；(C)评估PS2-2’-F修饰对适体和体内siRNA活性的影响；以及(D)充分表征PS2-2‘-F-RNA的药代动力学特性。处于第二阶段的AM还可能提供有限数量的研究级试剂用于市场贝塔测试。在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-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来开发用于各种人类疾病应用的改进的高效RNA药物。 与公共卫生相关：功能性RNA分子，如适配子和siRNAs，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药物需要进行化学修饰才能达到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，这将使生命科学界能够生产高效、高度稳定的二硫代磷酸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.