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、miRNAs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。为了达到这种潜力，这些分子必须具有很强的效力和高度的核酸酶抗性。未修饰的rna通常不满足这些要求。为了克服这些限制，人们探索了各种化学修饰。特别是，在使用2'- f -核糖和硫代酸骨架修饰（单独或联合）的各种体系中取得了一些成功。然而，进一步的改进是非常可取的。此外，硫代磷酸酯修饰是手性的，在每个主链取代处产生两个不同的异构体。因此，有必要改进可纳入功能性rna的化学修饰。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，从而合成二硫代磷2'- f -RNA （PS2-2'- f -RNA）。我们之前已经证明，在DNA适体的选定骨干位置上的PS2修饰增强了与靶蛋白的结合亲和力，而不会失去特异性。此外，sirna中选定的PS2修饰显著提高了基因沉默活性。因此，选定的PS2-2′-F-RNA修饰将显著提高2′-F-RNA适配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，消除了非对映异构体硫代rna的可变生化、生物物理和生物学特性。该一期项目将：1)开发生产四种2'- f -硫胺类化合物（ABz、CBz、GIbu和U）的化学工艺；2)优化PS2-2′- f - rna的合成；3)评价PS2-2′-F修饰对模型2′-F- rna适配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)评估PS2-2'-F修饰对适体和siRNA活性的体内影响；(d)充分表征PS2-2′-F-RNA的药代动力学特性。第二阶段的AM也可能提供有限数量的研究级试剂用于市场测试。在二期成功完成后，AM将与其现有的行业合作伙伴合作，将2'- f -硫酰胺商业化，并使整个生命科学界能够使用这些独特的试剂开发改进的高效RNA药物，用于各种人类疾病的应用。

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.