New Reagents for RNA-based Therapeutic Technologies

功能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。要达到这种潜力，这种分子必须具有很强的效力和高度的稳定性。未修饰的rna通常不能满足这些要求。通过单独或联合使用2'- o -甲基核糖（2'- ome）和硫代磷酸酯（PS）骨架修饰，已经在体外和体内取得了一些成功。然而，2'-OMe和PS修饰的rna都具有有限的体内稳定性和活性，这可能是有问题的。此外，含有PS修饰的RNA对磷具有手性，在每个PS取代处产生两个不同的异构体。因此，需要进一步改进。在该项目的第一阶段，我们展示了一种新方法的原理证明，该方法使用含有2'- ome -硫代磷酰亚胺（2'- ome -硫代磷酰亚胺）试剂制备的含有2'- ome -硫代磷酰亚胺（MS2）修饰的RNA。我们成功地在小尺度上合成了4种2'- ome -硫胺（A， C， G和U），并利用它们合成了多种含有MS2修饰的rna。值得注意的是，我们发现结合MS2修饰显著提高了靶VEGF蛋白的结合亲和力超过1000倍，从2 nM到< 1 pM。此外，我们发现含有MS2修饰的sirna在培养细胞中对多个基因靶标具有增强的基因沉默活性。为了实现这些新试剂的高潜力，该项目二期将重点关注以下目标：(1)增加2'- ome -硫胺的生产规模；(2)优化MS2-RNA体外和体内固相合成工艺；(3)确定MS2-RNA双链的热稳定性和结构；(4)验证所选VEGF ms2适配体的细胞结合亲和力和特异性；(5)开发配方ms2 - sirna，在小鼠转移性卵巢癌模型中提供更高的效力和抗肿瘤功效。该项目的成功完成将证明MS2- sirna在体外和体内的价值，并将使AM及其商业合作伙伴能够继续进行2'- ome -硫胺试剂的全面商业化，并为实现有效的MS2修饰rna为基础的治疗做出贡献。

DESCRIPTION: New Reagents for RNA-based Therapeutic Technologies Abstract 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 stable. Unmodified RNAs typically do not come close to meeting these requirements. Some success has been achieved in vitro and in vivo by using 2'-O- methyl-ribose (2'-OMe) and phosphorothioate (PS) backbone modifications, alone or in combination. However, both 2'-OMe and PS modified RNAs have limited in vivo stability and activity, which can be problematic. In addition, RNA containing PS modification(s) are chiral at phosphorus, resulting in two distinct isomers at each PS substitution. Therefore, there is a need for further improvements. In Phase I of this project, we demonstrated proof of principle for a new approach using RNA containing 2'-OMe-phosphorodithioate (MS2) modifications, prepared by using novel 2'-OMe-thiophosphoramidite (2'- OMe-thioamidite) reagents. We successfully synthesized the four 2'-OMe-thioamidites (A, C, G, and U) at small scale and used them to synthesize a variety of RNAs containing MS2 modifications. Significantly, we showed that incorporating MS2 modifications remarkably improved binding affinity toward the targeted VEGF protein more than 1000-fold, from 2 nM to < 1 pM. In addition, we showed that siRNAs containing MS2 modifications had increased gene silencing activity against multiple gene targets in cultured cells. To realize the high potential o these new reagents, Phase II of this project will focus on the following aims: (1) increase the scale of 2'-OMe-thioamidite production; (2) optimize protocols for solid-phase synthesis of MS2-RNA for in vitro and in vivo applications; (3) determine the thermal stability and structure of MS2-RNA duplexes; (4) validate the cellular binding affinity and specificity of the selected VEGF MS2-aptamers; (5) develop formulated MS2-siRNAs that provide increased potency and antitumor efficacy in a murine model of metastatic ovarian cancer. Successful completion of this project will demonstrate the value of MS2-siRNAs in vitro and in vivo, and will enable AM and its commercial partners to proceed with full commercialization of the 2'-OMe- thioamidite reagents and contribute toward the realization of effective MS2 modified RNA-based therapeutics.