New Reagents for RNA-based Therapeutic Technologies

产品说明：基于RNA的治疗技术的新试剂摘要功能性RNA分子如适体、siRNA、miRNA和相关化合物作为人类治疗剂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，这些分子必须是高度有效和高度稳定的。未经修饰的RNA通常不接近满足这些要求。通过单独或组合使用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在磷上是手性的，在每个PS取代处产生两种不同的异构体。因此，需要进一步改进。 在该项目的第一阶段，我们证明了使用含有2 '-OMe-二硫代磷酸酯（MS 2）修饰的RNA的新方法的原理证明，所述修饰通过使用新型2'-OMe-硫代亚磷酰胺（2 '-OMe-硫代亚酰胺）试剂制备。我们以小规模成功地合成了四种2 '-OMe-硫代酰胺（A、C、G和U），并使用它们合成了多种含有MS 2修饰的RNA。值得注意的是，我们表明，掺入MS 2修饰显著提高了对靶向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MS 2修饰的siRNA在培养的细胞中具有针对多个基因靶标的增加的基因沉默活性。为了实现这些新试剂的巨大潜力，该项目的第二阶段将集中于以下目标：（1）增加2 '-OMe-硫代酰胺的生产规模;（2）优化用于体外和体内应用的MS 2-RNA固相合成的方案;（3）确定MS 2-RNA双链体的热稳定性和结构;（4）确定MS 2-RNA双链体的热稳定性。（4）验证所选VEGF MS 2-适体的细胞结合亲和力和特异性;（5）开发配制的MS 2-siRNA，其在转移性卵巢癌的鼠模型中提供增加的效力和抗肿瘤功效。该项目的成功完成将证明MS 2-siRNA在体外和体内的价值，并将使AM及其商业合作伙伴能够进行2 '-OMe-硫代酰胺试剂的全面商业化，并有助于实现有效的基于MS 2修饰的RNA的治疗。

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.