Development of RNAi based sFLT1-targeting therapeutics for treatment of Preeclampsia

先兆子痫(PE)是导致早产的三大主要原因之一，占所有妊娠的5%-8%。 在全球范围内，PE和随后的子痫是母体、胎儿和新生儿的主要因素 发病率和死亡率。虽然PE的根本原因还没有完全弄清楚，但现在已经很好了 确定母亲的高血压、浮肿和蛋白尿的症状和体征是由 母亲血液中过多的抗血管生成蛋白。其中最主要的是“可溶性FMS样物质” 酪氨酸激酶1“蛋白(SFlt1)由胎盘产生。sflt1是膜的截短形式- 结合的血管内皮生长因子受体Flt1(又名VEGFR1)。当人体内异常高的 在母亲的循环系统中，它们会干扰她身体对血管内皮生长因子的反应能力。有选择性的 消除(过滤)母体循环sFlt1已被证明是一种成功的治疗策略 减少30%-40%的循环sFlt1，足以延长妊娠。 我们的愿望是利用RNA干扰(RNAi)开发一种简单且经济有效的PE治疗方法，以限制 胎盘组织中sFlt1蛋白过度表达。基于RNA-Seq和PAS-Seq数据，胎盘sFlt1 Flt1内含子内多聚腺苷酸化产生的三种截短的mRNA亚单位控制了flt1的表达 13和14.用RNAi化合物靶向这些异常表达的内含子区域可以选择性地 在不干扰全长Flt1蛋白的情况下沉默编码sFlt1蛋白的截短mRNA变体 表情。 我们已经开发出一种新型的RNA化学物质，它能够高度定向地将药物输送到滋养层细胞。 胎盘迷路，注射剂量的12%累积在胎盘内(单次注射SC或IV)。这里, 化合物浓度可达到100微克/克，是所需剂量的1000多倍 有效沉默(100 ng/克寡聚)。最重要的是，我们没有观察到可检测到的寡核苷酸 转移到胎儿，通过荧光显微镜和定量分析。 使用系统筛查，我们已经鉴定出一对功能超强、代谢完全稳定的 选择性靶向i13(sFlt1-i13-2283)和i15a(sflt1-i15a-2519)的疏水修饰siRNA 异构体以EC50和10 PM进入RISC，40-80 nM进入滋养层 送货。全身应用sFlt1-i13-2283可有效抑制小鼠sflt1-i13基因的表达 胎盘和肝/肾内皮细胞，循环中sFlt1蛋白减少40%以上，没有 对胎儿或产妇健康可观察到的不良影响。 当前提案的目标是生成足够的数据来移动我们目前确定的领先候选人 (sFlt-i13-2283/sflt1-i15a-2519)，为制定 简单且经济有效的PE治疗，这是一个极其重要的未得到满足的医疗需求。

Complicating 5-8% of all pregnancies, preeclampsia (PE) is one of the three main causes of premature birth. Across the globe, PE and subsequent eclampsia are major contributors to maternal, fetal and neonatal morbidity and mortality. Although the root causes of PE have yet to be fully understood, it is now well established that the maternal signs and symptoms of hypertension, edema and proteinuria are caused by an excess of anti-angiogenic proteins in the mother's bloodstream. Chief among these are "soluble fms-like tyrosine kinase 1" proteins (sFLT1s) produced by the placenta. sFLT1s are truncated forms of the membrane- bound vascular endothelial growth factor (VEGF) receptor FLT1 (aka, VEGFR1). When abnormally high in the mother's circulatory system, they can interfere with her body's ability to respond to VEGF. Selective elimination (filtration) of maternally circulating sFLT1 has been shown to be a successful strategy for treatment of PE, with a 30-40% reduction in circulating sFLT1 being sufficient to allow pregnancy extension. Our desire is to develop a simple and cost-effective PE therapeutic using RNA interference (RNAi) to limit excess placental expression of sFLT1 proteins. Based on RNA-Seq and PAS-Seq data, placental sFLT1 expression is dominated by three truncated mRNA isoforms generated by polyadenylation within Flt1 introns 13 and 14. Targeting these abnormally-expressed intronic regions with RNAi compounds enables selective silencing of the truncated mRNA variants that encode sFLT1 proteins without interfering with full-length FLT1 expression. We have developed a novel RNA chemistry that enables highly targeted delivery to trophoblasts in the placental labyrinth with up to 12% of injected dose accumulating in placentas (single SC or IV injection). Here, compound concentrations can achieve up to 100 µg/gram, over 1,000-fold above the dose required for effective silencing (100 ng/gram oligo). Most importantly, we have observed no detectable oligonucleotide transfer to the fetus, both by fluorescence microscopy and quantitative analysis. Using systematic screens, we have identified a pair of hyper-functional, fully-metabolically stabilized, hydrophobically modified siRNAs that selectively target the i13 (sFLT1-i13-2283) and i15a (sFLT1-i15a-2519) isoforms with EC50 <10 pM for RISC (RNA Induced Silencing Complex) entry and 40-80 nM for trophoblast delivery. Systemic administration of sFLT1-i13-2283 results in potent silencing of sFLT1-i13 mRNA in mouse placenta and liver/kidney endothelium and more than 40% reduction in circulating sFLT1 protein, with no observable adverse effects on fetal or maternal health. The goal of the current proposal is to generate sufficient data to move our currently identified lead candidate (sFLT-i13-2283/sFLT1-i15a-2519) toward formal GLP and IND-enabling studies for the development of a simple and cost-effective treatment of PE, a critically important unmet medical need.