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 g/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.
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. The goal of this grant is to advance a previously identified RNAi-based lead candidate (sFLT1-i13-2283/sFLT1-i15a-2519) toward the clinic as a novel, simple and cost-effective treatment for PE.
|Khvorova, Anastasia; Watts, Jonathan K (2017) The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol 35:238-248|