Engineering VEGF mRNA lipid nanoparticles to treat pre-eclampsia during pregnancy

Introduction: Pre-eclampsia is a placental disorder that affects 3–5% of all pregnancies and is a leading cause of maternal and fetal morbidity worldwide. Placental insufficiency disorders such as pre-eclampsia are caused by inadequate blood flow in the placenta that results in increased maternal blood pressure and restricted fetal growth. Therefore, improving vasodilation in the placenta can benefit both maternal and fetal health. With no drug available in the clinic to slow disease progression, engineering ionizable lipid nanoparticles (LNPs) for extrahepatic mRNA delivery to the placenta is an attractive therapeutic platform to treat pre-eclampsia. In this work, we utilize a high-throughput molecular barcoding approach to screen a library of 98 LNP formulations in vivo and identify a placenta-tropic LNP (LNP 55) that mediates more than 100-fold greater mRNA delivery to the placenta in pregnant mice than the FDA-approved DLin-MC3-DMA formulation. LNP 55 facilitates delivery to trophoblasts, endothelial cells, and immune cells in the placenta, the three key cell types responsible for regulating placental vasodilation during pre-eclampsia. In both inflammation- and hypoxia-induced murine models of pre-eclampsia, this LNP formulation encapsulating vascular endothelial growth factor (VEGF) mRNA rescues maternal blood pressure and restores a healthy, dilated placental vasculature. Together, these results demonstrate the potential of this modular LNP platform to encapsulate a variety of mRNA cargoes to treat placental disorders such as pre-eclampsia during pregnancy (Fig. 1a).

Materials and

Methods: A library of 98 LNPs with novel ionizable lipid materials and varied excipient compositions was formulated with barcoded DNA (b-DNA) via pipette mixing. The 98 LNP formulations were pooled and administered to non-pregnant and pregnant mice; 6 h following administration organs were collected, processed, and samples were prepared for next generation sequencing (NGS). Demultiplexing and subsequent data analysis identified lead LNP formulations for extrahepatic delivery to the placenta. Counter screening was performed to validate the efficacy of a lead placenta-tropic LNP for luciferase mRNA delivery to the placenta in pregnant mice. Finally, this placenta-tropic LNP encapsulating VEGF mRNA was evaluated in two murine models of pre-eclampsia, induced via systemic inflammation (i.p. LPS) or via hypoxia (i.v. sFlt-1 adenovirus). Maternal weight and blood pressure were recorded daily throughout gestation while fetal and placental weight, placental vasculature, and immune response were evaluated at the study endpoint before parturition.

Results, Conclusions, and Discussions: High-throughput in vivo DNA barcoding was used to assess the biodistribution of a 98 LNP library in non-pregnant and pregnant mice. Many of our LNP formulations with novel ionizable lipid materials enabled extrahepatic LNP delivery compared to industry standard formulations containing the C12-200 or DLin-MC3-DMA ionizable lipids (Fig. 1b). One lead placenta-tropic particle identified from high-throughput screening — ptLNP — was formulated encapsulating luciferase mRNA to assess functional protein expression in vivo. Consistent with the results from the high-throughput screen, ptLNP mediated potent luciferase expression to the placentas of pregnant mice, significantly more than liver-tropic C12-200 and DLin-MC3-DMA LNPs (Fig. 1c). ptLNP was then formulated with VEGF mRNA as a therapeutic for inflammation- and hypoxia-induced pre-eclampsia in pregnant mice. Following LNP administration on gestational day E12, maternal blood pressure was significantly lower than the untreated pre-eclamptic mice through the end of gestation (Fig. 1d). Hematoxylin and eosin (H&E) staining of placental tissue demonstrated enhanced vascularization following VEGF mRNA ptLNP treatment (Fig. 1e). Additionally, VEGF mRNA ptLNPs improved fetal weight compared to growth restricted fetuses from untreated pre-eclamptic mice (Fig. 1f). These results demonstrate the potential of placenta-tropic VEGF mRNA LNPs to treat pre-eclampsia during pregnancy. Future work will focus on using this ptLNP platform to encapsulate other therapeutic nucleic acid cargoes for treating a broad range of placental insufficiency disorders during pregnancy.

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