RNAi modulation of placental sFLT1 for the treatment of preeclampsia

Abstract

Preeclampsia is a placentally induced hypertensive disorder of pregnancy that is associated with substantial morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm preeclampsia result from excess circulating soluble vascular endothelial growth factor receptor FLT1 (sFLT1 or sVEGFR1) of placental origin. Here we identify short interfering RNAs (siRNAs) that selectively silence the three sFLT1 mRNA isoforms primarily responsible for placental overexpression of sFLT1 without reducing levels of full-length FLT1 mRNA. Full chemical stabilization in the context of hydrophobic modifications enabled productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduced circulating sFLT1 in pregnant mice (up to 50%). In a baboon preeclampsia model, a single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of preeclampsia. Our results demonstrate RNAi-based extrahepatic modulation of gene expression with nonformulated siRNAs in nonhuman primates and establish a path toward a new treatment paradigm for patients with preterm preeclampsia.

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Figure 1: Development of hydrophobically modified, chemically stabilized hsiRNA compounds against sFLT1.
Figure 2: Efficient silencing of sFLT1 by hsiRNAsFLT1-2283/2519 mixture in vitro.
Figure 3: Full chemical stabilization of cholesterol-conjugated hsiRNAs enables systemic delivery to placental labyrinth.
Figure 4: Efficient silencing of sFlt1 mRNA and protein by hsiRNAsFLT1 in pregnant mice.
Figure 5: hsiRNAsFLT1-2283/2519 treatment lowers sFLT1 serum levels and normalizes blood pressure and proteinuria in a baboon model of preeclampsia.

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Acknowledgements

We thank D. Conte for help revising the manuscript and Khvorova Laboratory members for their support. This project was funded by the National Institutes of Health (R01 HD086111 and S10 OD020012) and the Bill and Melinda Gates Foundation (OPP1086170).

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Contributions

A.A.T. managed the project, performed most of the experiments and drafted the manuscript. M.J.M., S.A.K. and A.K. conceived the study and led the project. M.R.H., D.E. and L.R. synthesized all compounds and developed and optimized protocols for gram-scale synthesis. A.H.C. and B.M.D.C.G. helped with in vivo studies. R.A.H. performed PNA assay analysis. A.A.-P. performed polyadenylation site sequence analysis and an initial siRNA screen. J.F.A. helped with the siRNA screen. A.L. performed primary cytotrophoblast isolations, ELISAs, toxicity studies and mouse pregnancy studies. Z.K.Z. performed placenta vascular immunohistochemistry. A.H., A.M., S.P., J.I., R.S. and R.O. developed the baboon preeclampsia model and performed all baboon experiments. A.A.T, A.K., M.J.M., S.A.K. and A.H. wrote the manuscript.

Corresponding authors

Correspondence to S Ananth Karumanchi or Melissa J Moore or Anastasia Khvorova.

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Competing interests

A.K. discloses ownership of stock in RXi Pharmaceuticals and Advirna. S.A.K. is a consultant to Thermofisher Scientific and owns stock in Aggamin Therapeutics. M.J.M. is employed by Moderna Therapeutics.

Integrated supplementary information

Supplementary Figure 1 Upregulation of alternative sFLT1 mRNAs in human placentas in preeclampsia.

Total RNA was isolated from 6 normal (N) and 11 preeclamptic (PE) human placentas and analyzed by Polyadenylation Site sequencing (PAS-Seq). Data for computation were derived from (Ashar et al, 2017). All three sFLT1 mRNA isoforms are contributing to sFLT1 overexpression during PE.

Supplementary Figure 2 Specific hsiRNA-mediated downregulation of sFLT1 mRNA isoforms in human placental cytotrophoblast (CTB).

a) Dose-response curves of hsiRNAPPIB silencing of PPIB mRNA in CTB (n=3 biologically independent experiments, mean +SD). b) Dose-response curves of hsiRNAsFLT1-2283 and hsiRNAsFLT1-2519 silencing of sFLT1-i13 and sFLT1-e15a mRNAs in CTB (n=3 biologically independent experiments, mean + s.d.). hsiRNANTC – non-targeting hsiRNA control. ****P<0.0001, **P<0.01, *P<0.05, two-way ANOVA.

Supplementary Figure 3 Full chemical stabilization of cholesterol-conjugated hsiRNAsFLT1-2283 enables efficient placenta accumulation.

a) Representative images of placenta distribution (24 hours post treatment) of partially and fully modified Cy3-hsiRNAsFLT1-2283 (red) following 10 mg/kg IV or SC injections. Nuclei stained with DAPI (blue). Scale bar, 1mm. All images were acquired at identical settings (n=3 biologically independent animals). b) Guide strand quantification by PNA hybridization-based assay in placentas from a (n=6 biologically independent samples). Boxes represent 25 to 75 percentiles and whiskers indicate minimal and maximal values (****P<0.0001, **P=0.015, unpaired t test, two-tailed).

Supplementary Figure 4 Efficient silencing of sFLT1 mRNA in liver and kidney by hsiRNAsFLT1-2283 in pregnant mice.

Pregnant CD1 mice were injected at day E14 and E15 with 2X20 mg/kg of hsiRNAsFLT1 -2283 (IV, tail vein) and sacrificed at day E19. a) sFLT1-i13 mRNA levels were measured using QuantiGene® assay (Affymetrix), normalized to housekeeping gene, and presented as % of PBS control (n=6 biologically independent animals, mean + s.d., ****P<0.0001, one-way ANOVA). b) Amount of guide strand accumulated in mouse tissues measured by PNA hybridization assay (n=6 biologically independent samples, boxes represent 25 to 75 percentiles and whiskers indicate minimal and maximal values, ****P<0.0001, one-way ANOVA).

Supplementary Figure 5 Newborn pup numbers and weights from pregnant mice treated with hsiRNA.

Pregnant CD1 mice were injected at day E14 and E15 with 2X20 mg/kg of hsiRNAsFLT1-2283, hsiRNANTC or PBS (IV, tail vein) and allowed to deliver pups on day E20. a-c). Newborn pups number and weights from mice injected with PBS or hsiRNAsFLT1 -2283 (n=9 biologically independent animals, boxes represent 25 to 75 percentiles and whiskers indicate minimal and maximal values, ns- non-significant, unpaired t test, two-tailed). d-e) Newborn pups number and weights from mice injected with PBS or hsiRNANTC (n=10 biologically independent animals, boxes represent 25 to 75 percentiles and whiskers indicate minimal and maximal values, ns- non-significant, unpaired t test, two-tailed).

Supplementary Figure 6 Efficient sFLT1 mRNA cleavage by hsiRNAsFLT1-2283 in mouse placenta (5′-RACE analysis).

Pregnant CD1 mice were injected at E14 and E15 with 2X20 mg/kg of hsiRNAsFLT1 -2283, hsiRNANTC or PBS (IV, tail vein) and sacrificed at E19. Total RNA was isolated from whole placenta and agarose gel of 5’-RACE PCR amplification with GR1 and GSP1 primers showed predicted cleavage product (~400 bp, indicated by Arrow). Representative image from two independent experiments. Corresponding sequencing result shown below.

Supplementary Figure 7 hsiRNAsFLT1-2283/2519 tissue concentrations after IV injection in pregnant mice.

Pregnant mice were injected with 2x20 mg/kg of hsiRNAsFLT1-2283/2519 at day E14 and E15 and tissues were collected at day E19. Milk was collected at post partum day P12. Guide strand concentration hsiRNAsFLT1-2283 and hsiRNAsFLT1-2519 were measured using PNA hybridization assay (n=2 biologically independent animals, mean +s.d.). # - milk samples concentrations are presented in ng/μl.

Supplementary Figure 8 Representative placental histology of mice treated with PBS, hsiRNA 2383 and 2283/2519 (2 × 20 mg/kg, IV, n = 4), showing anti-CD31 staining.

PBS treated placental labyrinth shows extensive staining for CD31 (upper panel), suggesting robust vascular network. Under higher magnifications (lower panel), PBS treated placental tissue displays small vessels filled with nucleated erythrocytes in close vicinity to maternal blood sinuses. There were no significant changes in the hsiRNA injected mice. D- decidua, Jz - junctional zone and L – labyrinth.

Supplementary Figure 9 Newborn baboon weights and centiles after hsiRNAsFLT1 treatment.

Pregnant baboons were injected with 20 mg/kg of hsiRNAsFLT1-2283/2519 around day 133 of gestation and newborns’ weights and centiles were recorded after delivery. ns- non-significant (n=9 biologically independent animals for control, n=3 for hsiRNA, P=0.1819, unpaired t test, two-tailed, boxes represent 25 to 75 percentiles and whiskers indicate minimal and maximal values). * - pregnancy was terminated before full term for unrelated conditions.

Supplementary Figure 10 hsiRNAsFLT1-2283/2519 blood, placenta and urine concentrations following a single IV injection in the baboon model of PE.

Pregnant baboons were injected with 20 mg/kg of hsiRNAsFLT1-2283/2519 around day 133 of gestation. Blood, urine and placenta biopsies were collected at the time points shows. Guide strand concentration hsiRNAsFLT1-2283 and hsiRNAsFLT1-2519 were measured using PNA hybridization assay (n=1).

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Supplementary Table 1

Chemical modification patterns and sequences of siRNAs (PDF 407 kb)

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Turanov, A., Lo, A., Hassler, M. et al. RNAi modulation of placental sFLT1 for the treatment of preeclampsia. Nat Biotechnol 36, 1164–1173 (2018). https://doi.org/10.1038/nbt.4297

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