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Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery.

Abstract5΄-Vinylphosphonate modification of siRNAs protects them from phosphatases, and improves silencing activity. Here, we show that 5΄-vinylphosphonate confers novel properties to siRNAs. Specifically, 5΄-vinylphosphonate (i) increases siRNA accumulation in tissues, (ii) extends duration of silencing in multiple organs and (iii) protects siRNAs from 5΄-to-3΄ exonucleases. Delivery of conjugated siRNAs requires extensive chemical modifications to achieve stability in vivo. Because chemically modified siRNAs are poor substrates for phosphorylation by kinases, and 5΄-phosphate is required for loading into RNA-induced silencing complex, the synthetic addition of a 5΄-phosphate on a fully modified siRNA guide strand is expected to be beneficial. Here, we show that synthetic phosphorylation of fully modified cholesterol-conjugated siRNAs increases their potency and efficacy in vitro, but when delivered systemically to mice, the 5΄-phosphate is removed within 2 hours. The 5΄-phosphate mimic 5΄-(E)-vinylphosphonate stabilizes the 5΄ end of the guide strand by protecting it from phosphatases and 5΄-to-3΄ exonucleases. The improved stability increases guide strand accumulation and retention in tissues, which significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. Moreover, we show that 5΄-(E)-vinylphosphonate stabilizes 5΄ phosphate, thereby enabling systemic delivery to and silencing in kidney and heart.

Preeclampsia (PE) is a placentally-induced hypertensive disorder of pregnancy that is associated with significant morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm PE 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. Full chemical stabilization in the context of hydrophobic modifications enables productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduces circulating sFLT1 in pregnant mice (up to 50%). In a baboon PE model, single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of PE. Our results demonstrate RNAi-based extra-hepatic modulation of gene expression with non-formulated siRNAs in non-human primates and establish a path toward a new treatment paradigm for patients with preterm PE.

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.