Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.

Phosphatidylcholine head group chemistry alters the extrahepatic accumulation of lipid-conjugated siRNA.

Small interfering RNAs (siRNAs) are revolutionizing the treatment of liver-associated indications. Yet, robust delivery to extrahepatic tissues remains a challenge. Conjugating lipids (e.g., docosanoic acid [DCA]) to siRNA supports extrahepatic delivery, but tissue accumulation remains lower than that achieved in liver by approved siRNA therapeutics. Early evidence suggests that functionalizing DCA with a head group (e.g., phosphatidylcholine [PC]) may enhance delivery to certain tissues. Here, we report the first systematic evaluation of the effect of PC head group chemistry on the extrahepatic distribution of DCA-conjugated siRNAs. We show that functionalizing DCA with a PC head group enhances siRNA accumulation in heart, muscle, lung, pancreas, duodenum, urinary bladder, and fat. Varying the size of the linker between the phosphate and choline moiety of the PC head group altered the extrahepatic accumulation of siRNA, with the optimal linker length being different for different tissues. Increasing PC head group valency also improved extrahepatic accumulation in a tissue-specific manner. This study demonstrates the structural impact of the PC moiety on the biodistribution of lipid-conjugated siRNA and introduces multiple novel PC variants for the chemical optimization of DCA-conjugated siRNA. These chemical variants can be used in the context of other lipids to increase the repertoire of conjugates for the extrahepatic distribution of siRNAs.

Enhancing siRNA efficacy in vivo with extended nucleic acid backbones.

Therapeutic small interfering RNA (siRNA) requires sugar and backbone modifications to inhibit nuclease degradation. However, metabolic stabilization by phosphorothioate (PS), the only backbone chemistry used clinically, may be insufficient for targeting extrahepatic tissues. To improve oligonucleotide stabilization, we report the discovery, synthesis and characterization of extended nucleic acid (exNA) consisting of a methylene insertion between the 5'-C and 5'-OH of a nucleoside. exNA incorporation is compatible with common oligonucleotide synthetic protocols and the PS backbone, provides stabilization against 3' and 5' exonucleases and is tolerated at multiple oligonucleotide positions. A combined exNA-PS backbone enhances resistance to 3' exonuclease by ~32-fold over the conventional PS backbone and by >1,000-fold over the natural phosphodiester backbone, improving tissue exposure, tissue accumulation and efficacy in mice, both systemically and in the brain. The improved efficacy and durability imparted by exNA may enable therapeutic interventions in extrahepatic tissues, both with siRNA and with other oligonucleotides such as CRISPR guide RNA, antisense oligonucleotides, mRNA and tRNA.

Dendritic amphiphilic siRNA: Selective albumin binding, in vivo efficacy, and low toxicity.

Although an increasing number of small interfering RNA (siRNA) therapies are reaching the market, the challenge of efficient extra-hepatic delivery continues to limit their full therapeutic potential. Drug delivery vehicles and hydrophobic conjugates are being used to overcome the delivery bottleneck. Previously, we reported a novel dendritic conjugate that can be appended efficiently to oligonucleotides, allowing them to bind albumin with nanomolar affinity. Here, we explore the ability of this novel albumin-binding conjugate to improve the delivery of siRNA in vivo. We demonstrate that the conjugate binds albumin exclusively in circulation and extravasates to various organs, enabling effective gene silencing. Notably, we show that the conjugate achieves a balance between hydrophobicity and safety, as it significantly reduces the side effects associated with siRNA interactions with blood components, which are commonly observed in some hydrophobically conjugated siRNAs. In addition, it reduces siRNA monocyte uptake, which may lead to cytokine/inflammatory responses. This work showcases the potential of using this dendritic conjugate as a selective albumin binding handle for the effective and safe delivery of nucleic acid therapeutics. We envision that these properties may pave the way for new opportunities to overcome delivery hurdles of oligonucleotides in future applications.

Chemical optimization of siRNA for safe and efficient silencing of placental sFLT1.

Preeclampsia (PE) is a rising, potentially lethal complication of pregnancy. PE is driven primarily by the overexpression of placental soluble fms-like tyrosine kinase 1 (sFLT1), a validated diagnostic and prognostic marker of the disease when normalized to placental growth factor (PlGF) levels. Injecting cholesterol-conjugated, fully modified, small interfering RNAs (siRNAs) targeting sFLT1 mRNA into pregnant mice or baboons reduces placental sFLT1 and ameliorates clinical signs of PE, providing a strong foundation for the development of a PE therapeutic. siRNA delivery, potency, and safety are dictated by conjugate chemistry, siRNA duplex structure, and chemical modification pattern. Here, we systematically evaluate these parameters and demonstrate that increasing 2'-O-methyl modifications and 5' chemical stabilization and using sequence-specific duplex asymmetry and a phosphocholine-docosanoic acid conjugate enhance placental accumulation, silencing efficiency and safety of sFLT1-targeting siRNAs. The optimization strategy here provides a framework for the chemical optimization of siRNAs for PE as well as other targets and clinical indications.