Single intravitreal administration of a tetravalent siRNA exhibits robust and efficient gene silencing in mouse and pig photoreceptors.

Inherited retinal dystrophies caused by dominant mutations in photoreceptor (PR) cell expressed genes are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets PR cell expressed genes. Here, we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best PR accumulation. A single intravitreal administration induces multimonths efficacy in rodent and porcine retinas while demonstrating a good safety profile. The data suggest that this configuration can treat retinal diseases caused by PR cell expressed genes with 1-2 intravitreal injections per year.

Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin.

Inhibition of Janus kinase (JAK) family enzymes is a popular strategy for treating inflammatory and autoimmune skin diseases. In the clinic, small molecule JAK inhibitors show distinct efficacy and safety profiles, likely reflecting variable selectivity for JAK subtypes. Absolute JAK subtype selectivity has not yet been achieved. Here, we rationally design small interfering RNAs (siRNAs) that offer sequence-specific gene silencing of JAK1, narrowing the spectrum of action on JAK-dependent cytokine signaling to maintain efficacy and improve safety. Our fully chemically modified siRNA supports efficient silencing of JAK1 expression in human skin explant and modulation of JAK1-dependent inflammatory signaling. A single injection into mouse skin enables five weeks of duration of effect. In a mouse model of vitiligo, local administration of the JAK1 siRNA significantly reduces skin infiltration of autoreactive CD8+ T cells and prevents epidermal depigmentation. This work establishes a path toward siRNA treatments as a new class of therapeutic modality for inflammatory and autoimmune skin diseases.

Single intravitreal administration of a tetravalent siRNA exhibits robust and efficient gene silencing in rodent and swine photoreceptors.

Inherited retinal dystrophies caused by dominant mutations in photoreceptor-expressed genes, are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets photoreceptor-expressed genes. Here we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best photoreceptor accumulation. A single intravitreal administration induces multi-months efficacy in rodent and porcine retinas while showing a good safety profile. The data suggest that this configuration can treat retinal diseases caused by photoreceptor-expressed genes with 1-2 intravitreal injections per year.

Therapeutic Oligonucleotides: An Outlook on Chemical Strategies to Improve Endosomal Trafficking.

The potential of oligonucleotide therapeutics is undeniable as more than 15 drugs have been approved to treat various diseases in the liver, central nervous system (CNS), and muscles. However, achieving effective delivery of oligonucleotide therapeutics to specific tissues still remains a major challenge, limiting their widespread use. Chemical modifications play a crucial role to overcome biological barriers to enable efficient oligonucleotide delivery to the tissues/cells of interest. They provide oligonucleotide metabolic stability and confer favourable pharmacokinetic/pharmacodynamic properties. This review focuses on the various chemical approaches implicated in mitigating the delivery problem of oligonucleotides and their limitations. It highlights the importance of linkers in designing oligonucleotide conjugates and discusses their potential role in escaping the endosomal barrier, a bottleneck in the development of oligonucleotide therapeutics.

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.

Engineered ionizable lipid siRNA conjugates enhance endosomal escape but induce toxicity in vivo.

Lipid conjugation supports delivery of small interfering RNAs (siRNAs) to extrahepatic tissues, expanding the therapeutic potential of siRNAs beyond liver indications. However, siRNA silencing efficacy in extrahepatic tissues remains inferior to that routinely achieved in liver, partially due to the low rate of endosomal escape following siRNA internalization. Improving siRNA endosomal release into cytoplasm is crucial to improving efficacy of lipid-conjugated siRNAs. Given the ability of ionizable lipids to enhance endosomal escape in a context of lipid nanoparticles (LNP), here, we provide the first report on the effect of an ionizable lipid conjugate on siRNA endosomal escape, tissue distribution, efficacy, and toxicity in vivo. After developing a synthetic route to covalently attach the ionizable lipid, DLin-MC3-DMA, to siRNAs, we demonstrate that DLin-MC3-DMA enhances endosomal escape in cell culture without compromising siRNA efficacy. In mice, DLin-MC3-DMA conjugated siRNAs exhibit a similar overall tissue distribution profile to the similarly hydrophobic cholesterol-conjugated siRNA. However, only DLin-MC3-DMA conjugated siRNAs accumulated in vascular compartments, suggesting an effect of conjugate structure on intratissue distribution. Interestingly, we observed non-specific modulation of gene expression in tissues with high accumulation of DLin-MC3-DMA siRNAs (>20 pmol/mg of tissue) while limited non-specific gene modulation has been observed in tissues with lower siRNA accumulation. These findings suggest modulating the nature of the conjugate is a promising strategy to alter siRNA intratissue and intracellular trafficking. Fine-tuning the nature of the conjugate to optimize endosomal escape while minimizing toxicity will be critical for the progression of therapeutic siRNA applications beyond the liver.

PK-modifying anchors significantly alter clearance kinetics, tissue distribution, and efficacy of therapeutics siRNAs.

Effective systemic delivery of small interfering RNAs (siRNAs) to tissues other than liver remains a challenge. siRNAs are small (∼15 kDa) and therefore rapidly cleared by the kidneys, resulting in limited blood residence times and tissue exposure. Current strategies to improve the unfavorable pharmacokinetic (PK) properties of siRNAs rely on enhancing binding to serum proteins through extensive phosphorothioate modifications or by conjugation of targeting ligands. Here, we describe an alternative strategy for enhancing blood and tissue PK based on dynamic modulation of the overall size of the siRNA. We engineered a high-affinity universal oligonucleotide anchor conjugated to a high-molecular-weight moiety, which binds to the 3' end of the guide strand of an asymmetric siRNA. Data showed a strong correlation between the size of the PK-modifying anchor and clearance kinetics. Large 40-kDa PK-modifying anchors reduced renal clearance by ∼23-fold and improved tissue exposure area under the curve (AUC) by ∼26-fold, resulting in increased extrahepatic tissue retention (∼3- to 5-fold). Furthermore, PK-modifying oligonucleotide anchors allowed for straightforward and versatile modulation of blood residence times and biodistribution of a panel of chemically distinct ligands. The effects were more pronounced for conjugates with low lipophilicity (e.g., N-Acetylgalactosamine [GalNAc]), where significant improvement in uptake by hepatocytes and dose-dependent silencing in the liver was observed.

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.

RNAi-based modulation of IFN-γ signaling in skin.

Aberrant activation of interferon (IFN)-γ signaling plays a key role in several autoimmune skin diseases, including lupus erythematosus, alopecia areata, vitiligo, and lichen planus. Here, we identify fully chemically modified small interfering RNAs (siRNAs) that silence the ligand binding chain of the IFN-γ receptor (IFNGR1), for the modulation of IFN-γ signaling. Conjugating these siRNAs to docosanoic acid (DCA) enables productive delivery to all major skin cell types local to the injection site, with a single dose of injection supporting effective IFNGR1 protein reduction for at least 1 month in mice. In an ex vivo model of IFN-γ signaling, DCA-siRNA efficiently inhibits the induction of IFN-γ-inducible chemokines, CXCL9 and CXCL10, in skin biopsies from the injection site. Our data demonstrate that DCA-siRNAs can be engineered for functional gene silencing in skin and establish a path toward siRNA treatment of autoimmune skin diseases.

Innovative developments and emerging technologies in RNA therapeutics.

RNA-based therapeutics are emerging as a powerful platform for the treatment of multiple diseases. Currently, the two main categories of nucleic acid therapeutics, antisense oligonucleotides and small interfering RNAs (siRNAs), achieve their therapeutic effect through either gene silencing, splicing modulation or microRNA binding, giving rise to versatile options to target pathogenic gene expression patterns. Moreover, ongoing research seeks to expand the scope of RNA-based drugs to include more complex nucleic acid templates, such as messenger RNA, as exemplified by the first approved mRNA-based vaccine in 2020. The increasing number of approved sequences and ongoing clinical trials has attracted considerable interest in the chemical development of oligonucleotides and nucleic acids as drugs, especially since the FDA approval of the first siRNA drug in 2018. As a result, a variety of innovative approaches is emerging, highlighting the potential of RNA as one of the most prominent therapeutic tools in the drug design and development pipeline. This review seeks to provide a comprehensive summary of current efforts in academia and industry aimed at fully realizing the potential of RNA-based therapeutics. Towards this, we introduce established and emerging RNA-based technologies, with a focus on their potential as biosensors and therapeutics. We then describe their mechanisms of action and their application in different disease contexts, along with the strengths and limitations of each strategy. Since the nucleic acid toolbox is rapidly expanding, we also introduce RNA minimal architectures, RNA/protein cleavers and viral RNA as promising modalities for new therapeutics and discuss future directions for the field.

Gene therapy with AR isoform 2 rescues spinal and bulbar muscular atrophy phenotype by modulating AR transcriptional activity.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset neuromuscular condition caused by an abnormal polyglutamine (polyQ) tract expansion in androgen receptor (AR) protein. SBMA is a disease with high unmet clinical need. Recent studies have shown that mutant AR-altered transcriptional activity is key to disease pathogenesis. Restoring the transcriptional dysregulation without affecting other AR critical functions holds great promise for the treatment of SBMA and other AR-related conditions; however, how this targeted approach can be achieved and translated into a clinical application remains to be understood. Here, we characterized the role of AR isoform 2, a naturally occurring variant encoding a truncated AR lacking the polyQ-harboring domain, as a regulatory switch of AR genomic functions in androgen-responsive tissues. Delivery of this isoform using a recombinant adeno-associated virus vector type 9 resulted in amelioration of the disease phenotype in SBMA mice by restoring polyQ AR-dysregulated transcriptional activity.

Docosanoic acid conjugation to siRNA enables functional and safe delivery to skeletal and cardiac muscles.

Oligonucleotide therapeutics hold promise for the treatment of muscle- and heart-related diseases. However, oligonucleotide delivery across the continuous endothelium of muscle tissue is challenging. Here, we demonstrate that docosanoic acid (DCA) conjugation of small interfering RNAs (siRNAs) enables efficient (~5% of injected dose), sustainable (>1 month), and non-toxic (no cytokine induction at 100 mg/kg) gene silencing in both skeletal and cardiac muscles after systemic injection. When designed to target myostatin (muscle growth regulation gene), siRNAs induced ~55% silencing in various muscle tissues and 80% silencing in heart, translating into a ~50% increase in muscle volume within 1 week. Our study identifies compounds for RNAi-based modulation of gene expression in skeletal and cardiac muscles, paving the way for both functional genomics studies and therapeutic gene modulation in muscle and heart.

The chemical structure and phosphorothioate content of hydrophobically modified siRNAs impact extrahepatic distribution and efficacy.

Small interfering RNAs (siRNAs) have revolutionized the treatment of liver diseases. However, robust siRNA delivery to other tissues represents a major technological need. Conjugating lipids (e.g. docosanoic acid, DCA) to siRNA supports extrahepatic delivery, but tissue accumulation and gene silencing efficacy are lower than that achieved in liver by clinical-stage compounds. The chemical structure of conjugated siRNA may significantly impact invivo efficacy, particularly in tissues with lower compound accumulation. Here, we report the first systematic evaluation of the impact of siRNA scaffold-i.e. structure, phosphorothioate (PS) content, linker composition-on DCA-conjugated siRNA delivery and efficacy in vivo. We found that structural asymmetry (e.g. 5- or 2-nt overhang) has no impact on accumulation, but is a principal factor for enhancing activity in extrahepatic tissues. Similarly, linker chemistry (cleavable versus stable) altered activity, but not accumulation. In contrast, increasing PS content enhanced accumulation of asymmetric compounds, but negatively impacted efficacy. Our findings suggest that siRNA tissue accumulation does not fully define efficacy, and that the impact of siRNA chemical structure on activity is driven by intracellular re-distribution and endosomal escape. Fine-tuning siRNA chemical structure for optimal extrahepatic efficacy is a critical next step for the progression of therapeutic RNAi applications beyond liver.

Hydrophobically Modified let-7b miRNA Enhances Biodistribution to NSCLC and Downregulates HMGA2 In Vivo.

MicroRNAs (miRNAs) have increasingly been shown to be involved in human cancer, and interest has grown about the potential use of miRNAs for cancer therapy. miRNA levels are known to be altered in cancer cells, including in non-small cell lung cancer (NSCLC), a subtype of lung cancer that is the most prevalent form of cancer worldwide and that lacks effective therapies. The let-7 miRNA is involved in the regulation of oncogene expression in cells and directly represses cancer growth in the lung. let-7 is therefore a potential molecular target for tumor therapy. However, applications of RNA interference for cancer research have been limited by a lack of simple and efficient methods to deliver oligonucleotides (ONs) to cancer cells. In this study, we have used in vitro and in vivo approaches to show that HCC827 cells internalize hydrophobically modified let-7b miRNAs (hmiRNAs) added directly to the culture medium without the need for lipid formulation. We identified functional let-7b hmiRNAs targeting the HMGA2 mRNA, one of the let-7 target genes upregulated in NSCLC, and show that direct uptake in HCC827 cells induced potent and specific gene silencing in vitro and in vivo. Thus, hmiRNAs constitute a novel class of ONs that enable functional studies of genes involved in cancer biology and are potentially therapeutic molecules.

The valency of fatty acid conjugates impacts siRNA pharmacokinetics, distribution, and efficacy in vivo.

Lipid-conjugated small-interfering RNAs (siRNAs) exhibit accumulation and gene silencing in extrahepatic tissues, providing an opportunity to expand therapeutic siRNA utility beyond the liver. Chemically engineering lipids may further improve siRNA delivery and efficacy, but the relationship between lipid structure/configuration and siRNA pharmacodynamics is unclear. Here, we synthesized a panel of mono-, di-, and tri-meric fatty acid-conjugated siRNAs to systematically evaluate the impact of fatty acid structure and valency on siRNA clearance, distribution, and efficacy. Fatty acid valency significantly altered the physicochemical properties of conjugated siRNAs, including hydrophobicity and micelle formation, which affected distribution. Trivalent lipid-conjugated siRNAs were predominantly retained at the site of injection with minimal systemic exposure, whereas monovalent lipid-conjugated siRNAs were quickly released into the circulation and accumulated primarily in kidney. Divalent lipid-conjugated siRNAs showed intermediate behavior, and preferentially accumulated in liver with functional distribution to lung, heart, and fat. The chemical structure of the conjugate, rather than overall physicochemical properties (i.e. hydrophobicity), predicted the degree of extrahepatic tissue accumulation necessary for productive gene silencing. Our findings will inform chemical engineering strategies for enhancing the extrahepatic delivery of lipophilic siRNAs.

Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo.

Small interfering RNA (siRNA)-based therapies are proving to be efficient for treating liver-associated disorders. However, extra-hepatic delivery remains challenging, limiting therapeutic siRNA utility. We synthesized a panel of fifteen lipid-conjugated siRNAs and systematically evaluated the impact of conjugate on siRNA tissue distribution and efficacy. Generally, conjugate hydrophobicity defines the degree of clearance and the liver-to-kidney distribution profile. In addition to primary clearance tissues, several conjugates achieve significant siRNA accumulation in muscle, lung, heart, adrenal glands and fat. Oligonucleotide distribution to extra-hepatic tissues with some conjugates was significantly higher than with cholesterol, a well studied conjugate, suggesting that altering conjugate structure can enhance extra-hepatic delivery. These conjugated siRNAs enable functional gene silencing in lung, muscle, fat, heart and adrenal gland. Required levels for productive silencing vary (5-200 µg/g) per tissue, suggesting that the chemical nature of conjugates impacts tissue-dependent cellular/intracellular trafficking mechanisms. The collection of conjugated siRNA described here enables functional gene modulation in vivo in several extra-hepatic tissues opening these tissues for gene expression modulation. A systemic evaluation of a panel of conjugated siRNA, as reported here, has not previously been investigated and shows that chemical engineering of lipid siRNAs is essential to advance the RNA therapeutic field.

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways.

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.

Exploring the Emergence of RNA Nucleosides and Nucleotides on the Early Earth.

Understanding how life began is one of the most fascinating problems to solve. By approaching this enigma from a chemistry perspective, the goal is to define what series of chemical reactions could lead to the synthesis of nucleotides, amino acids, lipids, and other cellular components from simple feedstocks under prebiotically plausible conditions. It is well established that evolution of life involved RNA which plays central roles in both inheritance and catalysis. In this review, we present historically important and recently published articles aimed at understanding the emergence of RNA nucleosides and nucleotides on the early Earth.

Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.

Effective transvascular delivery of therapeutic oligonucleotides to the brain presents a major hurdle to the development of gene silencing technologies for treatment of genetically defined neurological disorders. Distribution to the brain after systemic administrations is hampered by the low permeability of the blood-brain barrier (BBB) and the rapid clearance kinetics of these drugs from the blood. Here we show that transient osmotic disruption of the BBB enables transvascular delivery of hydrophobically modified small interfering RNA (hsiRNA) to the rat brain. Intracarotid administration of 25% mannitol and hsiRNA conjugated to phosphocholine-docosahexanoic acid (PC-DHA) resulted in broad ipsilateral distribution of PC-DHA-hsiRNAs in the brain. PC-DHA conjugation enables hsiRNA retention in the parenchyma proximal to the brain vasculature and enabled active internalization by neurons and astrocytes. Moreover, transvascular delivery of PC-DHA-hsiRNAs effected Htt mRNA silencing in the striatum (55%), hippocampus (51%), somatosensory cortex (52%), motor cortex (37%), and thalamus (33%) 1 week after administration. Aside from mild gliosis induced by osmotic disruption of the BBB, transvascular delivery of PC-DHA-hsiRNAs was not associated with neurotoxicity. Together, these findings provide proof-of-concept that temporary disruption of the BBB is an effective strategy for the delivery of therapeutic oligonucleotides to the brain.

Optimized Cholesterol-siRNA Chemistry Improves Productive Loading onto Extracellular Vesicles.

Extracellular vesicles are promising delivery vesicles for therapeutic RNAs. Small interfering RNA (siRNA) conjugation to cholesterol enables efficient and reproducible loading of extracellular vesicles with the therapeutic cargo. siRNAs are typically chemically modified to fit an application. However, siRNA chemical modification pattern has not been specifically optimized for extracellular vesicle-mediated delivery. Here we used cholesterol-conjugated, hydrophobically modified asymmetric siRNAs (hsiRNAs) to evaluate the effect of backbone, 5'-phosphate, and linker chemical modifications on productive hsiRNA loading onto extracellular vesicles. hsiRNAs with a combination of 5'-(E)-vinylphosphonate and alternating 2'-fluoro and 2'-O-methyl backbone modifications outperformed previously used partially modified siRNAs in extracellular vesicle-mediated Huntingtin silencing in neurons. Between two commercially available linkers (triethyl glycol [TEG] and 2-aminobutyl-1-3-propanediol [C7]) widely used to attach cholesterol to siRNAs, TEG is preferred compared to C7 for productive exosomal loading. Destabilization of the linker completely abolished silencing activity of loaded extracellular vesicles. The loading of cholesterol-conjugated siRNAs was saturated at ∼3,000 siRNA copies per extracellular vesicle. Overloading impaired the silencing activity of extracellular vesicles. The data reported here provide an optimization scheme for the successful use of hydrophobic modification as a strategy for productive loading of RNA cargo onto extracellular vesicles.