Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

Evaluation of GalNAc-siRNA Conjugate Activity in Pre-clinical Animal Models with Reduced Asialoglycoprotein Receptor Expression.

The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced.

Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.

Clinical Proof of Concept for a Novel Hepatocyte-Targeting GalNAc-siRNA Conjugate.

Advancement of RNAi-based therapeutics depends on effective delivery to the site of protein synthesis. Although intravenously administered, multi-component delivery vehicles have enabled small interfering RNA (siRNA) delivery and progression into clinical development, advances of single-component, systemic siRNA delivery have been challenging. In pre-clinical models, attachment of a triantennary N-acetylgalactosamine (GalNAc) ligand to an siRNA mediates hepatocyte uptake via the asialoglycoprotein receptor enabling RNAi-mediated gene silencing. In this phase 1 study, we assessed translation of this delivery approach by evaluating the safety, tolerability, pharmacokinetics, and pharmacodynamics of a GalNAc-siRNA conjugate, revusiran, targeting transthyretin (TTR). Subjects received a placebo or ascending doses of revusiran subcutaneously ranging from 1.25-10 mg/kg in the single and 2.5-10 mg/kg in the multiple ascending dose phases. Revusiran was generally well tolerated, with transient, mild to moderate injection site reactions the most common treatment-emergent adverse events. Doses of 2.5-10 mg/kg revusiran elicited a significant reduction of serum TTR versus the placebo (p < 0.01), with mean TTR reductions of approximately 90% observed with multiple dosing. These results demonstrate translation of this novel delivery platform, enabling clinical development of subcutaneously administered GalNAc-siRNAs for liver-based diseases.

Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

RNAi-mediated gene silencing in non-human primates.

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.

Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5'-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.

Mechanisms and optimization of in vivo delivery of lipophilic siRNAs.

Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery.

Ablation of Hepatic Production of the Acid-Labile Subunit in Bovine-GH Transgenic Mice: Effects on Organ and Skeletal Growth.

Growth hormone (GH) and insulinlike growth factor 1 (IGF-1) are anabolic hormones that facilitate somatic and skeletal growth and regulate metabolism via endocrine and autocrine/paracrine mechanisms. We hypothesized that excess tissue production of GH would protect skeletal growth and integrity in states of reduction in serum IGF-1 levels. To test our hypothesis, we used bovine GH (bGH) transgenic mice as a model of GH hypersecretion and ablated the liver-derived acid-labile subunit, which stabilizes IGF-1 complexes with IGF-binding protein-3 and -5 in circulation. We used a genetic approach to create bGH/als gene knockout (ALSKO) mice and small interfering RNA (siRNA) gene-silencing approach to reduce als or igf-1 gene expression. We found that in both models, decreased IGF-1 levels in serum were associated with decreased body and skeletal size of the bGH mice. Excess GH produced more robust bones but compromised mechanical properties in male mice. Excess GH production in tissues did not protect from trabecular bone loss in response to reductions in serum IGF-1 (in bGH/ALSKO or bGH mice treated with siRNAs). Reduced serum IGF-1 levels in the bGH mice did not alleviate the hyperinsulinemia and did not resolve liver or kidney pathologies that resulted from GH hypersecretion. We concluded that reduced serum IGF-1 levels decrease somatic and skeletal growth even in states of excess GH.

Preclinical evaluation of RNAi as a treatment for transthyretin-mediated amyloidosis.

ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis.

Optimized in vivo transfer of small interfering RNA targeting dermal tissue using in vivo surface electroporation.

Electroporation (EP) of mammalian tissue is a technique that has been used successfully in the clinic for the delivery of genetic-based vaccines in the form of DNA plasmids. There is great interest in platforms which efficiently deliver RNA molecules such as messenger RNA and small interfering RNA (siRNA) to mammalian tissue. However, the in vivo delivery of RNA enhanced by EP has not been extensively characterized. This paper details the optimization of electrical parameters for a novel low-voltage EP method to deliver oligonucleotides (both DNA and RNA) to dermal tissue in vivo. Initially, the electrical parameters were optimized for dermal delivery of plasmid DNA encoding green fluorescent protein (GFP) using this novel surface dermal EP device. While all investigated parameters resulted in visible transfection, voltage parameters in the 10 V range elicited the most robust signal. The parameters optimized for DNA, were then assessed for translation of successful electrotransfer of siRNA into dermal tissue. Robust tagged-siRNA transfection in skin was detected. We then assessed whether these parameters translated to successful transfer of siRNA resulting in gene knockdown in vivo. Using a reporter gene construct encoding GFP and tagged siRNA targeting the GFP message, we show simultaneous transfection of the siRNA to the skin via EP and the concomitant knockdown of the reporter gene signal. The siRNA delivery was accomplished with no evidence of injection site inflammation or local tissue damage. The minimally invasive low-voltage EP method is thus capable of efficiently delivering both DNA and RNA molecules to dermal tissue in a tolerable manner.

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.