Nucleic acid drug delivery with lipid nanoparticle (LNP) formulations has enabled the development of novel therapeutics and vaccines. LNP formulations are composed of both naturally occurring and synthetic lipid excipients. This perspective shares current practices in the nonclinical safety assessment of novel lipid excipients contained in LNP formulations and identifies gaps in current regulatory guidance on this topic. There is no globally harmonized regulatory guidance for the nonclinical safety assessment of novel excipients or guidance specific to safety testing of novel excipients in LNPs. Given the complexity of these LNP formulations, most nonclinical safety studies to support development are conducted with the drug product or with a LNP that contains non-active cargo. Three case studies (Onpattro®, Comirnaty®, and SpikeVax®) highlight that specific assessments may differ depending on the encapsulated modality, the intended use (e.g., therapeutic versus preventative vaccine), dose, and frequency of dosing. These case studies also suggest that regulatory agencies are open to scientific rationale to justify why certain tests should or should not be performed. As more products are approved, it will be important to understand how precedents set for approved products can be leveraged and what additional unique strategies may be applied to ensure nonclinical safety assessments are predictive, relevant, and meaningful for human safety. Proactive alignment with regulatory authorities will be critical in this context, especially as new approaches are proposed. Guidance documents may need to be revised or created as more experience is acquired to reflect the unique considerations for these novel excipients.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2′- O -hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer’s disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

RNA interference (RNAi) therapeutics are a new class of medicines that can address unmet medical needs by silencing disease-causing gene transcripts. While delivery of short interfering RNAs (siRNAs) to hepatocytes has yielded multiple drug approvals, novel delivery solutions are needed to expand the reach of RNAi therapeutics. Here we report that conjugation of 2'- O -hexadecyl (C16) to siRNAs enables efficient silencing in the central nervous system (CNS), eye, and lung of multiple nonclinical species with broad cell type specificity. Intrathecally delivered C16-siRNAs are active across CNS regions and cell types, with sustained silencing for at least three months, which is an especially important outcome considering the challenging dosing route. Similarly, intravitreal and intranasal administration of C16-siRNAs resulted in potent and sustained knockdown in the eye and lung, respectively. Efficient delivery facilitated through C16 conjugation to optimized siRNA designs has enabled candidate selection for investigational human clinical trials assessing therapeutic silencing beyond the liver with infrequent (e.g. bi-annual) dosing.

Xenobiotic-induced peripheral nerve damage is a growing concern. Identifying relative risks that a new drug may cause peripheral nerve injury over long periods of administration is gathering importance in the evaluation of animal models. Separating out age-related changes in peripheral nerves of rats caused by compression injury from drug-induced effects has been difficult. Biopsy of the sural nerve is utilized in humans for investigations of peripheral neuropathy, because it is largely removed from the effects of nerve compression. This study used transmission electron microscopy to identify incidental findings in the sural nerves and dorsal root ganglia of aged control rats over time. The goal was to establish a baseline understanding of the range of possible changes that could be noted in controls compared to rats treated with any new investigative drug. In this evaluation, most sural nerve fibers from aged control rats had few ultrastructural abnormalities of pathologic significance. However, glycogenosomes, polyglucosan bodies, swollen mitochondria, autolysosomes, split myelin, Schwann cell processes, and endoneural macrophages with phagocytosed debris (considered an indication of ongoing degenerative changes) were occasionally noted.

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 approximate to 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.

Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-acetylgalactosamine ligand to facilitate delivery to hepatocytes via uptake by the asialoglycoprotein receptors. Revusiran, in development for the treatment of hereditary transthyretin-mediated amyloidosis, was discontinued after an imbalance in deaths in the "ENDEAVOUR" phase 3 clinical trial. Nonclinical safety assessments included safety pharmacology, acute and repeat-dose toxicity, genotoxicity, and carcinogenicity. There were no effects on cardiovascular or respiratory function in monkeys after single doses of up to 100 mg/kg. No neurological effects were noted in monkeys in repeat-dose studies up to 300 mg/kg. Revusiran was well tolerated in repeat-dose mouse (weekly doses) and rat and monkey (five daily doses followed by weekly doses) toxicity studies. The no observed adverse effect level (NOAEL) in rats was 30 mg/kg based on reversible microscopic changes in liver that were accompanied by correlating elevations in clinical chemistry at higher doses. Dose-limiting toxicity was absent in monkeys, and the NOAEL was 200 mg/kg. There was no evidence of genotoxicity in vitro or in vivo at limit doses or carcinogenicity in a 2-year study in rats at doses up to 100 mg/kg. Overall, these results demonstrate that revusiran had a favorable nonclinical safety profile.

Short interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) are the most clinically advanced oligonucleotide-based platforms. A number of N-acetylgalactosamine (GalNAc)-conjugated siRNAs (GalNAc-siRNAs), also referred to as RNA interference (RNAi) therapeutics, are currently in various stages of development, though none is yet approved. While the safety of ASOs has been the subject of extensive review, the nonclinical safety profiles of GalNAc-siRNAs have not been reported. With the exception of sequence differences that confer target RNA specificity, GalNAc-siRNAs are largely chemically uniform, containing limited number of phosphorothioate linkages, and 2'-O-methyl and 2'-deoxy-2'-fluoro ribose modifications. Here, we present the outcomes of short-term (3-5 week) rat and monkey weekly repeat-dose toxicology studies of six Enhanced Stabilization Chemistry GalNAc-siRNAs currently in clinical development. In nonclinical studies at supratherapeutic doses, these molecules share similar safety signals, with histologic findings in the organ of pharmacodynamic effect (liver), the organ of elimination (kidney), and the reticuloendothelial system (lymph nodes). The majority of these changes are nonadverse, partially to completely reversible, correlate well with pharmacokinetic parameters and tissue distribution, and often reflect drug accumulation. Furthermore, all GalNAc-siRNAs tested to date have been negative in genotoxicity and safety pharmacology studies.

One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

Aim: There remains high unmet medical need for therapies to treat cardio/metabolic diseases. We validated in human trials, a platform for reducing the synthesis of genes expressed in the liver. The platform utilizes a GalNAc ligand attached to the 3’ end of the sense strand of an RNAi molecule to enable delivery specifically to the liver. Here we extend the platform to targets of interest in cardiovascular disease, including PCSK9, ANGPLT3 and ApoC3. METHODS: Chemically modified siRNAs were designed and were screened for potency in vitro . pM active siRNA molecules were developed targeting PCSK9, ANGPLT3 and ApoC3. The siRNAs were tested in either rodents or in non-human primates (NHPs) for activity. RESULTS: In NHPs a single dose of ALN-PCSsc at 6 mg/kg reduced PCSK9 levels up to 97% and LDL-C up to 67%. Moreover the nadir effect (without any rebound of LDL-C) lasted >30 days indicating that once a month or longer dosing frequency in clinic should be supported. Multidose studies in NHP at 2mg/kg reduced circulating PCSK9 levels up to 94% with a subsequent lowering of LDL-C up to 67%. The effects on both PCSK9 and LDL-C was also very durable with levels of LDL-C returning to baseline > 140 days post the last dose. Safety studies in rat at doses up to 225mg/kg (multi-dose) indicate that ALN-PCSsc is safe demonstrating a very wide therapeutic index. ALN-PCSsc was selected as a development candidate and is being advanced towards an IND ALN-ANGsc (an siRNA targeting ANGPTL3) was tested in two models of hyperlipidemia, the OB/OB mouse and the hCETP-ApoB mouse. In the Ob/Ob model, treatment with ALN-ANGsc at 3mg/kg resulted in a significant lowering ANGPLT3 protein (>95%), total cholesterol(>60%), and triglycerides (>85%). Finally, we have developed a prototype molecule targeting Apoc3 with an ED90 for ApoC3 protein of <2.5mg/kg showing 50% lowering of triglycerides in and db/db mouse model of hypertriglyceridemia. CONCLUSION: We have developed a modular, robust and durable platform for the delivery of RNAi therapeutics to the liver. This platform is administered as a small volume subcutaneous dose and has a remarkable duration of effect in rodent NHP models. We have extended this platform to several targets of high interest includingPCSK9,ANGPLT3 and ApoC3.

Introduction: Cardiovascular disease remains the top cause of mortality in the United States. We have developed, and validated in human trials, a platform for reducing the synthesis of genes expressed in the liver. The platform utilizes a GalNAc sugar ligand attached to the 3’end of the sense strand of an RNAi molecule to enable delivery specifically to the liver. Here we present data from this platform on multiple targets of interest in cardiovascular disease, including PCSK9, ANGPLT3 and ApoC3. Hypothesis: RNAi therapeutics targeting liver genes of interest (such as PCSK9, ApoC3 and Angptl3) will allow for control of lipid levels and reduce the risk of cardiovascular disease. Methods: Chemically modified siRNAs were designed using bioinformatic algorithms and were screened for potency in vitro . pM active siRNA molecules were developed targeting PCSK9, ANGPLT3 and ApoC3. Results: ALN-PCSsc: In NHPs a single dose of ALN-PCSsc at ≥ 6 mg/kg reduced circulating PCSK9 levels up to 97% and LDL-C up to 67%. Moreover the nadir effect ( where LDL-C is clamped) lasted ~90 days suggesting that once monthly to quarterly dosing could be supported. Multidose studies in NHP at 2 mg/kg reduced circulating PCSK9 levels up to 94% with a subsequent lowering of LDL-C up to 67%. Safety studies in rat and NHP at doses up to 300 mg/kg (multi-dose) showed that ALN-PCSsc was safe demonstrating potential for a very wide therapeutic index. ALN-PCSsc was selected as a development candidate and is being advanced towards an IND for the treatment of hypercholesterolemia. ALN-ANGsc: Was tested in two models of hyperlipidemia, the OB/OB mouse and the hCETP-ApoB mouse. In the Ob/Ob model, treatment with ALN-ANGsc at 3mg/kg resulted in a significant lowering ANGPLT3 protein (>95%), cholesterol (>60%) and triglycerides (>85%). ALN-ApoC3: Was tested in the db/db mouse model of hyperlipidemia. A robust lowering of plasma ApoC3 (>90%) lead to a >50% lowering of triglycerides. Screening for both programs is underway for a development candidates. Conclusions: We have developed a modular, robust and reliable platform for the delivery of RNAi therapeutics to the liver with a large therapeutic index. This small volume, subcutaneous, platform has a remarkable duration of effect.

Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9 result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment.Methods We did a randomised, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL cholesterol of 3.00 mmol/L or higher. Participants were randomly assigned in a 3: 1 ratio by computer algorithm to receive one dose of intravenous ALN-PCS (with doses ranging from 0.015 to 0.400 mg/kg) or placebo. The primary endpoint was safety and tolerability of ALN-PCS. Secondary endpoints were the pharmacokinetic characteristics of ALN-PCS and its pharmacodynamic effects on PCSK9 and LDL cholesterol. Study participants were masked to treatment assignment. Analysis was per protocol and we used ANCOVA to analyse pharmacodynamic endpoint data. This trial is registered with ClinicalTrials.gov, number NCT01437059.Findings Of 32 participants, 24 were randomly allocated to receive a single dose of ALN-PCS (0.015 mg/kg [n=3], 0.045 mg/kg [n=3], 0.090 mg/kg [n=3], 0.150 mg/kg [n=3], 0.250 mg/kg [n=6], or 0.400 mg/kg [n=6]) and eight to placebo. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (19 [79%] vs seven [88%]). ALN-PCS was rapidly distributed, with peak concentration and area under the curve (0 to last measurement) increasing in a roughly dose-proportional way across the dose range tested. In the group given 0.400 mg/kg of ALN-PCS, treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein (p<0.0001) and a mean 40% reduction in LDL cholesterol from baseline relative to placebo (p<0.0001).Interpretation Our results suggest that inhibition of PCSK9 synthesis by RNA interference (RNAi) provides a potentially safe mechanism to reduce LDL cholesterol concentration in healthy individuals with raised cholesterol. These results support the further assessment of ALN-PCS in patients with hypercholesterolaemia, including those being treated with statins. This study is the first to show an RNAi drug being used to affect a clinically validated endpoint (ie, LDL cholesterol) in human beings.

Proprotein convertases subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase (PC) family of subtilisin-like serine endoproteases that regulates low density lipoprotein receptor (LDLR) levels and function. Murine models and human genetic studies indicate that loss of PCSK9 protein increases LDLR levels while excess PCSK9 decreases LDLR levels. These changes in LDLR protein levels coincide with reciprocal changes in circulating levels of plasma LDL cholesterol (LDL-C). We have developed a highly potent RNA interference (RNAi) therapeutic, ALN-PCS, targeting both intra and extracellular PCSK9 for inhibition through an RNAi mechanism. Pre-clinical data in non-human primate models, indicate that a single intravenous dose of ALN-PCS results in rapid, dose dependent, and significant lowering of liver PCSK9 transcript, plasma PCSK9 protein and subsequently serum LDL-C and ApoB levels, without impacting serum HDL-C. Here we report on interim data from an ongoing Phase 1 trial of ALN-PCS being conducted as a randomized, single-blind, placebo-controlled, single-ascending dose study in healthy volunteer subjects with elevated baseline LDL-C (>116mg/dL) who are not on any lipid lowering therapy. The primary objective of the study is to evaluate the safety and tolerability of a single dose of ALN-PCS, with subjects being enrolled into sequential cohorts of increasing doses. Secondary objectives of the study include characterization of plasma and urine pharmacokinetics of ALN-PCS, assessment of pharmacodynamic effects of the drug on plasma PCSK9 protein levels, and evaluation of clinical efficacy as measured by LDL-C levels. Data from 20 subjects enrolled in five sequential dose cohorts ranging from 0.015 to 0.250 mg/kg in a 3:1 randomization of drug to placebo will be presented. ALN-PCS was safe and well tolerated in this study and there have been no serious adverse events related to study drug administration to date. There have been no drug-related discontinuations from the study and no liver enzyme elevations. A mild, transient rash that resolved spontaneously was observed in three subjects that received ALN-PCS, and in two that received placebo. To date, administration of ALN-PCS resulted in a rapid, dose-dependent, and durable silencing of PCSK9 protein levels in plasma of up to 66% relative to baseline, with a statistically significant mean reduction of 60% at day four in the current high dose group of 0.250 mg/kg (p<0.001). In addition, administration of ALN-PCS resulted in dose-dependent reductions in LDL-C of up to 50% relative to baseline, with a statistically significant mean reduction of 39% at day four (p<0.05) at the 0.250 mg/kg dose level. There was no significant decrease in high-density lipoprotein (HDL). Dosing of further cohorts at the current top dose (0.25 mg/kg) and at a higher dose is planned. Additional data will be presented as available.