Complement C3d/C4d Deposition on Platelets Correlates with 2'-O-Methoxyethyl Antisense Oligonucleotide-Induced Thrombocytopenia in Monkeys.

2'-O-Methoxyethyl antisense oligonucleotide (2'-MOE ASO)-induced severe thrombocytopenia (TCP) [platelet (PLT) count <50 K/μL] was observed in the Asian-sourced cynomolgus monkeys with low incidence (2%-4% at doses >5 mg/kg/week). The potential mechanisms for TCP were studied using the Mauritian-sourced cynomolgus monkeys, which were shown to be more susceptible to ASO-induced TCP, along with the Asian-sourced animals. ISIS 405879, a 2'-MOE ASO, induced severe TCP (PLT <50 K/μL) in seven of nine Mauritian-sourced monkeys but not in the Asian-sourced monkeys after 16 weeks of treatment at 40 mg/kg/week.

Correlations between preclinical BJAB assay ranking of antisense drugs and clinical trial adverse events.

This analysis sought to assess the clinical predictivity of an in vitro assay which utilized the human B-lymphoma BJAB cell line, for identification of antisense oligonucleotides (ASOs) with the potential to elicit innate immune activation in humans. Adverse events (AEs) from clinical trial data were analyzed based on prior clinical knowledge and network analysis of the clinical data to identify correlations with the BJAB assay. Clinically evaluated ASOs were ranked by the BJAB assay's mean log-fold increase in TNF expression levels.

Inflammatory Non-CpG Antisense Oligonucleotides Are Signaling Through TLR9 in Human Burkitt Lymphoma B Bjab Cells.

Nucleic acid-based phosphorothioate containing antisense oligonucleotides (PS-ASOs) have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing the degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed human peripheral blood mononuclear cell (hPBMC)-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource intensive and easily affected by subject variability.

Assessment of the Immunogenicity Potential for Oligonucleotide-Based Drugs.

Therapeutic oligonucleotides (ONs) have characteristics of both small molecules and biologics. Although safety assessment of ONs largely follows guidelines established for small molecules, the unique characteristics of ONs often require incorporation of concepts from the safety assessment of biologics. The assessment of immunogenicity for ON therapeutics is one area where the approach is distinct from either established small molecule or biologic platforms.

Early-Stage Identification and Avoidance of Antisense Oligonucleotides Causing Species-Specific Inflammatory Responses in Human Volunteer Peripheral Blood Mononuclear Cells.

A human peripheral blood mononuclear cell (PBMC)-based assay was developed to identify antisense oligonucleotide (ASO) with the potential to activate a cellular innate immune response outside of an acceptable level. The development of this assay was initiated when ISIS 353512 targeting the messenger ribonucleic acid for human C-reactive protein (CRP) was tested in a phase I clinical trial, in which healthy human volunteers unexpectedly experienced increases in interleukin-6 (IL-6) and CRP. This level of immune stimulation was not anticipated following rodent and nonhuman primate safety studies in which no evidence of exaggerated proinflammatory effects were observed.

Antisense Oligonucleotide-Related Macrovesicular Vacuolation of Hippocampal Neurons in Nonhuman Primates.

2'-methoxyethyl (MOE) antisense oligonucleotides (ASOs) tested in multidose intrathecal nonhuman primate (NHP) toxicity studies have consistently revealed the presence of single large vacuoles in pyramidal neurons of the hippocampus in the absence of any cellular response. Termed "macrovesicular," these vacuoles were characterized by immunohistochemistry and transmission electron microscopy which showed that these vacuoles are dilated lysosomes in neurons containing accumulated ASO. Additionally, two NHP studies were conducted to investigate the role of tissue fixation on their histogenesis.

Interspecies Scaling of Human Clearance and Plasma Trough Exposure for Antisense Oligonucleotides: A Retrospective Analysis of GalNAc3-Conjugated and Unconjugated-Antisense Oligonucleotides.

It is well documented and generally accepted that human clearance (CL) of unconjugated single-strand antisense oligonucleotides (ASOs) can be directly predicted from monkeys by body weight (BW) on a mg/kg dose basis. However, the scaling for triantennary -acetyl galactosamine (GalNAc)-conjugated ASOs has not been fully established. In this study, we retrospectively analyzed pharmacokinetic data from 9 GalNAc-conjugated and 12 unconjugated single-stranded ASOs (ten 2'-methoxyethyl and two 2', 4'-constrained ethyl ASOs) to identify an appropriate allometric scaling factor between the two species.

Sequence-specific 2'-O-methoxyethyl antisense oligonucleotides activate human platelets through glycoprotein VI, triggering formation of platelet-leukocyte aggregates.

Antisense oligonucleotides (ASO) are DNA-based, disease-modifying drugs. Clinical trials with 2'-O-methoxyethyl (2'MOE) ASO have shown dose- and sequence-specific lowering of platelet counts according to two phenotypes. Phenotype 1 is a moderate (but not clinically severe) drop in platelet count.

Safety, Pharmacokinetic, and Pharmacodynamic Evaluation of a 2'-(2-Methoxyethyl)-D-ribose Antisense Oligonucleotide-Triantenarry -Acetyl-galactosamine Conjugate that Targets the Human Transmembrane Protease Serine 6.

Cellular uptake of antisense oligonucleotides (ASOs) is one of the main determinants of in vivo activity and potency. A significant advancement in improving uptake into cells has come through the conjugation of ASOs to triantenarry -acetyl-galactosamine (GalNAc), a ligand for the asialoglycoprotein receptor on hepatocytes. The impact for antisense oligonucleotides, which are already taken up into hepatocytes, is a 10-fold improvement in potency in mice and up to a 30-fold potency improvement in humans, resulting in overall lower effective dose and exposure levels.

Immunogenicity Assessment of Inotersen, a 2'--(2-Methoxyethyl) Antisense Oligonucleotide in Animals and Humans: Effect on Pharmacokinetics, Pharmacodynamics, and Safety.

Inotersen (TEGSEDI™) is a 2'--(2-methoxyethyl)-modified antisense oligonucleotide, intended for treating hereditary transthyretin (TTR) amyloidosis with polyneuropathy. The potential immunogenicity (IM) response to inotersen was evaluated in chronic nonclinical safety studies and the pivotal phase 2/3 clinical study. The evaluation was designed to assess the characteristics of antidrug antibodies (ADAs) and their effects on the pharmacokinetics, pharmacodynamics, clinical efficacy, and safety in animals and humans.

Population Pharmacokinetic-Pharmacodynamic Modeling of Inotersen, an Antisense Oligonucleotide for Treatment of Patients with Hereditary Transthyretin Amyloidosis.

A population pharmacokinetic (PK) and pharmacodynamic (PD) model was developed for inotersen to evaluate exposure-response relationships and to optimize therapeutic dosing regimen in patients with hereditary transthyretin (TTR) amyloidosis polyneuropathy (hATTR-PN). Inotersen PK and TTR level (PD) data were composed of one Phase 1 study in healthy subjects, one Phase 2/3 study in hATTR patients, and its one open-label extension study. Effects of intrinsic and extrinsic factors (covariates) on PK and PK/PD of inotersen were evaluated using a full model approach.

Underlying Immune Disorder May Predispose Some Transthyretin Amyloidosis Subjects to Inotersen-Mediated Thrombocytopenia.

Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 10/L in 50% and ≥100 × 10/L in 80% of the subjects.

Impurity Qualification Toxicology Study for a 2'-O-Methoxyethyl-Modified Antisense Inhibitor in Mice.

Safety assessment of drug impurities is a routine part of the drug development process. For oligonucleotide-based drugs, impurities can arise from impurities in starting materials, as by-products of the manufacturing process or from degradation, and are generally structurally similar to the parent oligonucleotide. To study the potential impact of impurities, a representative batch of a 2'-O-methoxyethyl (MOE) antisense oligonucleotide (ASO) was compared to batches of drug that were enriched with nine of the common impurities encountered with the chemical class.

Comparison of the Class Effects of Antisense Oligonucleotides in CByB6F1-Tg(HRAS)2Jic and CD-1 Mice.

The 6-month Tg.rasH2 mouse carcinogenicity model provides an acceptable alternative to the 2-year carcinogenicity study in CD-1 mice. However, key questions related to the use of this model for testing antisense oligonucleotides (ASOs) include the similarity in the biologic response between mouse strains and the feasibility of using data from the CD-1 mouse to set doses and dose schedules for a Tg.

Investigation into the Mechanism(s) That Leads to Platelet Decreases in Cynomolgus Monkeys During Administration of ISIS 104838, a 2'-MOE-Modified Antisense Oligonucleotide.

ISIS 104838, a 2'-O-methoxyethyl (2'-MOE)-modified antisense oligonucleotide (ASO), causes a moderate, reproducible, dose-dependent, but selflimiting decrease in platelet (PLT) counts in monkeys and humans. To determine the etiology of PLT decrease in cynomolgus monkeys, a 12-week repeat dose toxicology study in 5 cynomolgus monkeys given subcutaneous injections of ISIS 104838 (30-60 mg/kg/week). Monkeys were also injected intravenously with 111Indium(In)-oxine-labeled PLTs to investigate PLT sequestration.

Chronic Toxicity Assessment of 2'-O-Methoxyethyl Antisense Oligonucleotides in Mice.

Advances in antisense oligonucleotide (ASO) chemistry and screening have enabled the design and selection of molecules that are optimized for a particular therapeutic application in terms of both potency and tolerability. The most-well studied of the chemically modified ASOs are single-stranded antisense inhibitors with phosphorothioate backbones and 2'-O-methoxyethyl modifications (2'-MOE ASO). The 2'-MOE chemical modification in the design of the ASO has conferred increased hybridization affinity, increased stability, and/or enhanced tissue residence time, resulting in better potency and pharmacokinetics.

2'-O-(2-Methoxyethyl) Nucleosides Are Not Phosphorylated or Incorporated Into the Genome of Human Lymphoblastoid TK6 Cells.

Nucleoside analogs with 2'-modified sugar moieties are often used to improve the RNA target affinity and nuclease resistance of therapeutic oligonucleotides in preclinical and clinical development. Despite their enhanced nuclease resistance, oligonucleotides could slowly degrade releasing nucleoside analogs that have the potential to become phosphorylated and incorporated into cellular DNA and RNA. For the first time, the phosphorylation and DNA/RNA incorporation of 2'-O-(2-methoxyethyl) (2'-O-MOE) nucleoside analogs have been investigated.

Assessment of the Drug Interaction Potential of Unconjugated and GalNAc-Conjugated 2'-MOE-ASOs.

Antisense oligonucleotides are metabolized by nucleases and drug interactions with small drug molecules at either the cytochrome P450 (CYP) enzyme or transporter levels have not been observed to date. Herein, a comprehensive in vitro assessment of the drug-drug interaction (DDI) potential was carried out with four 2'-O-(2-methoxyethyl)-modified antisense oligonucleotides (2'-MOE-ASOs), including a single triantennary N-acetyl galactosamine (GalNAc)-conjugated ASO. Several investigations to describe the DDI potential of a 2'-MOE-ASO conjugated to a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors are explored.

Reduction in ocular complement factor B protein in mice and monkeys by systemic administration of factor B antisense oligonucleotide.

Purpose: Age-related macular degeneration (AMD) is the leading cause of permanent vision loss among the elderly in many industrialized countries, and the complement system plays an important role in the pathogenesis of AMD. Inhibition of complement factor B, a key regulator of the alternative pathway, is implicated as a potential therapeutic intervention for AMD. Here we investigated the effect of liver factor B reduction on systemic and ocular factor B levels.

Lack of QT Prolongation for 2'-O-Methoxyethyl-Modified Antisense Oligonucleotides Based on Retrospective Exposure/Response Analysis of Ten Phase 1 Dose-Escalation Placebo-Controlled Studies in Healthy Subjects.

The potential of QT prolongation of ten 2'-O-methoxyethyl-modified (2'-MOE) antisense oligonucleotides (ASOs) was evaluated retrospectively via exposure/response (ER) analysis using data from Phase 1 clinical studies in healthy subjects. All Phase 1 studies were double-blind, placebo-controlled, single and multiple ascending dose studies designed to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of the ASOs in healthy subjects. The active doses in these studies ranged from 50 to 450 mg administered by subcutaneous (SC) injection in single and multiple ascending dose cohorts.

Assessment of the Effects of 2'-Methoxyethyl Antisense Oligonucleotides on Platelet Count in Cynomolgus Nonhuman Primates.

Decreases in platelet (PLT) counts observed in nonhuman primates (NHPs) given 2'-O-methoxyethyl modified antisense inhibitors (2'-MOE ASOs) have been reported, but the incidence and severity of the change vary considerably between sequences, studies, and animals. This article will broadly illustrate the spectrum of effects on PLT count in NHPs. From queries of an NHP safety database representing over 102 independent 2'-MOE ASOs, from 61 studies and >2200 NHPs, two patterns of PLT changes emerged.

Disposition and Pharmacokinetics of a GalNAc3-Conjugated Antisense Oligonucleotide Targeting Human Lipoprotein (a) in Monkeys.

Triantennary N-acetyl galactosamine (GalNAc)-conjugated antisense oligonucleotides (ASOs) have greatly improved potency due to receptor-mediated uptake into hepatocyte. The disposition and pharmacokinetics of ISIS 681257, a GalNAc-conjugated ASO, were studied in monkeys. Following subcutaneous (SC) injection, ISIS 681257 was rapidly absorbed into the systemic circulation, with peak plasma levels observed within hours after dosing.

Integrated Safety Assessment of 2'-O-Methoxyethyl Chimeric Antisense Oligonucleotides in NonHuman Primates and Healthy Human Volunteers.

The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2'-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2'-O-methoxyethyl chimeric antisense oligonucleotides.

Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys.

Triantennary N-acetyl galactosamine (GalNAc3) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey.