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The triantennary N -acetylgalactosamine (GalNAc 3 ) cluster has demonstrated the utility of receptor-mediated uptake of ligand-conjugated antisense drugs targeting RNA expressed by hepatocytes. GalNAc 3 -conjugated 2'- O -methoxyethyl (2'MOE) modified antisense oligonucleotides (ASOs) have demonstrated a higher potency than the unconjugated form to support lower doses for an equivalent pharmacological effect. We utilized the Ionis integrated safety database to compare four GalNAc 3 -conjugated and four same-sequence unconjugated 2'MOE ASOs. This assessment evaluated data from eight randomized placebo-controlled dose-ranging phase 1 studies involving 195 healthy volunteers (79 GalNAc 3 ASO, 24 placebo; 71 ASO, 21 placebo). No safety signals were identified by the incidence of abnormal threshold values in clinical laboratory tests for either ASO group. However, there was a significant increase in mean alanine transaminase levels compared with placebo in the upper dose range of the unconjugated 2'MOE ASO group. The mean percentage of subcutaneous injections leading to local cutaneous reaction was 30-fold lower in the GalNAc 3 -conjugated ASO group compared with the unconjugated ASO group (0.9% vs. 28.6%), with no incidence of flu-like reactions (0.0% vs. 0.7%). Three subjects (4.2%) in the unconjugated ASO group discontinued dosing. An improvement in the overall safety and tolerability profile of GalNAc 3 -conjugated 2'MOE ASOs is evident in this comparison of short-term clinical data in healthy volunteers.

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. Flu-like reactions (FLRs) and injection site reactions (ISRs), were chosen as AEs of interest, along with those Medical Dictionary for Regulatory Activities preferred terms identified using AE network analysis. Fifteen different 2'-O-methoxyethyl (2'MOE) modified ASOs were ranked by the incidence of each AE group in the integrated safety data from 35 clinical trials. ISRs are considered to be local to the injection site, whereas FLRs are reflected by systemic constitutional symptoms. The correlations identified in this analysis of integrated clinical data provide evidence that the ASO sequences selected by the BJAB assay have a lower likelihood of causing systemic inflammatory AEs associated with FLRs, but not ISRs., (© 2023 Ionis Pharmaceuticals, Inc. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Receptor-mediated delivery of an antisense oligonucleotide (ASO) using the ligand-conjugated antisense technology is establishing a new benchmark for antisense therapeutics. The triantennary N -acetylgalactosamine (GalNAc 3 ) cluster is the first conjugated ligand to yield a marked increase in ASO potency for RNA targets expressed by hepatocytes, compared to the unconjugated form. In this study, we present an integrated safety assessment of data available from randomized, placebo-controlled, phase 2 studies for six GalNAc 3 -conjugated 2'- O -methoxyethyl (2'MOE)-modified ASOs. The total study population included 642 participants (130 placebo; 512 ASO) with up to 1 year of exposure. The primary measures were the incidence of signals from standardized laboratory tests and the mean test results over time. The GalNAc 3 -conjugated ASOs were well tolerated with no class effect identified across all doses tested compared to placebo. These results extend prior observations from phase 1 studies, now with treatment up to 1 year.

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. The IL-6 increase induced by ISIS 353512 was caused by activation of B cells. The IL-6 induction was inhibited by chloroquine pretreatment of PBMCs and the nature of ASOs suggested that the response is mediated by a Toll-like receptor (TLR), in all likelihood TLR9. While assessing the inter PBMC donor variability, two classes of human PBMC responders to ISIS 353512 were identified (discriminator and nondiscriminators). The discriminator donor PBMCs were shown to produce low level of IL-6 after 24 h in culture, in the absence of ASO treatment. The PBMC assay using discriminator donors was shown to be reproducible, allowing to assess reliably the immune potential of ASOs by comparison to known benchmark ASO controls that were previously shown to be either safe or inflammatory in clinical trials. Clinical Trial registration numbers: NCT00048321 NCT00330330 NCT00519727.

Transthyretin-mediated amyloidosis (ATTR) is a rare, under-recognized, progressively debilitating, fatal disease caused by the aggregation and extracellular deposition of amyloid transthyretin (TTR) fibrils in multiple organs and tissues throughout the body. TTR is predominantly synthesized by the liver and normally circulates as a homotetramer, while misfolded monomers aggregate to form amyloid fibrils. One strategy to treat ATTR amyloidosis is to reduce the amount of TTR produced by the liver using drugs that directly target the TTR mRNA or gene. This narrative review focuses on how TTR gene silencing tools act to reduce TTR production, describing strategies for improved targeted delivery of these agents to hepatocytes where TTR is preferentially expressed. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), termed RNA silencers, cause selective degradation of TTR mRNA, while a TTR gene editing tool reduces TTR expression by introducing nonsense mutations into the TTR gene. Two strategies to facilitate tissue-specific delivery of these nucleic acid-based drugs employ endogenous receptors expressed by hepatocytes. Lipid nanoparticles (LNPs) that recruit apolipoprotein E support low-density lipoprotein receptor-mediated uptake of unconjugated siRNA and are now used for CRISPR gene editing tools. Additionally, conjugating N-acetylgalactosamine (GalNAc) moieties to ASOs or siRNAs facilitates receptor-mediated uptake by the asialoglycoprotein receptor. In summary, ATTR is a progressive disease with various clinical manifestations due to TTR aggregation, deposition, and amyloid formation. Receptor-targeted ligands (eg, GalNAc) and nanoparticle encapsulation (eg, LNPs) are technologies to deliver ASOs, siRNAs, and gene editing tools to hepatocytes, the primary location of TTR synthesis., (© 2022 The Authors. Journal of the Peripheral Nervous System published by Wiley Periodicals LLC on behalf of Peripheral Nerve Society.)

Background: Hereditary angioedema is characterized by recurrent and unpredictable swellings that are disabling and potentially fatal. Selective inhibition of plasma prekallikrein production by antisense oligonucleotide treatment (donidalorsen) may reduce the frequency of attacks and the burden of disease., Methods: In this phase 2 trial, we randomly assigned, in a 2:1 ratio, patients with hereditary angioedema with C1 inhibitor deficiency to receive four subcutaneous doses of either donidalorsen (80 mg) or placebo, with one dose administered every 4 weeks. The primary end point was the time-normalized number of investigator-confirmed angioedema attacks per month (attack rate) between week 1 (baseline) and week 17. Secondary end points included quality of life, as measured with the Angioedema Quality of Life Questionnaire (scores range from 0 to 100, with higher scores indicating worse quality of life), and safety., Results: A total of 20 patients were enrolled, of whom 14 were randomly assigned to receive donidalorsen and 6 to receive placebo. The mean monthly rate of investigator-confirmed angioedema attacks was 0.23 (95% confidence interval [CI], 0.08 to 0.39) among patients receiving donidalorsen and 2.21 (95% CI, 0.58 to 3.85) among patients receiving placebo (mean difference, -90%; 95% CI, -96 to -76; P<0.001). The mean change from baseline to week 17 in the Angioedema Quality of Life Questionnaire score was -26.8 points in the donidalorsen group and -6.2 points in the placebo group (mean difference, -20.7 points; 95% CI, -32.7 to -8.7). The incidence of mild-to-moderate adverse events was 71% among patients receiving donidalorsen and 83% among those receiving placebo., Conclusions: Among patients with hereditary angioedema, donidalorsen treatment resulted in a significantly lower rate of angioedema attacks than placebo in this small, phase 2 trial. (Funded by Ionis Pharmaceuticals; ISIS 721744-CS2 ClinicalTrials.gov number, NCT04030598.)., (Copyright © 2022 Massachusetts Medical Society.)

The development process of antisense oligonucleotides (ASOs) as therapeutic agents in humans has advanced through the implementation of chemical compound modifications as well as increasingly sophisticated toxicological preclinical screening techniques. The Ionis Integrated Safety Database was utilized to determine if advances in ASO screening and clinical lead identification methods have improved the tolerability profiles of 2'- O -methoxyethyl (2'MOE)-modified ASOs as a class, relative to the first 2'MOE ASO approved for use in humans, mipomersen. Tolerability was assessed by the incidence and percentage of subcutaneous doses leading to adverse events at the injection site or flu-like reactions (FLRs), as well as by the incidence of dose discontinuations due to these events. In randomized placebo-controlled phase 1 and phase 2 trials, the incidence of each measure of tolerability was lower in the test group of 12 ASOs (713 ASO-treated subjects) compared with the reference, mipomersen (266 ASO-treated subjects); with the most marked reduction in the incidence of FLRs (0.6% vs. 9.4%). A similar reduction in the incidence of dose discontinuation due to FLRs was also observed (0.2% vs. 0.9%). When compared with mipomersen, 8 of 12 ASOs showed significant improvements in their respective mean percentage of doses leading to adverse events at the injection site, whereas 7 ASOs showed a significant improvement in mean percentage of doses leading to FLRs. These results support an overall improvement in the tolerability profile in 2'MOE ASOs that entered development after mipomersen, in parallel with advances in the drug discovery screening process as well as the gains in clinical experience during development of each ASO.

When coined, the term "antisense" included oligonucleotides of any structure, with any chemical modification and designed to work through any post-RNA hybridization mechanism. However, in practice the term "antisense" has been used to describe single stranded oligonucleotides (ss ASOs) designed to hybridize to RNAswhile the term "siRNA" has come to mean double stranded oligonucleotides designed to activate Ago2. However, the two approaches share many common features. The medicinal chemistry developed for ASOs greatly facilitated the development of siRNA technology and remains the chemical basis for both approaches. Many of challenges faced and solutions achieved share many common features. In fact, because ss ASOs can be designed to activate Ago2, the two approaches intersect at this remarkably important protein. There are also meaningful differences. The pharmacokinetic properties are quite different and thus potential routes of delivery differ. ASOs may be designedto use a variety of post-RNA binding mechanismswhile siRNAs depend solely on the robust activity of Ago2. However, siRNAs and ASOs are both used for therapeutic purposes and both must be and can be understood in a pharmacological context. Thus, the goals of this review are to put ASOs in pharmacological context and compare their behavior as pharmacological agents to the those of siRNAs., (Copyright © 2020. Published by Elsevier Inc.)

Antisense technology is now beginning to deliver on its promise to treat diseases by targeting RNA. Nine single-stranded antisense oligonucleotide (ASO) drugs representing four chemical classes, two mechanisms of action and four routes of administration have been approved for commercial use, including the first RNA-targeted drug to be a major commercial success, nusinersen. Although all the approved drugs are for use in patients with rare diseases, many of the ASOs in late- and middle-stage clinical development are intended to treat patients with very common diseases. ASOs in development are showing substantial improvements in potency and performance based on advances in medicinal chemistry, understanding of molecular mechanisms and targeted delivery. Moreover, the ASOs in development include additional mechanisms of action and routes of administration such as aerosol and oral formulations. Here, we describe the key technological advances that have enabled this progress and discuss recent clinical trials that illustrate the impact of these advances on the performance of ASOs in a wide range of therapeutic applications. We also consider strategic issues such as target selection and provide perspectives on the future of the field.

Aims: Amyloidogenic transthyretin (ATTR) amyloidosis is a fatal disease characterized by progressive cardiomyopathy and/or polyneuropathy. AKCEA-TTR-L Rx (ION-682884) is a ligand-conjugated antisense drug designed for receptor-mediated uptake by hepatocytes, the primary source of circulating transthyretin (TTR). Enhanced delivery of the antisense pharmacophore is expected to increase drug potency and support lower, less frequent dosing in treatment., Methods and Results: AKCEA-TTR-L Rx demonstrated an approximate 50-fold and 30-fold increase in potency compared with the unconjugated antisense drug, inotersen, in human hepatocyte cell culture and mice expressing a mutated human genomic TTR sequence, respectively. This increase in potency was supported by a preferential distribution of AKCEA-TTR-L Rx to liver hepatocytes in the transgenic hTTR mouse model. A randomized, placebo-controlled, phase 1 study was conducted to evaluate AKCEA-TTR-L Rx in healthy volunteers (ClinicalTrials.gov: NCT03728634). Eligible participants were assigned to one of three multiple-dose cohorts (45, 60, and 90 mg) or a single-dose cohort (120 mg), and then randomized 10:2 (active : placebo) to receive a total of 4 SC doses (Day 1, 29, 57, and 85) in the multiple-dose cohorts or 1 SC dose in the single-dose cohort. The primary endpoint was safety and tolerability; pharmacokinetics and pharmacodynamics were secondary endpoints. All randomized participants completed treatment. No serious adverse events were reported. In the multiple-dose cohorts, AKCEA-TTR-L Rx reduced TTR levels from baseline to 2 weeks after the last dose of 45, 60, or 90 mg by a mean (SD) of -85.7% (8.0), -90.5% (7.4), and -93.8% (3.4), compared with -5.9% (14.0) for pooled placebo (P < 0.001). A maximum mean (SD) reduction in TTR levels of -86.3% (6.5) from baseline was achieved after a single dose of 120 mg AKCEA-TTR-L Rx ., Conclusions: These findings suggest an improved safety and tolerability profile with the increase in potency achieved by productive receptor-mediated uptake of AKCEA-TTR-L Rx by hepatocytes and supports further development of AKCEA-TTR-L Rx for the treatment of ATTR polyneuropathy and cardiomyopathy., (© 2020 Ionis Pharmaceuticals, INC. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Antisense technology is beginning to deliver on the broad promise of the technology. Ten RNA-targeted drugs including eight single-strand antisense drugs (ASOs) and two double-strand ASOs (siRNAs) have now been approved for commercial use, and the ASOs in phase 2/3 trials are innovative, delivered by multiple routes of administration and focused on both rare and common diseases. In fact, two ASOs are used in cardiovascular outcome studies and several others in very large trials. Interest in the technology continues to grow, and the field has been subject to a significant number of reviews. In this review, we focus on the molecular events that result in the effects observed and use recent clinical results involving several different ASOs to exemplify specific molecular mechanisms and specific issues. We conclude with the prospective on the technology., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Hereditary transthyretin-mediated amyloidosis (hATTR) manifests as multisystem dysfunction, including progressive polyneuropathy. Inotersen, an antisense oligonucleotide, improved the course of neuropathic impairment in patients with hATTR in the pivotal NEURO-TTR study (NCT01737398). To determine inotersen's impact on symptoms and patients' neuropathy experience, we performed a post hoc analysis of the Neuropathy Symptoms and Change (NSC) score., Methods: Stage 1 or 2 hATTR patients were randomized to receive weekly subcutaneous inotersen or placebo for 65 weeks. NSC score was assessed at baseline and 35 and 66 weeks., Results: At 66 weeks, inotersen-treated patients had symptom stabilization as compared with worsening in patients receiving placebo, based on total NSC score. There were also improvements in the subdomains of muscle weakness, sensory, pain, and autonomic symptoms, and for various individual items., Discussion: Inotersen treatment stabilized neuropathy symptoms, including autonomic symptoms, in patients with hATTR according to NSC score. Thus, the NSC may be an effective measure to assess neuropathy progression and patients' neuropathy experience in clinical practice., (© 2020 The Authors. Muscle & Nerve published by Wiley Periodicals LLC.)

Introduction: Inotersen, an antisense oligonucleotide inhibitor of transthyretin (TTR) protein production, demonstrated significant benefit versus placebo in the modified Neuropathy Impairment Score (NIS) +7 neurophysiologic tests (mNIS+7) in patients with hereditary TTR-mediated amyloidosis (hATTR) with polyneuropathy. This analysis assessed the mNIS+7 components by anatomic location and the lower limb function (LLF) test., Methods: Adults with hATTR in the NEURO-TTR trial (NCT01737398) were randomly assigned to receive weekly doses of subcutaneous inotersen 300 mg or placebo for 65 weeks. The mNIS+7 and LLF were assessed at 35 and 66 weeks., Results: All major mNIS+7 components (muscle weakness, muscle stretch reflexes, sensation) and the LLF showed significant efficacy in patients receiving inotersen versus placebo; however, NIS-reflexes (upper limb), touch pressure (upper and lower limbs), and heart rate during deep breathing did not show significant effects., Discussion: The results of this analysis reinforce the beneficial effect of inotersen on slowing neuropathy progression in patients with hATTR polyneuropathy., (© 2020 The Authors. Muscle & Nerve published by Wiley Periodicals LLC.)

Hereditary angioedema is characterized by recurrent and unpredictable episodes of subcutaneous and mucosal swelling that can be life threatening. IONIS-PKK-L Rx is a ligand-conjugated antisense oligonucleotide designed for receptor-mediated delivery to hepatocytes. In a compassionate-use pilot study, two patients with severe bradykinin-mediated angioedema were initially administered weekly subcutaneous injections of the unconjugated parent drug, IONIS-PKK Rx , for 12 to 16 weeks, after which they received IONIS-PKK-L Rx at a dose of 80 mg every 3 to 4 weeks for 7 to 8 months. Treatment was accompanied by a reduction in the angioedema attack rate. (Funded by Amsterdam UMC.)., (Copyright © 2020 Massachusetts Medical Society.)

Background: Diacylglycerol-O-acyltransferase 2 (DGAT2) is one of two enzyme isoforms that catalyse the final step in the synthesis of triglycerides. IONIS-DGAT2 Rx is an antisense oligonucleotide inhibitor of DGAT2 that is under clinical investigation for the treatment of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). The aim of this trial was to examine the safety, tolerability, and efficacy of IONIS-DGAT2 Rx versus placebo in reducing liver fat in patients with type 2 diabetes and NAFLD., Methods: This double-blind, randomised, placebo-controlled, phase 2 study consisted of a 2-week screening period, a run-in period of up to 4 weeks, a 13-week treatment period of once-weekly dosing, and a 13-week post-treatment follow-up period. The study was done at 16 clinical research sites in Canada, Poland, and Hungary. Eligible participants were aged 18-75 years, had a body-mass index at screening between 27 kg/m 2 and 39 kg/m 2 , haemoglobin A 1c (HbA 1c ) levels from 7·3% to 9·5%, and liver fat content 10% or greater before randomisation, and agreed to maintain a stable diet and exercise routine throughout the study. Enrolled participants were stratified on the basis of liver fat content during the run-in period (<20% or ≥20%) and then centrally randomised (2:1) to receive once weekly subcutaneous injection of 250 mg IONIS-DGAT2 Rx or placebo for 13 weeks. Participants, investigators, funder personnel, and the clinical research organisation staff, including central readers of MRI scans, were all masked to treatment identity. The primary endpoints were the safety, tolerability, and pharmacodynamic effect of IONIS-DGAT2 Rx on hepatic steatosis, according to absolute reduction from baseline in liver fat percentage as quantified by MRI-estimated proton density fat fraction and assessed in the per-protocol population. Pharmacodynamic performance was determined in the per-protocol population by the change in liver fat content from baseline to 2 weeks after the last dose. The per-protocol population included all randomised participants who received at least ten doses of study drug, with the first four doses administered in the first 5 weeks, did not miss more than three consecutive weekly doses, and who had no protocol deviations that might affect efficacy. All randomised participants who received at least one dose of study drug were included in the safety analysis. This study is registered with ClinicalTrials.gov, NCT03334214., Findings: Between Nov 3, 2017, and Nov 28, 2018, we screened 173 people for eligibility. 44 were enrolled and randomly assigned to receive either IONIS-DGAT2 Rx (29 participants) or placebo (15 participants). After 13 weeks of treatment, the mean absolute reduction from baseline was -5·2% (SD 5·4) in the IONIS-DGAT2 Rx group compared with -0·6% (6·1) in the placebo group (treatment difference -4·2%, 95% CI -7·8 to -0·5, p=0·026). Reductions in liver fat were not accompanied by hyperlipidaemia, elevations in serum aminotransferases or plasma glucose, changes in bodyweight, or gastrointestinal side-effects compared with placebo. Six serious adverse events occurred in four patients treated with IONIS-DGAT2 Rx . No serious adverse events were reported in the placebo group. One of four patients reported three serious adverse events: acute exacerbation of chronic obstructive pulmonary disease, cardiac arrest, and ischaemic cerebral infarction, each considered severe and not related to study drug. Three of four patients reported one serious adverse event of increased blood triglycerides (severe, unrelated to study drug), deep-vein thrombosis (severe, unlikely to be related to study drug), and acute pancreatitis (mild, unrelated to study drug)., Interpretation: Our results suggest that DGAT2 antisense inhibition could be a safe and efficacious strategy for treatment of NAFLD and support further investigation in patients with biopsy-proven NASH. Based on the pharmacological target, the response to treatment observed in this study population could extend to the broader population of patients with NAFLD., Funding: Ionis Pharmaceuticals., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Kallikrein is the key contact system mediator responsible for the conversion of high-molecular-weight kininogen into the inflammatory vasodilator peptide bradykinin, a process regulated by C1-esterase inhibitor (C1-INH). In hereditary angioedema (HAE), genetic mutations result in deficient or dysfunctional C1-INH and dysregulation of the contact system leading to recurrent, sometimes fatal, angioedema attacks. IONIS-PKK Rx is a second-generation 2'-O-(2-methoxyethyl)-modified chimeric antisense oligonucleotide, designed to bind and selectively reduce prekallikrein (PKK) mRNA in the liver. IONIS-PKK Rx demonstrated dose-dependent reduction of human prekallikrein hepatic mRNA and plasma protein in transgenic mice and dose- and time-dependent reductions of plasma PKK in Cynomolgus monkeys. Similar dose-dependent reductions of plasma PKK levels were observed in healthy human volunteers accompanied by decreases in bradykinin generation capacity with an acceptable safety and tolerability profile. These results highlight a novel and specific approach to target PKK for the treatment of HAE and other diseases involving contact system activation and overproduction of bradykinin.

Objective: To evaluate the safety and efficacy of IONIS-GCGR Rx , a 2'- O -methoxyethyl antisense oligonucleotide targeting the glucagon receptor (GCGR), and the underlying mechanism of liver transaminase increases in patients with type 2 diabetes on stable metformin therapy., Research Design and Methods: In three phase 2, randomized, double-blind studies, patients with type 2 diabetes on metformin received weekly subcutaneous injections of IONIS-GCGR Rx (50-200 mg) or placebo for 13 or 26 weeks., Results: Significant reductions in HbA 1c were observed after IONIS-GCGR Rx treatment versus placebo at week 14 (-2.0% 200 mg, -1.4% 100 mg, -0.3% placebo; P < 0.001) or week 27 (-1.6% 75 mg, -0.9% 50 mg, -0.2% placebo; P < 0.001). Dose-dependent increases in transaminases were observed with IONIS-GCGR Rx , which were attenuated at lower doses and remained mostly within the normal reference range at the 50-mg dose. There were no other significant safety observations and no symptomatic hypoglycemia or clinically relevant changes in blood pressure, LDL cholesterol, or other vital signs. At week 14, IONIS-GCGR Rx 100 mg did not significantly affect mean hepatic glycogen content compared with placebo (15.1 vs. -20.2 mmol/L, respectively; P = 0.093) but significantly increased hepatic lipid content (4.2 vs. -2.7%, respectively; P = 0.005) in the presence of transaminase increases., Conclusions: IONIS-GCGR Rx is a potent inhibitor of hepatic glucagon receptor expression with a potential to improve glycemic control at low weekly doses in combination with metformin. Significant reductions in HbA 1c occurred across the full-dose range tested, with minimal transaminase elevations at lower doses. Furthermore, novel results suggest that despite inhibition of glycogenolysis after GCGR antagonism, IONIS-GCGR Rx did not increase hepatic glycogen content., (© 2019 by the American Diabetes Association.)

Advances in medicinal chemistry have produced new chemical classes of antisense oligonucleotides (ASOs) with enhanced therapeutic properties. Conjugation of the triantennary N-acetylgalactosamine (GalNAc 3 ) moiety to the extensively characterized phosphorothioate (PS)-modified 2'-O-methoxyethyl (2'MOE) ASO exemplifies such an advance. This structure-activity optimized moiety effects receptor-mediated uptake of the ASO prodrug through the asialoglycoprotein receptor 1 to support selective targeting of RNAs expressed by hepatocytes. In this study we report the integrated assessment of data available from randomized placebo-controlled dose-ranging studies of this chemical class of ASOs administered systemically to healthy human volunteers. First, we compare the pharmacokinetic and pharmacodynamic profiles of a subset of the GalNAc 3 -conjugated PS-modified 2'MOE ASOs to the parent PS-modified 2'MOE ASOs for which plasma analytes are available. We then evaluate the safety profile of the full set of GalNAc 3 -conjugated PS-modified 2'MOE ASO conjugates by the incidence of signals in standardized laboratory tests and by the mean laboratory test results as a function of dose level over time. With hepatocyte targeted delivery, the ED 50 for the GalNAc 3 -conjugated PS-modified 2'MOE ASO subset ranges from 4 to 10 mg/week, up to 30-fold more potent than the parent PS-modified 2'MOE ASO. No GalNAc 3 -conjugated PS-modified 2'MOE ASO class effects were identified from the assessment of the integrated laboratory test data across all doses tested with either single or multidose regimens. The increase in potency supports an increase in the safety margin for this new chemical class of ASOs now under broad investigation in the clinic. Although the total exposure is limited in the initial phase 1 trials, ongoing and future investigations in patient populations will support evaluation of the effects of long-term exposure.

Background: Hereditary transthyretin amyloidosis is caused by pathogenic single-nucleotide variants in the gene encoding transthyretin ( TTR) that induce transthyretin misfolding and systemic deposition of amyloid. Progressive amyloid accumulation leads to multiorgan dysfunction and death. Inotersen, a 2'- O-methoxyethyl-modified antisense oligonucleotide, inhibits hepatic production of transthyretin., Methods: We conducted an international, randomized, double-blind, placebo-controlled, 15-month, phase 3 trial of inotersen in adults with stage 1 (patient is ambulatory) or stage 2 (patient is ambulatory with assistance) hereditary transthyretin amyloidosis with polyneuropathy. Patients were randomly assigned, in a 2:1 ratio, to receive weekly subcutaneous injections of inotersen (300 mg) or placebo. The primary end points were the change in the modified Neuropathy Impairment Score+7 (mNIS+7; range, -22.3 to 346.3, with higher scores indicating poorer function; minimal clinically meaningful change, 2 points) and the change in the score on the patient-reported Norfolk Quality of Life-Diabetic Neuropathy (QOL-DN) questionnaire (range, -4 to 136, with higher scores indicating poorer quality of life). A decrease in scores indicated improvement., Results: A total of 172 patients (112 in the inotersen group and 60 in the placebo group) received at least one dose of a trial regimen, and 139 (81%) completed the intervention period. Both primary efficacy assessments favored inotersen: the difference in the least-squares mean change from baseline to week 66 between the two groups (inotersen minus placebo) was -19.7 points (95% confidence interval [CI], -26.4 to -13.0; P<0.001) for the mNIS+7 and -11.7 points (95% CI, -18.3 to -5.1; P<0.001) for the Norfolk QOL-DN score. These improvements were independent of disease stage, mutation type, or the presence of cardiomyopathy. There were five deaths in the inotersen group and none in the placebo group. The most frequent serious adverse events in the inotersen group were glomerulonephritis (in 3 patients [3%]) and thrombocytopenia (in 3 patients [3%]), with one death associated with one of the cases of grade 4 thrombocytopenia. Thereafter, all patients received enhanced monitoring., Conclusions: Inotersen improved the course of neurologic disease and quality of life in patients with hereditary transthyretin amyloidosis. Thrombocytopenia and glomerulonephritis were managed with enhanced monitoring. (Funded by Ionis Pharmaceuticals; NEURO-TTR ClinicalTrials.gov number, NCT01737398 .).

RNA-targeted therapies represent a platform for drug discovery involving chemically modified oligonucleotides, a wide range of cellular RNAs, and a novel target-binding motif, Watson-Crick base pairing. Numerous hurdles considered by many to be impassable have been overcome. Today, four RNA-targeted therapies are approved for commercial use for indications as diverse as Spinal Muscular Atrophy (SMA) and reduction of low-density lipoprotein cholesterol (LDL-C) and by routes of administration including subcutaneous, intravitreal, and intrathecal delivery. The technology is efficient and supports approaching "undruggable" targets. Three additional agents are progressing through registration, and more are in clinical development, representing several chemical and structural classes. Moreover, progress in understanding the molecular mechanisms by which these drugs work has led to steadily better clinical performance and a wide range of mechanisms that may be exploited for therapeutic purposes. Here we summarize the progress, future challenges, and opportunities for this drug discovery platform., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To evaluate safety and efficacy of IONIS-PTP-1B Rx , a second-generation 2'- O -methoxyethyl antisense inhibitor of protein tyrosine phosphatase 1B, as add-on therapy in overweight patients with type 2 diabetes inadequately controlled with metformin with or without sulfonylurea therapy., Research Design and Methods: In this phase II, double-blind, randomized, placebo-controlled, multicenter trial, overweight and obese patients (BMI ≥27 kg/m 2 ) with type 2 diabetes (HbA 1c ≥7.5% [58 mmol/mol] and ≤10.5% [91 mmol/mol]) on a stable dose of metformin alone or with sulfonylurea were randomized 2:1 to IONIS-PTP-1B Rx 200 mg ( n = 62) or placebo ( n = 30) once weekly for 26 weeks., Results: Mean baseline HbA 1c was 8.6% (70 mmol/mol) and 8.7% (72 mmol/mol) in placebo and active treatment, respectively. At week 27, IONIS-PTP-1B Rx reduced mean HbA 1c levels by -0.44% (-4.8 mmol/mol; P = 0.074) from baseline and improved leptin (-4.4 ng/mL; P = 0.007) and adiponectin (0.99 μg/mL; P = 0.026) levels compared with placebo. By week 36, mean HbA 1c was significantly reduced (-0.69% [-7.5 mmol/mol]; P = 0.034) and accompanied by reductions in fructosamine (-33.2 μmol/L; P = 0.005) and glycated albumin (-1.6%; P = 0.031) versus placebo. Despite both treatment groups receiving similar lifestyle counseling, mean body weight significantly decreased from baseline to week 27 with IONIS-PTP-1B Rx versus placebo (-2.6 kg; P = 0.002) independent of HbA 1c reduction ( R 2 = 0.0020). No safety concerns were identified in the study., Conclusions: Compared with placebo, IONIS-PTP-1B Rx treatment for 26 weeks produced prolonged reductions in HbA 1c , improved medium-term glycemic parameters, reduced leptin and increased adiponectin levels, and resulted in a distinct body weight-reducing effect., (© 2018 by the American Diabetes Association.)

Systemically administered 2'-O-methoxyethyl (2'MOE) antisense oligonucleotides (ASOs) accumulate in the kidney and metabolites are cleared in urine. The effects of eleven 2'MOE ASOs on renal function were assessed in 2,435 patients from 32 phase 2 and phase 3 trials. The principle analysis was on data from 28 randomized placebo-controlled trials. Mean levels of renal parameters remained within normal ranges over time across dose groups. Patient-level meta-analyses demonstrated a significant difference between placebo-treated and 2'MOE ASO-treated patients at doses >175 mg/week in the percentage and absolute change from baseline for serum creatinine and estimated glomerular filtration rate. However, these changes were not clinically significant or progressive. No dose-related effects were observed in the incidence of abnormal renal test results in the total population of patients, or subpopulation of diabetic patients or patients with renal dysfunction at baseline. The incidence of acute kidney injury [serum creatinine ≥0.3 mg/dL (26.5 μM) increases from baseline or ≥1.5 × baseline] in 2'MOE ASO-treated patients (2.4%) was not statistically different from placebo (1.7%, P = 0.411). In conclusion, in this database, encompassing 32 clinical trials and 11 different 2'MOE ASOs, we found no evidence of clinically significant renal dysfunction up to 52 weeks of randomized-controlled treatment.

Background: Epidemiologic and genomewide association studies have linked loss-of-function variants in ANGPTL3, encoding angiopoietin-like 3, with low levels of plasma lipoproteins., Methods: We evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side-effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins., Results: The treated mice had dose-dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low-density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple-dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, P<0.01 for all doses vs. placebo) and in levels of triglycerides (reductions of 33.2 to 63.1%), LDL cholesterol (1.3 to 32.9%), very-low-density lipoprotein cholesterol (27.9 to 60.0%), non-high-density lipoprotein cholesterol (10.0 to 36.6%), apolipoprotein B (3.4 to 25.7%), and apolipoprotein C-III (18.9 to 58.8%). Three participants who received the antisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events., Conclusions: Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in mice. Use of the same strategy to target human ANGPTL3 reduced levels of atherogenic lipoproteins in humans. (Funded by Ionis Pharmaceuticals; ClinicalTrials.gov number, NCT02709850 .).

A thorough analysis of clinical trial data in the Ionis integrated safety database (ISDB) was performed to determine if there is a class effect on platelet numbers and function in subjects treated with 2'-O-methoxyethyl (2'MOE)-modified antisense oligonucleotides (ASOs). The Ionis ISDB includes over 2,600 human subjects treated with 16 different 2'MOE ASOs in placebo-controlled and open-label clinical trials over a range of doses up to 624 mg/week and treatment durations as long as 4.6 years. This analysis showed that there is no class generic effect on platelet numbers and no incidence of confirmed platelet levels below 50 K/μL in subjects treated with 2'MOE ASOs. Only 7 of 2,638 (0.3%) subjects treated with a 2'MOE ASO experienced a confirmed postbaseline (BSLN) platelet count between 100 and 50 K/μL. Three of sixteen 2'MOE ASOs had >10% incidence of platelet decreases >30% from BSLN, suggesting that certain sequences may associate with clinically insignificant platelet declines. Further to these results, we found no evidence that 2'MOE ASOs alter platelet function, as measured by the lack of clinically relevant bleeding in the presence or absence of other drugs that alter platelet function and/or number and by the results from trials conducted with the factor XI (FXI) ASO.

Recent studies have led to a greater appreciation of the diverse roles RNAs play in maintaining normal cellular function and how they contribute to disease pathology, broadening the number of potential therapeutic targets. Antisense oligonucleotides are the most direct means to target RNA in a selective manner and have become an established platform technology for drug discovery. There are multiple molecular mechanisms by which antisense oligonucleotides can be used to modulate RNAs in cells, including promoting the degradation of the targeted RNA or modulating RNA function without degradation. Antisense drugs utilizing various antisense mechanisms are demonstrating therapeutic potential for the treatment of a broad variety of diseases. This review focuses on some of the advances that have taken place in translating antisense technology from the bench to the clinic.

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. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied.

Objective: To determine the effects of volanesorsen (ISIS 304801), a second-generation 2'-O-methoxyethyl chimeric antisense inhibitor of apolipoprotein (apo)C-III, on triglyceride (TG) levels and insulin resistance in patients with type 2 diabetes., Research Design and Methods: A randomized, double-blind, placebo-controlled trial was performed in 15 adult patients with type 2 diabetes (HbA1c >7.5% [58 mmol/mol]) and hypertriglyceridemia (TG >200 and <500 mg/dL). Patients were randomized 2:1 to receive volanesorsen 300 mg or placebo for a total of 15 subcutaneous weekly doses. Glucose handling and insulin sensitivity were measured before and after treatment using a two-step hyperinsulinemic-euglycemic clamp procedure., Results: Treatment with volanesorsen significantly reduced plasma apoC-III (-88%, P = 0.02) and TG (-69%, P = 0.02) levels and raised HDL cholesterol (HDL-C) (42%, P = 0.03) compared with placebo. These changes were accompanied by a 57% improvement in whole-body insulin sensitivity (P < 0.001). Importantly, we found a strong relationship between enhanced insulin sensitivity and both plasma apoC-III (r = -0.61, P = 0.03) and TG (r = -0.68, P = 0.01) suppression. Improved insulin sensitivity was sufficient to significantly lower glycated albumin (-1.7%, P = 0.034) and fructosamine (-38.7 μmol/L, P = 0.045) at the end of dosing and HbA1c (-0.44% [-4.9 mmol/mol], P = 0.025) 3 months postdosing., Conclusions: Volanesorsen reduced plasma apoC-III and TG while raising HDL-C levels. Importantly, glucose disposal, insulin sensitivity, and integrative markers of diabetes also improved in these patients after short-term treatment., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Background: Lipoprotein(a) (Lp[a]) is a risk factor for cardiovascular disease and calcific aortic valve stenosis. No effective therapies to lower plasma Lp(a) concentrations exist. We have assessed the safety, pharmacokinetics, and pharmacodynamics of ISIS-APO(a)Rx, a second-generation antisense drug designed to reduce the synthesis of apolipoprotein(a) (apo[a]) in the liver., Methods: In this randomised, double-blind, placebo-controlled, phase 1 study at the PAREXEL Clinical Pharmacology Research Unit (Harrow, Middlesex, UK), we screened for healthy adults aged 18-65 years, with a body-mass index less than 32·0 kg/m(2), and Lp(a) concentration of 25 nmol/L (100 mg/L) or more. Via a randomisation technique, we randomly assigned participants to receive a single subcutaneous injection of ISIS-APO(a)Rx (50 mg, 100 mg, 200 mg, or 400 mg) or placebo (3:1) in the single-dose part of the study or to receive six subcutaneous injections of ISIS-APO(a)Rx (100 mg, 200 mg, or 300 mg, for a total dose exposure of 600 mg, 1200 mg, or 1800 mg) or placebo (4:1) during a 4 week period in the multi-dose part of the study. Participants, investigators, and study staff were masked to the treatment assignment, except for the pharmacist who prepared the ISIS-APO(a)Rx or placebo. The primary efficacy endpoint was the percentage change from baseline in Lp(a) concentration at 30 days in the single-dose cohorts and at 36 days for the multi-dose cohorts. Safety and tolerability was assessed 1 week after last dose and included determination of the incidence, severity, and dose relation of adverse events and changes in laboratory variables, including lipid panel, routine haematology, blood chemistry, urinalysis, coagulation, and complement variables. Other assessments included vital signs, a physical examination, and 12-lead electrocardiograph. This trial is registered with European Clinical Trials Database, number 2012-004909-27., Findings: Between Feb 27, 2013, and July 15, 2013, 47 (23%) of 206 screened volunteers were randomly assigned to receive ISIS-APO(a)Rx as a single-dose or multi-dose of ascending concentrations or placebo. In the single-dose study, we assigned three participants to receive 50 mg ISIS-APO(a)Rx, three participants to receive 100 mg ISIS-APO(a)Rx, three participants to receive 200 mg ISIS-APO(a)Rx, three participants to receive 400 mg ISIS-APO(a)Rx, and four participants to receive placebo. All 16 participants completed treatment and follow-up and were included in the pharmacodynamics, pharmacokinetics, and safety analyses. For the multi-dose study, we assigned eight participants to receive six doses of 100 mg ISIS-APO(a)Rx, nine participants to receive six doses of 200 mg ISIS-APO(a)Rx, eight participants to receive six doses of 300 mg ISIS-APO(a)Rx, and six participants to receive six doses of placebo. Whereas single doses of ISIS-APO(a)Rx (50-400 mg) did not decrease Lp(a) concentrations at day 30, six doses of ISIS-APO(a)Rx (100-300 mg) resulted in dose-dependent, mean percentage decreases in plasma Lp(a) concentration of 39·6% from baseline in the 100 mg group (p=0·005), 59·0% in the 200 mg group (p=0·001), and 77·8% in the 300 mg group (p=0·001). Similar reductions were observed in the amount of oxidized phospholipids associated with apolipoprotein B-100 and apolipoprotein(a). Mild injection site reactions were the most common adverse events., Interpretation: ISIS-APO(a)Rx results in potent, dose-dependent, selective reductions of plasma Lp(a). The safety and tolerability support continued clinical development of ISIS-APO(a)Rx as a potential therapeutic drug to reduce the risk of cardiovascular disease and calcific aortic valve stenosis in patients with elevated Lp(a) concentration., Funding: Isis Pharmaceuticals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Apolipoprotein C-III (APOC3) is a key regulator of plasma triglyceride levels. Elevated triglyceride levels are associated with a risk of adverse cardiovascular events and pancreatitis. ISIS 304801 is a second-generation antisense inhibitor of APOC3 synthesis., Methods: We conducted a randomized, double-blind, placebo-controlled, dose-ranging, phase 2 study to evaluate ISIS 304801 in untreated patients with fasting triglyceride levels between 350 mg per deciliter (4.0 mmol per liter) and 2000 mg per deciliter (22.6 mmol per liter) (ISIS 304801 monotherapy cohort), as well as in patients receiving stable fibrate therapy who had fasting triglyceride levels between 225 mg per deciliter (2.5 mmol per liter) and 2000 mg per deciliter (ISIS 304801-fibrate cohort). Eligible patients were randomly assigned to receive either ISIS 304801, at doses ranging from 100 to 300 mg, or placebo, once weekly for 13 weeks. The primary outcome was the percentage change in APOC3 level from baseline., Results: A total of 57 patients were treated in the ISIS 304801 monotherapy cohort (41 received active agent, and 16 received placebo), and 28 patients were treated in the ISIS 304801-fibrate cohort (20 received active agent, and 8 received placebo). The mean (±SD) baseline triglyceride levels in the two cohorts were 581±291 mg per deciliter (6.6±3.3 mmol per liter) and 376±188 mg per deciliter (4.2±2.1 mmol per liter), respectively. Treatment with ISIS 304801 resulted in dose-dependent and prolonged decreases in plasma APOC3 levels when the drug was administered as a single agent (decreases of 40.0±32.0% in the 100-mg group, 63.8±22.3% in the 200-mg group, and 79.6±9.3% in the 300-mg group, vs. an increase of 4.2±41.7% in the placebo group) and when it was administered as an add-on to fibrates (decreases of 60.2±12.5% in the 200-mg group and 70.9±13.0% in the 300-mg group, vs. a decrease of 2.2±25.2% in the placebo group). Concordant reductions of 31.3 to 70.9% were observed in triglyceride levels. No safety concerns were identified in this short-term study., Conclusions: We found that treatment with ISIS 304801 was associated with significant lowering of triglyceride levels, among patients with a broad range of baseline levels, through selective antisense inhibition of APOC3 synthesis. (Funded by Isis Pharmaceuticals; ClinicalTrials.gov number, NCT01529424.).

Mipomersen (Kynamro(®)), a second-generation 2'-O-methoxyethyl chimeric antisense oligonucleotide (ASO), inhibits the synthesis of apolipoprotein B (apoB) and is indicated in the US as an adjunct therapy for homozygous familial hypercholesterolemia (HoFH) at a dose of 200 mg subcutaneously (SC) once weekly. The pharmacokinetic (PK) properties of mipomersen are generally consistent across all species studied, including mouse, rat, monkey, and humans. After SC administration, mipomersen is rapidly and extensively absorbed. It has an apparent plasma and tissue terminal elimination half-life of approximately 30 days. Mipomersen achieves steady-state tissue concentrations within approximately 4-6 months of once-weekly dosing. It does not exhibit PK-based drug-drug interactions with other concomitant medications, either involving competition for plasma protein binding or alterations in disposition of any evaluated drugs. Furthermore, mipomersen does not prolong the corrected QT (QTc) interval. There have been no ethnic- or gender-related differences in PK observed. In clinical trials, both as a single agent and in the presence of maximal lipid-lowering therapy, mipomersen has demonstrated significant dose-dependent reductions in all measured apoB-containing atherogenic lipoproteins. Overall, mipomersen has well-characterized PK and pharmacodynamic properties in both animals and humans, and is an efficacious adjunct treatment for patients with HoFH.

The familial chylomicronemia syndrome is a genetic disorder characterized by severe hypertriglyceridemia and recurrent pancreatitis due to a deficiency in lipoprotein lipase (LPL). Currently, there are no effective therapies except for extreme restriction in the consumption of dietary fat. Apolipoprotein C-III (APOC3) is known to inhibit LPL, although there is also evidence that APOC3 increases the level of plasma triglycerides through an LPL-independent mechanism. We administered an inhibitor of APOC3 messenger RNA (mRNA), called ISIS 304801, to treat three patients with the familial chylomicronemia syndrome and triglyceride levels ranging from 1406 to 2083 mg per deciliter (15.9 to 23.5 mmol per liter). After 13 weeks of study-drug administration, plasma APOC3 levels were reduced by 71 to 90% and triglyceride levels by 56 to 86%. During the study, all patients had a triglyceride level of less than 500 mg per deciliter (5.7 mmol per liter) with treatment. These data support the role of APOC3 as a key regulator of LPL-independent pathways of triglyceride metabolism.

Background: C-reactive protein (CRP) binds to damaged cells, activates the classical complement pathway, is elevated in multiple inflammatory conditions, and provides prognostic information on risk of future atherosclerotic events. It is controversial, however, as to whether inhibiting CRP synthesis would have any direct anti-inflammatory effects in humans., Methods and Results: A placebo-controlled study was used to evaluate the effects of ISIS 329993 (ISIS-CRPR x) on the acute-phase response after endotoxin challenge in 30 evaluable subjects. Healthy adult males were randomly allocated to receive 6 injections over a 22-day period of placebo or active therapy with ISIS 329993 at 400- or 600-mg doses. Eligible subjects were subsequently challenged with a bolus of endotoxin (2 ng/kg). Inflammatory and hematological biomarkers were measured before and serially after the challenge. ISIS-CRPR x was well tolerated with no serious adverse events. Median CRP levels increased more than 50-fold from baseline 24 hours after endotoxin challenge in the placebo group. In contrast, the median increase in CRP levels was attenuated by 37% (400 mg) and 69% (600 mg) in subjects pretreated with ISIS-CRPR x (P<0.05 vs. placebo). All other aspects of the acute inflammatory response were similar between treatment groups., Conclusion: Pretreatment of subjects with ISIS-CRPR x selectively reduced the endotoxin-induced increase in CRP levels in a dose-dependent manner, without affecting other components of the acute-phase response. These data demonstrate the specificity of antisense oligonucleotides and provide an investigative tool to further define the role of CRP in human pathological conditions., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Mipomersen, an apolipoprotein B synthesis inhibitor, demonstrated significant reductions in low-density lipoprotein (LDL) cholesterol, non-high density lipoprotein cholesterol, and apolipoprotein B in 4 phase 3 studies at the FDA-approved subcutaneous dose of 200 mg once weekly., Methods and Results: A short-term phase 1 study in healthy volunteers was conducted to evaluate the relative bioavailability, safety, and tolerability of mipomersen in 2 test dose regimens in reference to the 200 mg weekly dose regimen. Eighty-four adults were randomized to 1 of 3 cohorts (30 mg once daily, 70 mg 3 times weekly, or 200 mg once weekly) and then mipomersen or placebo (3:1 ratio) for 3 weeks of treatment. Comparable mipomersen post-distribution phase plasma concentrations were observed across the 3 dose regimens suggesting similar tissue exposure. Injection site reactions were reported, but did not lead to treatment discontinuation. The median incidence of these responses per injection was decreased by lowering the dose. Signals from a diverse panel of systemic inflammation markers were essentially indistinguishable between dose regimens and placebo treatment. The one exception was a modest transient post-dose elevation of C-reactive protein (CRP) in the mipomersen 200 mg weekly group. This elevation was not associated with an increase in other proinflammatory markers., Conclusions: This study demonstrated a similar drug exposure and overall safety profile between the 3 dosing regimens. Exploratory assessment of a diverse panel of biomarkers found no indication of a systemic inflammatory response to mipomersen treatment. These results support assessment of alternative dose regimens in longer-term studies., Clinical Trial Registration Url: http://www.clinicaltrials.gov. Unique identifier: NCT01061814.

Antisense oligonucleotides and small interfering RNAs, which suppress the translation of specific mRNA target proteins, are emerging as important therapeutic modalities for the treatment of cardiovascular disease. Over the last 25 years, the advances in all aspects of antisense technology, as well as a detailed understanding of the mechanism of action of antisense drugs, have enabled their use as therapeutic agents. These advancements culminated in the FDA approval of the first chronically administered cardiovascular antisense therapeutic, mipomersen, which targets hepatic apolipoprotein B mRNA. This review provides a brief history of antisense technology, highlights the progression of mipomersen from preclinical studies to multiple Phase III registration trials, and gives an update on the status of other cardiovascular antisense therapeutics currently in the clinic.

Rationale: Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic., Objective: To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels., Methods and Results: Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations., Conclusions: Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.

Observational studies of patients with established rheumatoid arthritis (RA) document a positive correlation between C-reactive protein (CRP) blood concentration and worsening of RA symptoms, but whether this association is causal or not is not known. Using CRP transgenic mice (CRPTg) with collagen-induced arthritis (CIA; a rodent model of RA), we explored causality by testing if CRP lowering via treatment with antisense oligonucleotides (ASOs) targeting human CRP mRNA was efficacious and of clinical benefit. We found that in CRPtg with established CIA, ASO-mediated lowering of blood human CRP levels improved the clinical signs of arthritis. In addition, in healthy human volunteers the ASO was well tolerated and efficacious i.e., treatment achieved significant CRP lowering. ASOs targeting CRP should provide a specific and effective way to lower human CRP levels, which might be an effective therapy in patients with established RA.Molecular Therapy - Nucleic Acids (2012) 1, e52; doi:10.1038/mtna.2012.44; published online 13 November 2012.

Aims: A randomized, double-blind, placebo-controlled study was conducted to investigate the safety and efficacy of mipomersen, an apolipoprotein B-100 (apoB) synthesis inhibitor, in patients who are statin intolerant and at high risk for cardiovascular disease (CVD)., Methods and Results: Thirty-three subjects, not receiving statin therapy because of statin intolerance, received a weekly subcutaneous dose of 200 mg mipomersen or placebo (2:1 randomization) for 26 weeks. The primary endpoint was per cent change in LDL cholesterol (LDL-c) from the baseline to Week 28. The other efficacy endpoints were per cent change in apoB and lipoprotein a [Lp(a)]. Safety was determined using the incidence of treatment-emergent adverse events (AEs) and clinical laboratory evaluations. After 26 weeks of mipomersen administration, LDL-c was reduced by 47 ± 18% (P < 0.001 vs. placebo). apoB and Lp(a) were also significantly reduced by 46 and 27%, respectively (P < 0.001 vs. placebo). Four mipomersen (19%) and two placebo subjects (17%) discontinued dosing prematurely due to AEs. Persistent liver transaminase increases ≥ 3× the upper limit of normal were observed in seven (33%) subjects assigned to mipomersen. In selected subjects, liver fat content was assessed, during and after treatment, using magnetic resonance spectroscopy. Liver fat content in these patients ranged from 0.8 to 47.3%. Liver needle biopsy was performed in two of these subjects, confirming hepatic steatosis with minimal inflammation or fibrosis., Conclusion: The present data suggest that mipomersen is a potential therapeutic option in statin-intolerant patients at high risk for CVD. The long-term follow-up of liver safety is required., Clinical Trial Registration: ClinicalTrials.gov identifier: NCT00707746.

Aims: Mipomersen, an apolipoprotein (apo) B synthesis inhibitor, has been shown to produce potent reductions in apoB and LDL-cholesterol levels in animal models as well as healthy human volunteers. A randomized, double-blind, placebo-controlled, dose-escalation study was designed to evaluate the efficacy and safety of mipomersen monotherapy with or without dose loading in subjects with mild-to-moderate hyperlipidaemia., Methods and Results: Fifty subjects with LDL-cholesterol levels between 119 and 266 mg/dL were enrolled into five cohorts at a 4:1 randomization ratio of active to placebo. Two 13-week dose regimens were evaluated at doses ranging from 50 to 400 mg/week. Mipomersen produced dose-dependent reductions in all apoB containing lipoproteins. In the 200 and 300 mg/week dose cohorts, mean reductions from baseline in LDL cholesterol were -45 ± 10% (P= 0.000) and -61 ± 8% (P= 0.000), corresponding to a -46 ± 11% (P= 0.000) and -61 ± 7% (P= 0.000) decrease in apoB levels. Triglyceride levels were also lowered with median reductions up to 53% (P= 0.021). The most common adverse events were injection site reactions. Seven of 40 subjects (18%) showed consecutive transaminase elevations >3× upper limit of normal. Five of these subjects received 400 mg/week, four of whom had apoB levels below the limit of detection. As a consequence, the 400 mg/week cohort was discontinued., Conclusions: Mipomersen administered as monotherapy in subjects with mild-to-moderate hyperlipidaemia produced potent reductions in all apoB-containing lipoproteins. Higher doses were associated with hepatic transaminase increases.

A randomized, double-blind, placebo-controlled, dose-escalation study was conducted to examine the efficacy and safety of mipomersen (ISIS 301012), an antisense inhibitor of apolipoprotein B, when added to conventional lipid-lowering therapy for patients with heterozygous familial hypercholesterolemia. A total of 44 patients were enrolled and were separated into 4 cohorts, with doses ranging from 50 to 300 mg (4:1 active treatment/placebo ratio). Patients received 8 doses subcutaneously during a 6-week treatment period. Patients assigned to the 300-mg dose continued for an additional 7 weeks with once-per-week dosing. The primary efficacy end point was the percentage of change from baseline to week 7 in low-density lipoprotein (LDL) cholesterol. Safety was assessed using the laboratory test results and according to the incidence, severity, and relation of adverse events to drug dose. Mipomersen produced significant reductions in LDL cholesterol and other atherogenic apolipoprotein B-containing lipoproteins. After 6 weeks of treatment, the LDL cholesterol level was reduced by 21% from baseline in the 200-mg/week dose group (p <0.05) and 34% from baseline in the 300-mg/week dose group (p <0.01), with a concomitant reduction in apolipoprotein B of 23% (p <0.05) and 33% (p <0.01), respectively. Injection site reactions were the most common adverse event. Elevations in liver transaminase levels (> or =3 times the upper limit of normal) occurred in 4 (11%) of 36 patients assigned to active treatment; 3 of these patients were in the highest dose group. In conclusion, mipomersen has an incremental LDL cholesterol lowering effect when added to conventional lipid-lowering therapy., (Copyright 2010 Elsevier Inc. All rights reserved.)

Objectives: The aim of this study was to evaluate the efficacy and safety of mipomersen in hypercholesterolemic subjects taking stable statin therapy., Background: Mipomersen is an apolipoprotein (apo) B synthesis inhibitor that has demonstrated significant reductions in apo B and low-density lipoprotein (LDL) cholesterol in Phase 1 clinical trials in healthy volunteers., Methods: A randomized, placebo-controlled, dose-escalation Phase 2 study was designed to evaluate the effects of mipomersen in hypercholesterolemic subjects taking stable statin therapy. Seventy-four subjects were enrolled sequentially into 1 of 6 dose cohorts at a 4:1 (active/placebo) ratio. Subjects received 7 doses of 30 to 400 mg over 5 weeks in the first 5 cohorts and 15 doses of 200 mg over 13 weeks in the sixth cohort. Pre-specified end points included percentage change from baseline in apo B and LDL cholesterol. Safety was assessed with laboratory test results and by the incidence and severity of adverse events., Results: The apo B and LDL cholesterol were reduced by 19% to 54% and 21% to 52%, respectively, at doses of 100 mg/week mipomersen and higher in the 5-week treatment cohorts. Efficacy seemed to increase upon treatment for 13 weeks at a dose of 200 mg/week. Injection site reactions (mild to moderate erythema [90%]) and hepatic transaminase increases (17%) were the most common adverse events, leading to discontinuation in 2 subjects and 1 subject, respectively. In the 13-week treatment cohort, 5 of 10 subjects (50%) had elevations >or=3x the upper limit of normal, 4 of which persisted on 2 consecutive occasions., Conclusions: Mipomersen might hold promise for treatment of patients not reaching target LDL cholesterol levels on stable statin therapy. Further studies are needed to address the mechanisms and clinical relevance of transaminase changes after mipomersen administration. (Dose-Escalating Safety Study in Subjects on Stable Statin Therapy; NCT00231569)., (Copyright (c) 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Several strategies have been pursued to increase the extent of exon 7 inclusion during splicing of SMN2 (survival of motor neuron 2) transcripts, for eventual therapeutic use in spinal muscular atrophy (SMA), a genetic neuromuscular disease. Antisense oligonucleotides (ASOs) that target an exon or its flanking splice sites usually promote exon skipping. Here we systematically tested a large number of ASOs with a 2'-O-methoxy-ethyl ribose (MOE) backbone that hybridize to different positions of SMN2 exon 7, and identified several that promote greater exon inclusion, others that promote exon skipping, and still others with complex effects on the accumulation of the two alternatively spliced products. This approach provides positional information about presumptive exonic elements or secondary structures with positive or negative effects on exon inclusion. The ASOs are effective not only in cell-free splicing assays, but also when transfected into cultured cells, where they affect splicing of endogenous SMN transcripts. The ASOs that promote exon 7 inclusion increase full-length SMN protein levels, demonstrating that they do not interfere with mRNA export or translation, despite hybridizing to an exon. Some of the ASOs we identified are sufficiently active to proceed with experiments in SMA mouse models.

Synthetic small interfering RNA (siRNA) mediated silencing of a specific gene is emerging as a powerful tool for gene regulation. However, their utility is limited for therapeutic applications primarily due to poor stability. The 2',5'-linked oligonucleotides are known to be more stable to nucleolytic degradation than 3',5'-linked oligonucleotides. The 2',5'-linkage is tolerated in the sense strand of the siRNA duplex. However, the 2',5'-linkage is not tolerated in the antisense strand of the siRNA duplex.

Background: Alicaforsen is an antisense oligonucleotide inhibitor of intercellular adhesion molecule 1 protein expression with activity in subjects with ulcerative colitis and pouchitis., Aim: To compare the effects of alicaforsen enema to standard of care mesalazine (mesalamine) enema in subjects with mild to moderate active left-sided ulcerative colitis., Method: A randomized, double-blind, active-controlled multicentre clinical trial. Subjects received a nightly enema of 120 mg alicaforsen (n=55), 240 mg alicaforsen (n=50), or 4 g mesalazine (n=54) for 6 weeks, followed by a 24-week monitoring period. The primary end point was Disease Activity Index at week 6. Clinical improvement, remission and relapse were secondary end points., Results: No significant difference was observed between treatment arms in the primary end point. However, the median duration of response to alicaforsen enema treatment was two- to threefold longer (128 and 146 days) in comparison with mesalazine (54 days). Complete mucosal healing occurred in 24% of the 240 mg alicaforsen group, when compared with 17% in the mesalazine., Conclusions: Alicaforsen enema demonstrated an acute response and safety profile similar to mesalazine enema, but was differentiated by a more durable response. The extended length of remission suggests that alicaforsen enema treatment may have a disease modifying effect.

Background: Alicaforsen is a phosphorothioate-modified antisense oligodeoxynucleotide designed to sequence-specifically reduce intercellular adhesion molecule 1 messenger RNA levels., Aims: To determine the systemic and local bioavailability of alicaforsen, and its activity when administered as a once daily enema in subjects with active ulcerative colitis. METHODS An open-label study was conducted to assess the relative absorption (local and systemic pharmacokinetics) and pharmacologic activity of alicaforsen enema in subjects with active ulcerative colitis. Fifteen subjects received nightly enemas of alicaforsen (240 mg) for a treatment period of 6 weeks. Alicaforsen concentrations in plasma and colonic tissue biopsies were determined. Disease activity index and multiple measurements including endoscopy were used to assess alicaforsen activity in these subjects., Results: Plasma concentrations of parent alicaforsen represented < 0.6% mean bioavailability when compared with historical intravenous area under the plasma concentration-time curves. Concentrations of the intact oligonucleotide in mucosal colonic tissue biopsies were orders of magnitude higher than those observed in plasma. A 46% reduction in mean Disease Activity Index and 33% rate of remission as defined by complete mucosal healing were observed at the end of treatment. Conclusion These data confirm that alicaforsen enema provides local treatment for a local disease with little meaningful systemic exposure.

Background: Alicaforsen is an antisense oligonucleotide designed to inhibit expression of human intercellular adhesion molecule 1. Previous clinical studies have demonstrated activity of alicaforsen enema in ulcerative colitis and pouchitis., Aim: To determine the minimally effective dosing regimen of alicaforsen enema in subjects with mild to moderate left-sided ulcerative colitis., Methods: Randomized, placebo-controlled, double-blind, two-dose ranging multicentre study. One hundred and twelve subjects were equally randomized to receive one of four alicaforsen enema regimens or placebo daily for 6 weeks. Primary end point was Disease Activity Index at week 6. Secondary end points included evaluation of clinical improvement, relapse rates and durability of response. Analysis of data were performed on the intent-to-treat population., Results: No significant difference was observed between treatment arms and placebo in the primary end point. A prolonged reduction in mean% Disease Activity Index relative to baseline was observed in the daily 240 mg alicaforsen enema treatment arm in comparison with placebo from week 18 (51% vs. 18%, P=0.04) to week 30 (50% vs. 11%, P=0.03)., Conclusions: Alicaforsen enema was safe and well tolerated at all doses studied. The durability of the response to alicaforsen enema treatment may suggests a disease-modifying effect.

Diabetic retinopathy and acromegaly are diseases associated with excess action of GH and its effector IGF-I, and there is a need for improved therapies. We have designed an optimised 2'-O-(2-methoxyethyl)-modified phosphorothioate oligodeoxynucleotide, ATL 227446, and demonstrated its ability to suppress GH receptor mRNA in vitro. Subcutaneous injections of ATL 227446 reduced GH receptor mRNA levels, GH binding activity and serum IGF-I levels in mice after seven days of dosing. The reduction in serum IGF-I could be sustained for over ten weeks of dosing at therapeutically relevant levels, during which there was also a significant decrease in body weight gain in antisense-treated mice relative to saline and mismatch control-treated mice. The findings indicate that administration of an antisense oligonucleotide to the GH receptor may be applicable to human diseases in which suppression of GH action provides therapeutic benefit.

RNA interference (RNAi) is a process by which short interfering RNAs (siRNAs) direct the degradation of complementary single-strand RNAs. In this study, we investigated the effects of full-strand phosphorothioate (PS) backbone and 2'-O-methyl (2'-OMe) sugar modifications on RNAi-mediated silencing. In contrast to previous reports, we have identified active siRNA duplexes containing full 2'-OMe-modified sense strands that display comparable activity to the unmodified analog of similar sequence. The structure of these modified siRNAs is the predominant determinant of their activity, with sequence and backbone composition being secondary. We further show, by using biotin-tagged siRNAs and affinity-tagged hAgo2/eIF2C2, that activity of siRNA duplexes containing full 2'-OMe substitutions in the sense strand is mediated by the RNA-induced silencing complex (RISC) and that strand-specific loading (or binding) to hAgo2 may be modulated through selective incorporation of these modifications.

A systematic study on the effect of 2'-sugar modifications (2'-F (2'-F-2'-deoxy-nucleoside residues), 2'-O-Me (2'-O-methyl-nucleoside residues), and 2'-O-MOE [2'-O-(2-methoxyethyl)]-nucleoside residues) in the antisense and sense strands of short interference RNA (siRNA) was performed in HeLa cells. The study of the antisense strand of siRNAs demonstrated that activity depends on the position of the modifications in the sequence. The siRNAs with modified ribonucleotides at the 5'-end of the antisense strand were less active relative to the 3'-modified ones. The 2'-F sugar was generally well-tolerated on the antisense strand, whereas the 2'-O-Me showed significant shift in activity depending on the position of modification. The 2'-O-MOE modification in the antisense strand resulted in less active siRNA constructs regardless of placement position in the construct. The incorporation of the modified residues, e.g., 2'-O-Me and 2'-O-MOE, in the sense strand of siRNA did not show a strong positional preference. These results may provide guidelines to design effective and stable siRNAs for RNA interference mediated therapeutic applications.