Search

Background: Peritoneal metastasis of gastric cancer is closely associated with dismal prognosis. In previous preclinical proof-of-concept studies, an amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotide (ASO), designated ASO-4733 that targets the gene encoding synaptotagmin XIII (SYT13), inhibited cellular functions required for the formation of peritoneal metastasis of gastric cancer cells. ASO-4733 achieved therapeutic effects when intra-abdominally administered to mouse xenograft models. Here, we conducted an analysis of Syt13-deficient mice to determine the pharmacokinetics and toxicity of intra-abdominal administration of ASO-4733., Methods: The effects of Syt13-deficiency in mice were determined. Good Laboratory Practice toxicity tests and the toxicokinetics of intra-abdominal administration of ASO-4733 were conducted in cynomolgus monkeys and rats. The pharmacokinetics of ASO-4733 administered intravenously or intra-abdominally to rats were investigated., Results: Syt13-deficient mice exhibited normal reproduction, organ functions, and motor functions. Weekly intra-abdominal administration of ASO-4733 (125 mg/kg), corresponding to a 50-fold increase of the estimated clinical dose for 4 weeks, was well tolerated by cynomolgus monkeys. In rats, off-target toxicity (not attributable to hybridization) was observed after weekly intra-abdominal administration of ASO-4733. Blood concentrations of ASO-4733 were lower and rose more slowly after intra-abdominal administration compared with intravenous administration., Conclusions: The preclinical profile of intra-abdominal administration of ASO-4733 demonstrated its suitability for entry into clinical trials of patients with peritoneal metastasis of gastric cancer., (© 2024. The Author(s) under exclusive licence to The International Gastric Cancer Association and The Japanese Gastric Cancer Association.)

Introduction: Nucleic acid (NA)-based therapeutics have shown great potential for downregulating or augmenting gene expression, and for promising applications, e.g. , protein-replacement therapy and vaccination, a comprehensive understanding of the requirements for their targeted delivery to specific tissues or cells is needed., Areas Covered: In this review, we discuss clinical applications of four representative types of NA-based therapeutics, i.e. antisense oligonucleotides, small interfering RNA, messenger RNA, and circular RNA, with a focus on the lipid nanoparticle (LNP) technology used for intracellular delivery. The in vivo fate of LNPs is discussed to improve the understanding of trafficking of nanomedicines at the systemic and cellular levels. In addition, NA-based vaccines are discussed, focusing on targeting antigen-presenting cells and immune activation., Expert Opinion: Optimization of delivery systems for NA-based therapeutics is mainly focused on the standard requirements of prolonged systemic circulation and enhancing endosomal escape. Depending on the final destination in specific target tissues or cells, strategies should be adjusted to achieve the desired biodistribution of NA-based payloads. More studies relating to the pharmacokinetics of both cargo and carrier are encouraged, because their in vivo fates may differ, considering the possibility of premature cargo release before reaching the target.

Antisense oligonucleotides (AONs) are a versatile tool for treating inherited retinal diseases. However, little is known about how different chemical modifications of AONs can affect their biodistribution, toxicity, and uptake in the retina. Here, we addressed this question by comparing splice-switching AONs with three different chemical modifications commonly used in a clinical setting (2'O-methyl-phosphorothioate (2-OMe/PS), 2'O-methoxyethyl-phosphoriate (2-MOE/PS), and phosphorodiamidite morpholino oligomers (PMO)). These AONs targeted genes exclusively expressed in certain types of retinal cells. Overall, studies in vitro and in vivo in C57BL/6J wild-type mouse retinas showed that 2-OMe/PS and 2-MOE/PS AONs have comparable efficacy and safety profiles. In contrast, octa-guanidine-dendrimer-conjugated in vivo PMO-oligonucleotides (ivPMO) caused toxicity. This was evidenced by externally visible ocular phenotypes in 88.5% of all ivPMO-treated animals, accompanied by severe alterations at the morphological level. However, delivery of unmodified PMO-AONs did not cause any toxicity, although it clearly reduced the efficacy. We conducted the first systematic comparison of different chemical modifications of AONs in the retina. Our results showed that the same AON sequence with different chemical modifications displayed different splicing modulation efficacies, suggesting the 2'MOE/PS modification as the most efficacious in these conditions. Thereby, our work provides important insights for future clinical applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Hexanucleotide repeat expansion of C9orf72 is a common genetic cause of amyotrophic lateral sclerosis (ALS). No C9orf72-targeted treatments are available. BIIB078 is an investigational antisense oligonucleotide targeting C9orf72 sense RNA. We aimed to assess the safety, tolerability, and pharmacokinetics of BIIB078 in participants with C9orf72-associated ALS., Methods: This phase 1, randomised controlled trial was done at 22 sites in six countries (Canada, Ireland, Netherlands, Switzerland, UK, and USA). Adults with ALS and a pathogenic repeat expansion in C9orf72 were randomly assigned within six cohorts, via Interactive Response Technology in a 3:1 ratio per cohort, to receive BIIB078 (5 mg, 10 mg, 20 mg, 35 mg, 60 mg, or 90 mg in cohorts 1-6, respectively) or placebo, via an intrathecal bolus injection. The treatment period consisted of three loading doses of study treatment, administered approximately once every 2 weeks, followed by monthly maintenance doses during a treatment period of about 3 months for cohorts 1-3 and about 6 months for cohorts 4-6. Patients and investigators were masked to treatment assignment. The primary endpoint was the incidence of adverse events and serious adverse events. This trial was registered with ClinicalTrials.gov (NCT03626012) and is completed., Findings: Between Sept 10, 2018, and Nov 17, 2021, 124 patients were screened for inclusion in the study. 18 patients were excluded and 106 participants were enrolled and randomly assigned to receive 5 mg (n=6), 10 mg (n=9), 20 mg (n=9), 35 mg (n=19), 60 mg (n=18), or 90 mg (n=18) of BIIB078, or placebo (n=27). 58 (55%) of 106 patients were female. All patients received at least one dose of study treatment and were included in all analyses. All participants had at least one adverse event; most adverse events were mild or moderate in severity and did not lead to treatment discontinuation. The most common adverse events in BIIB078-treated participants were falls, procedural pain, headache, and post lumbar puncture syndrome. 14 (18%) of 79 patients who received any dose of BIIB078 reported serious adverse events, compared with nine (33%) of 27 patients who received placebo. Five participants who received BIIB078 and three participants who received placebo had fatal adverse events: respiratory failure in a participant who received 10 mg BIIB078, ALS worsening in two participants who received 35 mg BIIB078, traumatic intracerebral haemorrhage in one participant who received 35 mg BIIB078, pulmonary embolism in one participant who received 60 mg BIIB078, and respiratory failure in three participants who received placebo. All deaths were assessed as not related to the study treatment by the reporting investigator., Interpretation: On the basis of these phase 1 study results, including secondary and exploratory findings showing no reduction in neurofilament levels and no benefit on clinical outcomes relative to the placebo cohort, BIIB078 clinical development has been discontinued. However, these results will be informative in furthering our understanding of the complex pathobiology of C9orf72-associated ALS., Funding: Biogen., Competing Interests: Declaration of interests LHvdB: advisory board for Amylyx, Biogen, Cytokinetics, Denali, QurAlis, Corcept, Novartis, Ferrer, and Sanofi. JDR: advisory board for Expansion Therapeutics and Sanofi and receives support from AbbVie Foundation, ALS Association, ALS Finding a Cure Foundation, American Airlines, Answer ALS Foundation, Aviators Against ALS, Bruce Edwards Foundation, Calico, Caterpillar Foundation, Chan Zuckerberg Initiative, Fishman Family Foundation, F Prime, Glaxo Smith Kline, Microsoft, M Armstrong, Muscular Dystrophy Association, National Football League, National Institutes of Health, Stay, Strong Vs ALS, Team Gleason, The Judith and Jean Pape Adams Charitable Foundation, Travelers Insurance, and the US Department of Defense. PJS: advisory board member for Aclipse Therapeutics, Benevolent AI, Biogen, Bristol Meyers Squibb, Darby Rimmer Foundation, Lilly, Novartis, Quell Therapeutics, QurAlis, Samsara Therapeutics, and Pangea Botanica; receives research support from US Department of Defense, EU Horizon 2020, EU Innovative Medicines Initiative, Fight MND, LifeArc, the Medical Research Council, MND Association, My Name'5 Doddie Foundation, NIHR, Pfizer, Quell Therapeutics, SwanBio, and Wolfson Foundation; and has received support for clinical trials participation from Alexion, Biogen, the EU Horizon programme, and UK NIHR. SB: received research funding from National Institutes of Health, American Academy of Neurology, American Association for Electrodiagnostic and Neuromuscular Medicine (AANEM), Muscular Dystrophy Association, OrphAI Therapeutics, Biogen, Ionis, Medicinova, Novartis, Orion, Voyager Therapeutics, and Denali Therapeutics; honoraria for patient and clinician educational activities related to ALS from AANEM Foundation, McCourt Foundation, and Medscape; institutional consulting or advisory board fees from OrphAI Therapeutics and Biogen; and platform trial coordination centre activities from HEALEY ALS. MB: consultant to Alector, Annexon, Arrowhead, Biogen, Denali, Eli Lilly, Novartis, Roche, Sanofi, UniQure, and Woolsey; clinical trial site investigator for Biogen and Orphazyme; and intellectual property licensed to Biogen and Orphazyme. RCB: advisory board for MT Pharma, has a consulting role with Biogen, has Equity in Neuroquestions LLC, and receives a recurring annual gift from a patient's family for research on neuralgic amyotrophy. AG: ad hoc consultant for genetic testing for Biogen; consultant on ALS trial design for Alexion, AL-S Pharma, Calico, Cytokinetics, and Sanofi; and CMO at QurAlis. JDG: institution was contracted by Biogen as a trial site and was paid for those services; received grants from Muscular Dystrophy Association and National Institutes of Health The Amyotrophic Lateral Sclerosis Association and received commercial sponsorships for clinical trials from Amylyx, Biogen, Cytokinetics, and Neuralstem. OH: funding from Science Foundation Ireland (grants SFI 16/RC/3948 and 20/SP/8953); received consulting fees from Accelsior, Biogen, Cytokinetics, Denali, Novartis, Orion, and Wave Pharmaceuticals; and is Editor-in-Chief of the journal Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration. VL: institution was contracted by Biogen as a trial site and was paid for those services; and Chair of independent data monitoring committee at Vico Therapeutics and member of independent data monitoring committees at QurAlis and Cyclo Therapeutics. TM: consultant to Amylyx, Biogen, Cytokinetics, and QurAlis. BO: serves as a consultant for Columbia University/Tsumura, MediciNova, Biogen, UniQure, Amylyx, and Mitsubishi and has research grants from Columbia University/Tsumura, Biogen, MediciNova, Cytokinetics, Mitsubishi, Calico, Novartis, Sanofi, Ashvattha, and TARGET ALS. GLP: medical advisory roles for Amylyx, Avanir, Biogen, Catalyst, Cytokinetics, MT Pharma, Otsuka, and the US Department of Defense. JR: serves on advisory boards to Libra, Golgi, and Transposon and consultant to Verge, Iris, and Zydus. MW: consultant to Allmiral, Biogen, Mitsubishi Tanabe Pharma, Neuraxpharm, and Novartis. LZ: received honorarium for being on a Biogen advisory board. PJ-n and FR: paid employees of Ionis Pharmaceuticals. LL, DG, MM, JI, SS, SG, and SF: employees of and may hold stock in Biogen. TAF, ALG, and SE: employees of Biogen at the time the study was conducted. CES: declares no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfR mu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfR mu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1 , across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfR mu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.

RNA interference (RNAi) is a biological process that evolved to protect eukaryotic organisms from foreign genes delivered by viruses. This process has been adapted as a powerful tool to treat numerous diseases through the delivery of small-interfering RNAs (siRNAs) to target cells to alter aberrant gene expression.Antibody-oligonucleotide conjugates (AOCs) are monoclonal antibodies with complexed siRNA or antisense oligonucleotides (ASOs) that have emerged to address some of the challenges faced by naked or chemically conjugated siRNA, which include rapid clearance from systemic circulation and lack of selective delivery of siRNA to target cells.It is essential to characterise the ADME properties of an AOC during development to optimise distribution to target tissues, to minimise the impact of biotransformation on exposure, and to characterise the PK/PD relationship to guide translation. However, owing to the complexity of AOC structure, this presents significant bioanalytical challenges, and multiple bioanalytical measurements are required to investigate the pharmacokinetics and biotransformation of the antibody, linker, and siRNA payload.In this paper, we describe an analytical workflow that details in vivo characterisation of AOCs through measurement of their distinct molecular components to provide the basis for greater understanding of their ADME properties. Although the approaches herein can be applied to in vitro characterisation of AOCs, this paper will focus on in vivo applications. This workflow relies on high-resolution mass spectrometry as the principal means of detection and leverages chromatographic, affinity-based, and enzymatic sample preparation steps.

Background: Vupanorsen is a GalNAc 3 -conjugated antisense oligonucleotide targeting angiopoietin-like 3 (ANGPTL3) mRNA shown to reduce atherogenic lipoproteins in individuals with dyslipidemia., Objectives: The aim of this study was to satisfy Chinese regulatory requirements and support ethnic sensitivity assessment by evaluating pharmacokinetics (PK), pharmacodynamics (PD), and safety of vupanorsen in healthy Chinese adults with elevated triglycerides (TG)., Methods: In this phase I, parallel-cohort, open-label study, 18 Chinese adults with elevated fasting TG (≥ 90 mg/dL) were randomized 1:1 to receive a single subcutaneous dose of vupanorsen 80 mg or 160 mg. PK parameters, PD markers (including ANGPTL3, TG, non-high-density lipoprotein cholesterol [non-HDL-C]), and safety were assessed., Results: Absorption of vupanorsen was rapid (median time to maximum concentration [T max ]: 2.0 h for both doses), followed by a multiphasic decline (mean terminal half-life 475.9 [80 mg] and 465.2 h [160 mg]). Exposure (area under curve [AUC] and maximum plasma concentration [C max ]) generally increased in a greater than dose-proportional manner from 80 mg to 160 mg. Time-dependent reductions in ANGPTL3 and lipid parameters were observed. Mean percentage change from baseline for the 80-mg and 160-mg doses, respectively, were - 59.7% and - 69.5% for ANGPTL3, - 41.9% and - 52.5% for TG, and - 23.2% and - 25.4% for non-HDL-C. No serious or severe adverse events (AEs), deaths, or discontinuations due to AEs were reported. Three participants experienced treatment-related AEs; all were mild and resolved by end of study., Conclusions: This study provided the first clinical vupanorsen data in China. In Chinese participants with elevated TG, PK and PD parameters were consistent with those reported previously in non-Chinese participants, including in Japanese individuals. No safety concerns were noted., Trial Registration: ClinicalTrials.gov identifier: NCT04916795., (© 2024. The Author(s).)

Aims: The aim of this study was to characterize the population pharmacokinetics of AZD8233, an antisense oligonucleotide (ASO) that targets the PCSK9 transcript to reduce hepatocyte PCSK9 protein production and plasma levels. AZD8233 utilizes generation 2.5 S-constrained ethyl motif (cET) chemistry and is conjugated to a triantennary N-acetylgalactosamine (GalNAc3) ligand for targeted hepatocyte uptake., Methods: A non-linear mixed-effect modelling approach utilizing NONMEM software was applied to AZD8233 concentration-time data from 3416 samples in 219 participants from four phase 1-2 studies, one in healthy volunteers (NCT03593785) and three in patients with dyslipidaemia (NCT04155645, NCT04641299 and NCT04823611)., Results: The final model described the AZD8233 plasma concentration-time profile from four phase 1-2 studies in healthy volunteers or participants with dyslipidaemia, covering a dose range of 4 to 120 mg. The pharmacokinetics of AZD8233 were adequately described by a two-compartment model with first-order absorption. The supra-proportional increase in maximum plasma concentration (C max ) across the observed dose range was described by non-linear Michaelis-Menten elimination (maximum elimination rate, 9.9 mg/h [12% relative standard error]; concentration yielding half-maximal elimination rate, 4.8 mg/L [18% relative standard error]). Body weight, sex, estimated glomerular filtration rate and disease status (healthy participant vs. patient with dyslipidaemia) were identified as factors affecting exposure to AZD8233., Conclusions: Covariate analysis showed body weight to be the main factor affecting exposure to AZD8233, which largely explained the higher C max observed in the Asian population relative to non-Asians., (© 2024 AstraZeneca. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aim: To evaluate a novel antisense oligonucleotide drug targeting human IGF-1R in preclinical and phase I studies of liver cancer. Materials & methods: The tolerability and safety of an investigational new drug were evaluated in a dose-escalation trial involving 17 patients with advanced liver cancer after preclinical assessment of pharmacokinetics and pharmacodynamics. Results: The drug exposure levels in the phase I trial were determined by the in vivo efficacy with pharmacokinetics evaluation in rats and rhesus monkeys. This clinical study showed that the maximum tolerated dose was 3.96 mg/kg, and the dose-limiting toxicity dose was 4.4 mg/kg. Conclusion: The drug was safe and tolerable in patients with advanced liver cancer. Clinical Trial Registration: ChiCTR2100044235 (www.chictr.org.cn).

Background: Antisense oligonucleotides (ASOs) have been conjugated to various moieties, such as peptides, antibodies or Fab regions of antibodies, to enhance their delivery to target tissues. The quantitation of free ASO (ASO payload) is critical to characterize its pharmacokinetics/pharmacodynamics (PK/PD) properties and biodistribution after delivery of the peptide/antibody/Fab ASO conjugates. Results: We developed a hybridization-based LC-MS/MS methodology for quantification of free ASO in tissues in the presence of Fab-ASO and ASO with linker (ASO-linker). Conclusion: The developed method was applied to measure accurately the free ASO concentrations in liver and gastrocnemius in mice that were dosed with Fab-ASO. This methodology has also been applied to free ASO bioanalysis for other antibody-ASO and Fab-ASO conjugates in various tissues and plasma/serum samples.

Quantitative bioanalysis is essential when establishing pharmacokinetic properties during the drug development process. To overcome challenges of sensitivity, specificity and process complexity associated with the conventional analysis of antisense oligonucleotides (ASOs), a new approach to nonenzymatic hybridization assays using probe alteration-linked self-assembly reaction (PALSAR) technology as a signal amplifier was evaluated. PALSAR quantification of ASOs in mouse tissue and plasma was able to achieve a high sensitivity ranging from 1.5 to 6 pg/ml, intra-/interday accuracies in the range of 86.8-119.1% and 88.1-113.1%, respectively, and precision of ≤17.2%. Furthermore, crossreactivity of 3'n-1, a metabolite with a single base difference, was <1%. Our approach provides an auspicious method for distinguishing metabolites and detecting ASOs with high sensitivity and specificity.

Objectives: We assessed the safety, tolerability, pharmacokinetics, preliminary antitumour activity and pharmacodynamics of danvatirsen, an antisense oligonucleotide targeting signal transducer and activator of transcription 3 (STAT3), monotherapy and danvatirsen plus durvalumab, an antiprogrammed cell death ligand 1 monoclonal antibody, in patients with advanced solid malignancies., Design: Phase 1, open-label study with two cohorts., Setting: Two centres in Japan., Participants: Japanese individuals aged ≥20 years, with histologically confirmed solid malignancies, except for hepatocellular carcinoma, refractory to standard therapy., Interventions: In cohort 1, patients received danvatirsen monotherapy; in cohort 2, patients received danvatirsen plus durvalumab combination therapy., Primary and Secondary Outcome Measures: The primary endpoint was safety and tolerability based on adverse events (AEs). Secondary endpoints were pharmacokinetics, immunogenicity, antitumour activity and pharmacodynamics., Results: Eleven patients were assigned to treatment and included in the analysis. Danvatirsen dose reductions were only required in cohort 2 for hepatic function abnormal (alanine aminotransferase (ALT)/ aspartate aminotransferase (AST)/gamma-glutamyl transferase (γGT) increased), neutrophil count decreased and platelet count decreased. One patient experienced grade 3 ALT/AST increased and new appearance of eosinophilia as a dose-limiting toxicity. AEs were reported in 90.9% (10/11) patients. Commonly reported AEs causally related to the danvatirsen were platelet count decreased (60% (3/5)) and ALT/AST/γGT increased (50% (3/6)) in cohorts 1 and 2, respectively; none was causally related to durvalumab. One serious AE occurred in cohort 1 (pancreatitis; unrelated to study treatment). One case of ALT/AST/γGT increased occurred in cohort 2, leading to discontinuation. No AEs led to death. Danvatirsen did not accumulate in plasma after multiple dosing. In cohort 2, three patients had disease control at 12 weeks and one had unconfirmed partial response. STAT3 expression tended to decrease regardless of monotherapy or combination therapy., Conclusions: Danvatirsen was well tolerated by Japanese patients with advanced solid tumours as monotherapy and combined with durvalumab. No new safety signals arose., Trial Registration Number: NCT03394144; ClinicalTrials.gov., Competing Interests: Competing interests: TN has received grants from AstraZeneca K.K., during the conduct of the study; grants from Taiho Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., MSD K.K., Ono Pharmaceutical Co., Ltd., Bristol-Myers Squibb K.K., Eli Lilly Japan K.K., and Sumitomo Dainippon Pharma Co., Ltd., outside the submitted work. TF is an employee of, and reports stock ownership in, AstraZeneca K.K. NY is an employee of AstraZeneca K.K. KS is an employee of, and reports stock ownership in, AstraZeneca K.K. KM is an employee of, and reports stock ownership in, AstraZeneca K.K. YK has received grants and honoraria from Taiho Pharmaceutical Co., Ltd., and Ono Pharmaceutical Co., Ltd.; honoraria from Bayer Yakuhin, Ltd., Sanofi K.K., Eli Lilly Japan K.K., and Bristol-Myers Squibb K.K.; and grants from Takeda Pharmaceutical Co., Ltd., AstraZeneca K.K., Daiichi Sankyo Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., Amgen K.K., Chugai Pharmaceutical Co., Ltd., AbbVie GK, and GlaxoSmithKline K.K., outside the submitted work., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Antisense oligonucleotide (ASO), an emerging modality in drug research and development, demands accurate and sensitive bioanalysis to understand its pharmacokinetic and pharmacodynamic properties. Results: By combining the advantages of both ligand binding and liquid chromatography-mass spectrometry/tandem mass (LC-MS/MS), hybridization LC-MS/MS methods were successfully developed and validated/qualified in a good lab practice (GLP) environment for the quantitation of an ASO drug candidate in monkey serum, cerebrospinal fluid (CSF) and tissues in the range of 0.5-500 ng/ml. Special treatment of CSF samples was employed to mitigate nonspecific binding, improve long-term storage stability and enable the usage of artificial CSF as a more accessible surrogate matrix. The method was also qualified and applied to ASO quantitation in various monkey tissue samples using a cocktail tissue homogenate as a surrogate matrix. Conclusion: This work was the first reported GLP validation and application of ASO bioanalysis using the hybridization LC-MS/MS platform.

The development of quantitative models for prediction of drug pharmacokinetics based on in vitro data has transformed early drug discovery. Drug unbound fraction (ƒ u ) characterization is a key consideration in pharmacokinetic and pharmacodynamic (PK/PD) modeling, assuming only unbound drug can interact with the target, and therefore has direct implications in the efficacy and potential toxicity of the drug. The current study describes the implementation of a hybridization liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform for the direct quantitation of antisense oligonucleotide (ASO) ƒ u The method provides substantial improvements, including minimal matrix effects and high specificity when compared with previously used oligonucleotide ƒ u detection methods such as ligand binding assays or liquid scintillation. The hybridization LC-MS/MS platform was integrated with ultracentrifugation, ultrafiltration, and equilibrium dialysis, and method performance for each technique was evaluated. Although ASO protein binding has been previously characterized in plasma, there were no studies that quantitated ASO ƒ u in brain or cerebral spinal fluid (CSF). As ASOs continue to undergo clinical trials for neurologic and neuromuscular indications, ƒ u characterization in brain and CSF can provide invaluable information about ASO distribution and target engagement in the central nervous system, therefore providing support for in vivo PK/PD data characterization. SIGNIFICANCE STATEMENT: A novel hybridization LC-MS/MS-based approach was successfully developed for the determination of ASO in vitro protein binding in plasma, and for the first time brain and CSF. Ultrafiltration, equilibrium dialysis, and ultracentrifugation were assessed for the separation of unbound ASO from biological matrices. The hybridization LC-MS/MS platform provided unique advantages, including minimal matrix effects and high specificity, compared with traditional ligand binding assays or liquid scintillation approaches, which enabled efficient and reliable in vitro protein binding assay., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Nusinersen is approved for the treatment of spinal muscular atrophy. The most common approved dosing regimen is four intrathecal loading doses of nusinersen 12 mg; the first three are administered at 14-day intervals followed by a fourth dose 30 days later, and then 12-mg maintenance doses are administered every 4 months thereafter. Interruption of nusinersen treatment in the maintenance dosing phase might occur for a number of clinical reasons., Objective: The objective of this report is to describe dosing regimens that allow for the most rapid restoration of steady-state concentrations of nusinersen in the cerebrospinal fluid (CSF) following a treatment interruption during maintenance dosing., Methods: Population pharmacokinetic models using integrated pharmacokinetic data from ten nusinersen clinical trials that included a broad range of participants with spinal muscular atrophy treated with intrathecal nusinersen were used to investigate different durations of treatment interruptions during maintenance treatment. Potential dosing regimens for re-initiation of nusinersen were evaluated, with the goal of achieving the quickest restoration of steady-state nusinersen CSF concentrations without exceeding maximal CSF exposures observed during the initial loading period., Results: Our pharmacokinetic modeling indicates the following regimen will lead to optimal restoration of nusinersen CSF levels after treatment interruption: two doses of nusinersen should be administered at 14-day intervals following treatment interruptions of ≥ 8 to < 16 months since the last dose, and three doses of nusinersen at 14-day intervals for treatment interruptions of ≥ 16 to < 40 months since the last maintenance dose, with subsequent maintenance dosing every 4 months in both instances. After treatment interruptions of ≥ 40 months, the full loading regimen will rapidly restore nusinersen CSF levels., Conclusions: Prolonged treatment interruptions lead to suboptimal CSF levels of nusinersen. The optimal regimen to restore nusinersen CSF levels depends on the interval since the last maintenance dose was administered., (© 2022. The Author(s).)

Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.

Drug properties of antisense oligonucleotides (ASOs) differ significantly from those of traditional small-molecule therapeutics. In this review, we focus on ASO disposition, mainly as characterized by distribution and biotransformation, of nonconjugated and conjugated ASOs. We introduce ASO chemistry to allow the following in-depth discussion on bioanalytical methods and determination of distribution and elimination kinetics at low concentrations over extended periods of time. The resulting quantitative data on the parent oligonucleotide, and the identification and quantification of formed metabolites define the disposition. Proper quantitative understanding of disposition is pivotal for nonclinical to clinical predictions, supports communication with health agencies, and increases the probability of delivering optimal ASO therapy to patients., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Antisense oligonucleotides (ASOs) are promising therapeutic agents for a variety of neurodegenerative and neuromuscular disorders, e.g., Alzheimer's, Parkinson's and Huntington's diseases, spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), caused by genetic abnormalities or increased protein accumulation. The blood-brain barrier (BBB) represents a challenge to the delivery of systemically administered ASOs to the relevant sites of action within the central nervous system (CNS). Intrathecal (IT) delivery, in which drugs are administered directly into the cerebrospinal fluid (CSF) space, enables to bypass the BBB. Several IT-administered ASO therapeutics have already demonstrated clinical effect, e.g., nusinersen (SMA) and tofersen (ALS). Due to novelty of IT dosing for ASOs, very limited pharmacokinetic (PK) data is available and only a few modeling reports have been generated. The objective of this work is to advance fundamental understanding of whole-body distribution of IT-administered ASOs. We propose a physiologically-based pharmacokinetic modeling approach to describe the distribution along the neuroaxis based on PK data from non-human primate (NHP) studies. We aim to understand the key processes that drive and limit ASO access to the CNS target tissues. To elucidate the trade-off between parameter identifiability and physiological plausibility of the model, several alternative model structures were chosen and fitted to the NHP data. The model analysis of the NHP data led to important qualitative conclusions that can inform projection to human. In particular, the model predicts that the maximum total exposure in the CNS tissues, including the spinal cord and brain, is achieved within two days after the IT injection, and the maximum amount absorbed by the CNS tissues is about 4% of the administered IT dose. This amount greatly exceeds the CNS exposures delivered by systemic administration of ASOs. Clearance from the CNS is controlled by the rate of transfer from the CNS tissues back to CSF, whereas ASO degradation in tissues is very slow and can be neglected. The model also describes local differences in ASO concentration emerging along the spinal CSF canal. These local concentrations need to be taken into account when scaling the NHP model to human: due to the lengthier human spinal column, inhomogeneity along the spinal CSF may cause even higher gradients and delays potentially limiting ASO access to target CNS tissues., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

IONIS-FXI RX (BAY2306001) is an antisense oligonucleotide that inhibits the synthesis of coagulation factor XI (FXI) and has been investigated in healthy volunteers and patients with end-stage renal disease (ESRD). FXI-LICA (BAY2976217) shares the same RNA sequence as IONIS-FXI RX but contains a GalNAc-conjugation that facilitates asialoglycoprotein receptor (ASGPR)-mediated uptake into hepatocytes. FXI-LICA has been studied in healthy volunteers and is currently investigated in patients with ESRD on hemodialysis. We present a model-informed bridging approach that facilitates the extrapolation of the dose-exposure-FXI relationship from IONIS-FXI RX to FXI-LICA in patients with ESRD and, thus, supports the selection of FX-LICA doses being investigated in patients with ESRD. A two-compartment pharmacokinetic (PK) model, with mixed first- and zero-order subcutaneous absorption and first-order elimination, was combined with an indirect response model for the inhibitory effect on the FXI synthesis rate via an effect compartment. This PK/pharmacodynamic model adequately described the median trends, as well as the interindividual variabilities for plasma drug concentration and FXI activity in healthy volunteers of IONIS-FXI RX and FXI-LICA, and in patients with ESRD of IONIS-FXI RX . The model was then used to predict dose-dependent steady-state FXI activity following repeat once-monthly doses of FXI-LICA in a virtual ESRD patient population. Under the assumption of similar ASGPR expression in patients with ESRD and healthy volunteers, doses of 40 mg, 80 mg, and 120 mg FXI-LICA are expected to cover the target range of clinical interest for steady-state FXI activity in the phase IIb study of FXI-LICA in patients with ESRD undergoing hemodialysis., (© 2021 Bayer AG, Research & Development. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Objective: The present study was to explore the feasibility of developing positron molecular probes for the metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), to evaluate the distribution and pharmacokinetics, and to explore whether the probe can be used for the imaging of malignant tumours with high MALAT-1 expression in vivo., Methods: [68Ga]Ga labelling of MALAT-1 antisense oligonucleotides ([68Ga]Ga-MALAT-1-ASO) was synthesized by the conjugation of MALAT-1-NOTA-ASO and [68Ga] Ga3+. The radiochemical purity was shown by radio-HPLC. Pharmacokinetic studies and cellular uptake studies were performed. The biodistribution and metabolism of [68Ga] Ga-MALAT-1-ASO in normal ICR and MHCC-LM3 xenograft-bearing nude mice were studied in vitro and in vivo., Results: [68Ga]Ga-MALAT-1-ASO was obtained in 98% radiochemical yield from a 10-min synthesis with 100 ± 50 MBq/nmol specific activity and >99% radiochemical purity. The Log D was -2.53 ± 0.19. The tracer displayed excellent stability in vitro. 68Ga-MALAT-1 ASO showed satisfactory binding ability to MHCC-LM3 cells; the biodistribution of [68Ga]Ga-MALAT-1-ASO in MHCC-LM3 tumour-bearing mice demonstrated specific uptake of the radiotracer (3.04 ± 0.11%ID/g). Micro-PET images of the MHCC-LM3 cell xenograft mouse model provided further evidence to support the hypothesis that [68Ga]Ga-MALAT-1-ASO can target tumours in vivo., Conclusions: We conclude that [68Ga]Ga labelling of MALAT-1 ASO is a convenient approach. The high accumulation of [68Ga]Ga-MALAT-1-ASO for tumours expressing MALAT-1 suggests that this radio compound may be used as a potential positron molecular probe. Molecular structure optimization studies need to be more in-depth to further reduce its background uptake and enhance tumour targeting., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The blood-brain barrier (BBB) is the most restrictive and complicated barrier that keeps most biomolecules and drugs from the brain. An efficient brain delivery strategy is urgently needed for the treatment of brain diseases. Based on the studies of brain-targeting extracellular vesicles (EVs), the potential of using small apoptotic bodies (sABs) from brain metastatic cancer cells for brain-targeting drug delivery is explored. It is found that anti-TNF- α  antisense oligonucleotide (ASO) combined with cationic konjac glucomannan (cKGM) can be successfully loaded into sABs via a transfection/apoptosis induction process and that the sABs generated by B16F10 cells have an extraordinarily high brain delivery efficiency. Further studies suggest that ASO-loaded sABs (sCABs) are transcytosed by b. End3 (brain microvascular endothelial cells, BMECs) to penetrate the BBB, which is mediated by CD44v6, and eventually taken up by microglial cells in the brain. In a Parkinson's disease (PD) mouse model, sCABs dramatically ameliorate PD symptoms via the anti-inflammatory effect of ASO. This study suggests that sABs from brain metastatic cancer cells are excellent carriers for brain-targeted delivery, as they have not only an extraordinary delivery efficiency but also a much higher scale-up production potential than other EVs., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

RNA-based therapeutics have emerged as one of the most powerful therapeutic options used for the modulation of gene/protein expression and gene editing with the potential to treat neurodegenerative diseases. However, the delivery of nucleic acids to the central nervous system (CNS), in particular by the systemic route, remains a major hurdle. This review will focus on the strategies for systemic delivery of therapeutic nucleic acids designed to overcome these barriers. Pathways and mechanisms of transport across the blood-brain barrier which could be exploited for delivery are described, focusing in particular on smaller nucleic acids including antisense oligonucleotides (ASOs) and small interfering RNA (siRNA). Approaches used to enhance delivery including chemical modifications, nanocarrier systems, and target selection (cell-specific delivery) are critically analyzed. Learnings achieved from a comparison of the successes and failures reported for CNS delivery of ASOs versus siRNA will help identify opportunities for a wider range of nucleic acids and accelerate the clinical translation of these innovative therapies.

Conjugation with lipophilic ligands such as cholesterol and α-tocopherol dramatically improves the delivery and efficacy of antisense oligonucleotides (ASOs) in the liver. To estimate the hepatic ASO concentration and the efficacy of ASOs conjugated with lipophilic ligands in mice, we constructed a pharmacokinetic-pharmacodynamic (PK-PD) model that consisted of a two-linear compartment model for the plasma and the hepatic ASO concentration, and two indirect response models for the hepatic apolipoprotein B (Apo-B) mRNA and plasma total cholesterol. The model provided a good fit of the hepatic ASO concentration although it showed an overprediction of Apo-B mRNA and an underprediction of the plasma total cholesterol within 2-fold at a later time after single intravenous administration of ASOs conjugated with lipophilic ligands. In addition, the model simulations indicated that the efficacy at a dose regimen of ASOs conjugated with lipophilic ligands (0.2 mg/kg, once a week) in mice was comparable to that at an effective dose of unchanged ASO (2.5 mg/kg, once a week). Although further studies are required to refine the parameters of the PK-PD model, this approach could be used to guide dose-ranging pharmacological studies for ASOs conjugated with lipophilic ligands in mice., (© 2021 John Wiley & Sons Ltd.)

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 N -acetyl-galactosamine (GalNAc 3 ), 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. 2'-Methoxyethyl-modified antisense oligonucleotide conjugated to GalNAc 3 (ISIS 702843) is specific for human transmembrane protease serine 6 and is currently in clinical trials for the treatment of β -thalassemia. This report summarizes a chronic toxicity study of ISIS 702843 in nonhuman primates (NHPs), including pharmacokinetic and pharmacology assessments. Suprapharmacologic doses of ISIS 702843 were well tolerated in NHPs after chronic dosing, and the data indicate that the overall safety profile is very similar to that of the unconjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) ASOs. Notably, the GalNAc 3 moiety did not cause any new toxicities nor exacerbate the known nonspecific class effects of the 2'-MOE ASOs. This observation was confirmed with multiple GalNAc 3 -MOE conjugates by querying a data base of monkey studies containing both GalNAc 3 -conjugated and unconjugated 2'-MOE ASOs. SIGNIFICANCE STATEMENT: This report documents the potency, pharmacology, and overall tolerability profile of a triantenarry N -acetyl-galactosamine (GalNAc 3 )-conjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) antisense oligonucleotide (ASO) specific to transmembrane protease serine 6 after chronic treatment in the cynomolgus monkey. Collective analysis of 15 independent GalNAc 3 -conjugated and unconjugated 2'-MOE ASOs shows the consistency in the dose response and character of hepatic and platelet tolerability across sequences that will result in much larger safety margins for the GalNAc 3 -conjugated 2'-MOE ASOs when compared with the unconjugated 2'-MOE ASOs given the increased potency., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Polyglutamine (polyQ) diseases, such as Huntington's disease and several types of spinocerebellar ataxias, are dominantly inherited progressive neurodegenerative disorders and characterized by the presence of expanded CAG trinucleotide repeats in the respective disease locus of the patient genomes. Patients with polyQ diseases currently need to rely on symptom-relieving treatments because disease-modifying therapeutic interventions remain scarce. Many disease-modifying therapeutic agents are now under clinical testing for treating polyQ diseases, but their delivery to the brain is often too invasive (e.g., intracranial injection) or inefficient, owing to in vivo degradation and clearance by physiological barriers (e.g., oral and intravenous administration). Nanoparticles provide a feasible solution for improving drug delivery to the brain, as evidenced by an increasing number of preclinical studies that document the efficacy of nanomedicines for polyQ diseases over the past 5-6 years. In this review, we present the pathogenic mechanisms of polyQ diseases, the common animal models of polyQ diseases for evaluating the efficacy of nanomedicines, and the common administration routes for delivering nanoparticles to the brain. Next, we summarize the recent preclinical applications of nanomedicines for treating polyQ diseases and improving neurological conditions in vivo , placing emphasis on antisense oligonucleotides, small peptide inhibitors, and small molecules as the disease-modifying agents. We conclude with our perspectives of the burgeoning field of "nanomedicines for polyQ diseases", including the use of inorganic nanoparticles and potential drugs as next-generation nanomedicines, development of higher-order animal models of polyQ diseases, and importance of "brain-nano" interactions.

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial-both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2'-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology., Competing Interests: The authors declare no competing interest.

Herein, we report a novel strategy to enhance the antisense activity and the pharmacokinetics of therapeutic oligonucleotides. Through the DNA hybridization chain reaction, DNA hairpins modified with poly(ethylene glycol) (PEG) form a bottlebrush architecture consisting of a double-stranded DNA backbone, PEG side chains, and antisense overhangs. The assembled structure exhibits high PEG density on the surface, which suppresses unwanted interactions between the DNA and proteins (e.g., enzymatic degradation) while allowing the antisense overhangs to hybridize with the mRNA target and thereby deplete target protein expression. We show that these PEGylated bottlebrushes targeting oncogenic KRAS can achieve much higher antisense efficacy compared with unassembled hairpins with or without PEGylation and can inhibit the proliferation of lung cancer cells bearing the G12C mutant KRAS gene. Meanwhile, these structures exhibit elevated blood retention times in vivo due to the biological stealth properties of PEG and the high molecular weight of the overall assembly. Collectively, this self-assembly approach bears the characteristics of a simple, safe, yet highly translatable strategy to improve the biopharmaceutical properties of therapeutic oligonucleotides.

Introduction: Antisense oligonucleotides (ASOs) represent a class of drugs which can be rationally designed to complement the coding or non-coding regions of target RNA transcripts. They could modulate pre-messenger RNA splicing, induce mRNA knockdown, or block translation of disease-causing genes, thereby slowing disease progression. The pharmacokinetics of intravitreal delivery may enable ASOs to be effective in the treatment of inherited retinal diseases., Areas Covered: We review the current status of clinical trials of ASO therapies for inherited retinal diseases, which have demonstrated safety, viable durability, and early efficacy. Future applications are discussed in the context of alternative genetic approaches, including gene augmentation and gene editing., Expert Opinion: Early efficacy data suggest that the splicing-modulating ASO, sepofarsen, is a promising treatment for Leber congenital amaurosis associated with the common c.2991+1655A>G mutation in CEP290 . However, potential variability in clinical response to ASO-mediated correction of splicing defect on one allele in patients who are compound heterozygotes needs to be assessed. ASOs hold great therapeutic potential for numerous other inherited retinal diseases with common deep-intronic and dominant gain-of-function mutations. These would complement viral vector-mediated gene augmentation which is generally limited by the size of the transgene and to the treatment of recessive diseases.

Inotersen (TEGSEDI™) is a 2'- O -(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. No immunogenic response was observed after long-term treatment with inotersen in mice. In monkeys, the incidence rate of IM to inotersen appeared to be dose dependent, with 28.6%-50.0% of animals developing ADAs after 36 weeks of treatment. This was characterized as late onset (median onset of 185 days) with low titers (median titer of 8, or 400 if minimum required dilution of 50 is included). The overall incidence rate of patients who developed ADAs was 30% after 65 weeks of treatment with median onset of 203 days and median peak titer of 300. IM had minimal effect on plasma peak ( C max ) and total exposure (i.e. area under curve, AUC) of inotersen, but showed elevated plasma trough levels in both IM-positive animals and humans. However, ADAs had no effect on tissue exposure, TTR messenger RNA, or plasma TTR levels in the long-term monkey study. Similarly, IM showed no effect on plasma TTR levels in clinical studies. Thus, ADAs antibodies were binding antibodies, but not neutralizing antibodies. Finally, no association was observed between IM and toxicity findings (eg, platelet, complement activation, and histopathology findings) in the inotersen 9-month monkey study. In humans, no difference was observed in hematology, including platelets, kidney function tests, or incidence of adverse events between IM-positive and -negative patients. Overall, IM showed no effect on toxicity or safety of inotersen evaluated in both monkeys and humans. ClinicalTrials.gov Identifier: NCT01737398.

Despite recent advances, targeted delivery of therapeutic oligonucleotide to extra-hepatic tissues continues to be a challenging endeavor and efficient ligand-receptor systems need to be identified. To determine the feasibility of using neurotensin to improve the productive uptake of antisense oligonucleotides (ASO), we synthesized neurotensin-ASO conjugates and evaluated their cellular uptake and activity in cells and in mice. We performed a comprehensive structure-activity relationship study of the conjugates and determined the influence of ASO charge, ASO length, peptide charge, linker chemistry and ligand identity on receptor binding and internalization. We identified a modified neurotensin peptide capable of improving the cellular uptake and activity of gapmer ASOs in sortilin expressing cells (sixfold) and in spinal cord in mice (twofold). Neurotensin conjugation also improved the potency of morpholino ASO designed to correct splicing of survival motor neuron pre-mRNA in the cortex and striatum after intracerebroventricular injection. Neurotensin-mediated targeted delivery represents a possible approach for enhancing the potency of ASOs with diverse nucleic acid modifications.

Quantitative bioanalysis in plasma and tissues samples is required to study the pharmacokinetic and pharmacodynamic properties of antisense oligonucleotides (ASOs). To overcome intrinsic drawbacks in specificity, sensitivity, and throughput of traditional ligand-binding assay (LBA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, an alternative bioanalytical method was developed by combining oligonucleotide hybridization and LC-MS/MS technologies. Target ASOs were extracted from biological samples by hybridization with biotinylated sense-strand oligonucleotides coupled to streptavidin magnetic beads. Using ion-pairing chromatography and tandem mass spectrometry, this method demonstrated high sensitivity (0.5 ng/mL using 100 μL of plasma), high specificity, wide linear range, complete automation, and generic applications in tests with multiple ASOs. The typical challenge of sensitivity drop in traditional ion-pairing LC-MS/MS was for the first time overcome by the introduction of a ternary pump system. Due to the high specificity, quantitation in various biological matrixes was achieved using calibration standards in plasma, largely improving efficiency and consistency. Another major advantage was the capability of simultaneous quantitation of ASO metabolites. The hybridization LC-MS/MS was considered an improved alternative for quantitation of ASOs and metabolites in plasma and tissue samples, showing a great potential to replace traditional LBA and LC-MS/MS methods.

Background: Antisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bacteria effectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes, but there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efficiency in bacteria., Results: Td was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Td could penetrate into bacteria more effectively with the help of Lipofectamine 2000 (LP2000) at a 0.125 μL/μg ratio to Td, but the same concentration of LP2000 had no apparent effect on linear DNA. Furthermore, linkage modes between ASOs and Td influenced gene-knockdown efficiency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efficiency than the overhung structure., Conclusions: This study established an effective antisense delivery system based on loop-armed Td, which opens opportunities for developing antisense antibiotics.

Background: Tofersen is an antisense oligonucleotide that mediates the degradation of superoxide dismutase 1 (SOD1) messenger RNA to reduce SOD1 protein synthesis. Intrathecal administration of tofersen is being studied for the treatment of amyotrophic lateral sclerosis (ALS) due to SOD1 mutations., Methods: We conducted a phase 1-2 ascending-dose trial evaluating tofersen in adults with ALS due to SOD1 mutations. In each dose cohort (20, 40, 60, or 100 mg), participants were randomly assigned in a 3:1 ratio to receive five doses of tofersen or placebo, administered intrathecally for 12 weeks. The primary outcomes were safety and pharmacokinetics. The secondary outcome was the change from baseline in the cerebrospinal fluid (CSF) SOD1 concentration at day 85. Clinical function and vital capacity were measured., Results: A total of 50 participants underwent randomization and were included in the analyses; 48 participants received all five planned doses. Lumbar puncture-related adverse events were observed in most participants. Elevations in CSF white-cell count and protein were reported as adverse events in 4 and 5 participants, respectively, who received tofersen. Among participants who received tofersen, one died from pulmonary embolus on day 137, and one from respiratory failure on day 152; one participant in the placebo group died from respiratory failure on day 52. The difference at day 85 in the change from baseline in the CSF SOD1 concentration between the tofersen groups and the placebo group was 2 percentage points (95% confidence interval [CI], -18 to 27) for the 20-mg dose, -25 percentage points (95% CI, -40 to -5) for the 40-mg dose, -19 percentage points (95% CI, -35 to 2) for the 60-mg dose, and -33 percentage points (95% CI, -47 to -16) for the 100-mg dose., Conclusions: In adults with ALS due to SOD1 mutations, CSF SOD1 concentrations decreased at the highest concentration of tofersen administered intrathecally over a period of 12 weeks. CSF pleocytosis occurred in some participants receiving tofersen. Lumbar puncture-related adverse events were observed in most participants. (Funded by Biogen; ClinicalTrials.gov number, NCT02623699; EudraCT number, 2015-004098-33.)., (Copyright © 2020 Massachusetts Medical Society.)

Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1-/-) and FcRn knockout (FcRn-/-)., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Introduction : Reduction of low-density-lipoprotein cholesterol (LDL-C) and other apolipoprotein B (apoB)-containing lipoproteins reduces cardiovascular (CV) events and greater reductions have greater benefits. Current lipid treatments cannot always achieve desirable LDL-C targets and additional or alternative treatments are often needed. Areas covered : In this article, we review the pharmacokinetics of the available and emerging treatments for hypercholesterolemia and focus on recently approved drugs and those at a late stage of development. Expert opinion : Statin pharmacokinetics are well known and appropriate drugs and doses can usually be chosen for individual patients to achieve LDL-C targets and avoid adverse effects and drug-drug interactions. Ezetimibe, icosapent ethyl and the monoclonal antibodies evolocumab and alirocumab have established efficacy and safety. Newer oral agents including pemafibrate and bempedoic acid have generally favorable pharmacokinetics supporting use in a wide range of patients. RNA-based therapies with antisense oligonucleotides are highly specific for their targets and those inhibiting apoB, apoCIII, angiopoietin-like protein 3 and lipoprotein(a) have shown promising results. The small-interfering RNA inclisiran has the notable advantage that a single subcutaneous administration may be effective for up to 6 months. The CV outcome trial results and long term safety data are eagerly awaited for these new agents.

: Antisense oligonucleotides (ASOs) are synthetically prepared short single-stranded deoxynucleotide sequences that have been validated as therapeutic agents and as a valuable tool in molecular driving biology. ASOs can block the expression of specific target genes via complementary hybridization to mRNA. Due to their high specificity and well-known mechanism of action, there has been a growing interest in using them for improving vaccine efficacy. Several studies have shown that ASOs can improve the efficacy of vaccines either by inducing antigen modification such as enhanced expression of immunogenic molecules or by targeting certain components of the host immune system to achieve the desired immune response. However, despite their extended use, some problems such as insufficient stability and low cellular delivery have not been sufficiently resolved to achieve effective and safe ASO-based vaccines. In this review, we analyze the molecular bases and the research that has been conducted to demonstrate the potential use of ASOs in vaccines., Competing Interests: The authors declare no conflict of interest.

Despite proven efficacy of pharmacotherapies targeting primarily global neurohormonal dysregulation, heart failure (HF) is a growing pandemic with increasing burden. Treatments mechanistically focusing at the cardiomyocyte level are lacking. MicroRNAs (miRNA) are transcriptional regulators and essential drivers of disease progression. We previously demonstrated that miR-132 is both necessary and sufficient to drive the pathological cardiomyocytes growth, a hallmark of adverse cardiac remodelling. Therefore, miR-132 may serve as a target for HF therapy. Here we report further mechanistic insight of the mode of action and translational evidence for an optimized, synthetic locked nucleic acid antisense oligonucleotide inhibitor (antimiR-132). We reveal the compound's therapeutic efficacy in various models, including a clinically highly relevant pig model of HF. We demonstrate favourable pharmacokinetics, safety, tolerability, dose-dependent PK/PD relationships and high clinical potential for the antimiR-132 treatment scheme.

Oligonucleotide drugs (ODs) have gained increasing attention owing to their promising therapeutic potential. One major obstacle that ODs have been facing is the lack of appropriate in vitro validation systems that can predict in vivo activity and toxicity. We have devised a transfection method called CEM (Ca 2+ -enrichment method), where the simple enrichment of calcium ion with calcium chloride in culture medium potentiates the activity of various types of naked oligonucleotides including gapmers, siRNA, and phosphorodiamidate morpholino antisense oligonucleotides (PMO) in many cultured cell lines with limited cytotoxicity. We here describe a precise procedure of the method. Besides the benefit of the CEM's predictive power to accurately estimate in vivo activity of ODs of your interest in drug discovery and development settings, this cost-efficient, easy-to-access method can be a robust laboratory technique to modulate gene expressions with ODs with a variety of mechanisms of action.

Prolonged circulation and modulation of the pharmacokinetic profile are important to improve the clinical potential of antisense oligonucleotides (ASOs). Gapmer ASOs demonstrate excellent nuclease stability and robust gene silencing activity without the requirement of transfection agents. A major challenge for in vivo applications, however, is the short blood circulatory half-life. This work describes utilization of the long circulation of serum albumin to increase the blood residence time of gapmer ASOs. The method introduces fatty acid modifications into the gapmer ASOs design to exploit the binding and transport property of serum albumin for endogenous ligands. The level of albumin-gapmer ASOs interaction, blood circulatory half-life and biodistribution was dependent on number, position, and fatty acid type (palmitic or myristic acid) within the gapmer ASO sequence and either phosphorothioate or phosphodiester backbone modifications. This work offers a strategy to optimize gapmer ASO pharmacokinetics by a proposed endogenous assembly process with serum albumin that can be tuned by gapmer ASO design modifications.

Delivery to the target site and adversities related to off-target exposure have made the road to clinical success and approval of antisense oligonucleotide (AON) therapies challenging. Various classes of AONs have distinct chemical features and pharmacological properties. Understanding the similarities and differences in pharmacokinetics (PKs) among AON classes is important to make future development more efficient and may facilitate regulatory guidance of AON development programs. For the class of 2'- O -methyl phosphorothioate (2OMe PS) RNA AONs, most nonclinical and clinical PK data available today are derived from development of exon skipping therapies for Duchenne muscular dystrophy (DMD). While some publications have featured PK aspects of these AONs, no comprehensive overview is available to date. This article presents a detailed review of absorption, distribution, metabolism, and excretion of 2OMe PS AONs, compiled from publicly available data and previously unpublished internal data on drisapersen and related exon skipping candidates in preclinical species and DMD patients. Considerations regarding drug-drug interactions, toxicokinetics, and pharmacodynamics are also discussed. From the data presented, the picture emerges of consistent PK properties within the 2OMe PS class, predictable behavior across species, and a considerable overlap with other single-stranded PS AONs. A level of detail on muscle as a target tissue is provided, which was not previously available. Furthermore, muscle biopsy samples taken in DMD clinical trials allowed confirmation of the applicability of interspecies scaling approaches commonly applied in the absence of clinical target tissue data.

Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.

Background: Eluforsen (previously known as QR-010) is a 33-mer antisense oligonucleotide under development for oral inhalation in cystic fibrosis (CF) patients with the delta F508 mutation. Previous work has shown that eluforsen restores CF transmembrane conductance regulator (CFTR) function in vitro and in vivo . To be effective, eluforsen has first to reach its primary target, the lung epithelial cells. Therefore, it has to diffuse through the CF airway surface layer (ASL), which in CF is characterized by the presence of thick and viscous mucus, impaired mucociliary clearance, and persistent infections. The goal of this study was to assess delivery of eluforsen through CF-like ASL. Methods and Results: First, air-liquid interface studies with cultured primary airway epithelial cells revealed that eluforsen rapidly diffuses through CF-like mucus at clinically relevant doses when nebulized once or repeatedly, over a range of testing doses. Furthermore, eluforsen concentrations remained stable in CF patient sputum for at least 48 hours, and eluforsen remained intact in the presence of various inhaled CF medications for at least 24 hours. When testing biodistribution of eluforsen after orotracheal administration in vivo , no differences in lung, liver, trachea, and kidney eluforsen concentration were observed between mice with a CF-like lung phenotype (ENaC-overexpressing mice) and control wild-type (WT) littermates. Also, eluforsen was visualized in the airway epithelial cell layer of CF-like muco-obstructed mice and WT littermates. Finally, studies of eluforsen uptake and binding to bacteria prevalent in CF lungs, and diffusion through bacterial biofilms showed that eluforsen was stable and not absorbed by, or bound to bacteria. In addition, eluforsen was found to be able to penetrate Pseudomonas aeruginosa biofilms. Conclusions: The thickened and concentrated CF ASL does not constitute a significant barrier for delivery of eluforsen, and feasibility of oral inhalation of eluforsen is supported by these data.

AntimiR is an antisense oligonucleotide that has been developed to silence microRNA (miRNA) for the treatment of intractable diseases. Enhancement of its in vivo efficacy and improvement of its toxicity are highly desirable but remain challenging. We here design heteroduplex oligonucleotide (HDO)-antimiR as a new technology comprising an antimiR and its complementary RNA. HDO-antimiR binds targeted miRNA in vivo more efficiently by 12-fold than the parent single-stranded antimiR. HDO-antimiR also produced enhanced phenotypic effects in mice with upregulated expression of miRNA-targeting messenger RNAs. In addition, we demonstrated that the enhanced potency of HDO-antimiR was not explained by its bio-stability or delivery to the targeted cell, but reflected an improved intracellular potency. Our findings provide new insights into biology of miRNA silencing by double-stranded oligonucleotides and support the in vivo potential of this technology based on a new class of for the treatment of miRNA-related diseases., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)