Search

The efficient synthesis, antiviral activity, and duplex-stabilizing properties of both isomers of the 2'-fluoro analogue of Northern methanocarbathymidine (N-MCT), 2 and 3, are reported. We show that 2'-F incorporation on the N-MCT scaffold has a strong stabilizing effect on duplex thermal stability. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tau plays a key role in Alzheimer's disease (AD) pathophysiology, and accumulating evidence suggests that lowering tau may reduce this pathology. We sought to inhibit MAPT expression with a tau-targeting antisense oligonucleotide (MAPT Rx ) and reduce tau levels in patients with mild AD. A randomized, double-blind, placebo-controlled, multiple-ascending dose phase 1b trial evaluated the safety, pharmacokinetics and target engagement of MAPT Rx . Four ascending dose cohorts were enrolled sequentially and randomized 3:1 to intrathecal bolus administrations of MAPT Rx or placebo every 4 or 12 weeks during the 13-week treatment period, followed by a 23 week post-treatment period. The primary endpoint was safety. The secondary endpoint was MAPT Rx pharmacokinetics in cerebrospinal fluid (CSF). The prespecified key exploratory outcome was CSF total-tau protein concentration. Forty-six patients enrolled in the trial, of whom 34 were randomized to MAPT Rx and 12 to placebo. Adverse events were reported in 94% of MAPT Rx -treated patients and 75% of placebo-treated patients; all were mild or moderate. No serious adverse events were reported in MAPT Rx -treated patients. Dose-dependent reduction in the CSF total-tau concentration was observed with greater than 50% mean reduction from baseline at 24 weeks post-last dose in the 60 mg (four doses) and 115 mg (two doses) MAPT Rx groups. Clinicaltrials.gov registration number: NCT03186989 ., (© 2023. The Author(s).)

We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson-Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics. ONs containing 12- and 13-membered rings exhibited faster on-rates (up to 5-fold) and off-rates (up to 161-fold). In contrast, ONs using the larger ring size macrocycles generally exhibited smaller changes in binding kinetics relative to unmodified DNA. Interestingly, several of the analogues retained significant binding affinity for RNA based on their dissociation constants, despite being modestly destabilizing in the thermal denaturation experiments, highlighting the potential utility of measuring dissociation constants versus duplex thermal stability when evaluating novel nucleic acid analogues. Overall, our results provide additional insights into the ability of backbone-constrained macrocyclic nucleic acid analogues to modulate hybridization kinetics of modified ONs with RNA.

Although the COVID-19 pandemic began over three years ago, the virus responsible for the disease, SARS-CoV-2, continues to infect people across the globe. As such, there remains a critical need for development of novel therapeutics against SARS-CoV-2. One technology that has remained relatively unexplored in COVID-19 is the use of antisense oligonucleotides (ASOs)-short single-stranded nucleic acids that bind to target RNA transcripts to modulate their expression. In this study, ASOs targeted against the SARS-CoV-2 genome and host entry factors, ACE2 and TMPRSS2, were designed and tested for their ability to inhibit cellular infection by SARS-CoV-2. Using our previously developed SARS-CoV-2 bioassay platform, we screened 180 total ASOs targeting various regions of the SARS-CoV-2 genome and validated several ASOs that potently blocked SARS-CoV-2 infection in vitro. Notably, select ASOs retained activity against both the WA1 and B.1.1.7 (commonly known as alpha) variants. Screening of ACE2 and TMPRSS2 ASOs showed that targeting of ACE2 also potently prevented infection by the WA1 and B.1.1.7 SARS-CoV-2 viruses in the tested cell lines. Combined with the demonstrated success of ASOs in other disease indications, these results support further research into the development of ASOs targeting SARS-CoV-2 and host entry factors as potential COVID-19 therapeutics., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.W., D.G., E.S., S.F., E.E.S., and S.G. are employees of Ionis Pharmaceuticals. The remaining authors have no competing interests. We did not receive any financial support from Ionis Pharmaceuticals; they just provided the antisense oligonucleotides for the study (ASOs)., (Copyright: © 2023 Qiao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association ( k a ) and dissociation ( k d ). Single-stranded ONs are highly flexible and can adopt multiple conformations in solution, some of which may not be conducive for hybridization. We investigated if restricting rotation around the sugar-phosphate backbone, by tethering two adjacent backbone phosphonate esters using hydrocarbon bridges, can modulate hybridization kinetics of the modified ONs for complementary RNA. Given the large number of possible analogues with different tether lengths and configurations at the phosphorus atoms, we employed molecular dynamic simulations to optimize the size of the hydrocarbon bridge to guide the synthetic efforts. The backbone-constrained nucleotide trimers with stereodefined configurations at the contiguous backbone phosphorus atoms were assembled using a ring-closing metathesis reaction, then incorporated into oligonucleotides by an in situ synthesis of the phosphoramidites followed by coupling to solid supports. Evaluation of the modified oligonucleotides revealed that 15-membered macrocyclic-constrained analogues displayed similar or slightly improved on-rates but significantly increased off-rates compared to unmodified DNA ONs, resulting in reduced duplex stability. In contrast, LNA ONs with conformationally preorganized furanose rings showed similar on-rates to DNA ONs but very slow off-rates, resulting in net improvement in duplex stability. Furthermore, the experimental data generally supported the molecular dynamics simulation results, suggesting that this strategy can be used as a predictive tool for designing the next generation of constrained backbone ON analogues with improved hybridization properties.

The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O -isopropyl and O -tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.

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.)

Parkinson's disease (PD) is a prevalent neurodegenerative disease with no approved disease-modifying therapies. Multiplications, mutations, and single nucleotide polymorphisms in the SNCA gene, encoding α-synuclein (aSyn) protein, either cause or increase risk for PD. Intracellular accumulations of aSyn are pathological hallmarks of PD. Taken together, reduction of aSyn production may provide a disease-modifying therapy for PD. We show that antisense oligonucleotides (ASOs) reduce production of aSyn in rodent preformed fibril (PFF) models of PD. Reduced aSyn production leads to prevention and removal of established aSyn pathology and prevents dopaminergic cell dysfunction. In addition, we address the translational potential of the approach through characterization of human SNCA-targeting ASOs that efficiently suppress the human SNCA transcript in vivo. We demonstrate broad activity and distribution of the human SNCA ASOs throughout the nonhuman primate brain and a corresponding decrease in aSyn cerebral spinal fluid (CSF) levels. Taken together, these data suggest that, by inhibiting production of aSyn, it may be possible to reverse established pathology; thus, these data support the development of SNCA ASOs as a potential disease-modifying therapy for PD and related synucleinopathies.

We recently showed that site-specific incorporation of 2'-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5'-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5'-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2'-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5'-Me and R-5'-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5'-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.)

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.

Background: The intrathecal (IT) dosing route introduces drugs directly into the CSF to bypass the blood-brain barrier and gain direct access to the CNS. We evaluated the use of convective forces acting on the cerebrospinal fluid as a means for increasing rostral delivery of IT dosed radioactive tracer molecules and antisense oligonucleotides (ASO) in the monkey CNS. We also measured the cerebral spinal fluid (CSF) volume in a group of cynomolgus monkeys., Methods: There are three studies presented, in each of which cynomolgus monkeys were injected into the IT space with radioactive tracer molecules and/or ASO by lumbar puncture in either a low or high volume. The first study used the radioactive tracer 64 Cu-DOTA and PET imaging to evaluate the effect of the convective forces. The second study combined the injection of the radioactive tracer 99m Tc-DTPA and ASO, then used SPECT imaging and ex vivo tissue analysis of the effects of convective forces to bridge between the tracer and the ASO distributions. The third experiment evaluated the effects of different injection volumes on the distribution of an ASO. In the course of performing these studies we also measured the CSF volume in the subject monkeys by Magnetic Resonance Imaging., Results: It was consistently found that larger bolus dose volumes produced greater rostral distribution along the neuraxis. Thoracic percussive treatment also increased rostral distribution of low volume injections. There was little added benefit on distribution by combining the thoracic percussive treatment with the high-volume injection. The CSF volume of the monkeys was found to be 11.9 ± 1.6 cm 3 ., Conclusions: These results indicate that increasing convective forces after IT injection increases distribution of molecules up the neuraxis. In particular, the use of high IT injection volumes will be useful to increase rostral CNS distribution of therapeutic ASOs for CNS diseases in the clinic.

Reduction of matched and nearly complementary unintended transcripts was evaluated for 96 antisense oligonucleotides (ASOs) and 832 nearly matched unintended transcripts. The ASOs were 16-20 nucleotide "gapmers" with a gap of 8-10 DNA residues and 2'-O-methoxy-ethyl or constrained-ethyl substitutions in the wings. Most unintended transcripts were not reduced or were reduced with a potency more than 10-fold weaker than the intended transcript. For the unintended transcripts that were reduced, a strong correlation between relative potency of the intended versus the unintended transcript with predicted free energy of hybridization was observed. These results suggest ASO selectivity should be evaluated by testing for reduction of the unintended transcripts predicted to bind most stably to the ASO.

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.)

Knockout of the memory suppressor gene histone deacetylase 2 (Hdac2) in mice elicits cognitive enhancement, and drugs that block HDAC2 have potential as therapeutics for disorders affecting memory. Currently available HDAC2 catalytic activity inhibitors are not fully isoform specific and have short half-lives. Antisense oligonucleotides (ASOs) are drugs that elicit extremely long-lasting, specific inhibition through base pairing with RNA targets. We utilized an ASO to reduce Hdac2 messenger RNA (mRNA) in mice and determined its longevity, specificity, and mechanism of repression. A single injection of the Hdac2-targeted ASO in the central nervous system produced persistent reduction in HDAC2 protein and Hdac2 mRNA levels for 16 weeks. It enhanced object location memory for 8 weeks. RNA sequencing (RNA-seq) analysis of brain tissues revealed that the repression was specific to Hdac2 relative to related Hdac isoforms, and Hdac2 reduction caused alterations in the expression of genes involved in extracellular signal-regulated kinase (ERK) and memory-associated immune signaling pathways. Hdac2-targeted ASOs also suppress a nonpolyadenylated Hdac2 regulatory RNA and elicit direct transcriptional suppression of the Hdac2 gene through stalling RNA polymerase II. These findings identify transcriptional suppression of the target gene as a novel mechanism of action of ASOs., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2'-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2'-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

Prion disease is a fatal, incurable neurodegenerative disease of humans and other mammals caused by conversion of cellular prion protein (PrP; PrPC) into a self-propagating neurotoxic conformer (prions; PrPSc). Strong genetic proofs of concept support lowering PrP expression as a therapeutic strategy. Antisense oligonucleotides (ASOs) can provide a practical route to lowering one target mRNA in the brain, but their development for prion disease has been hindered by three unresolved questions from prior work: uncertainty about mechanism of action, unclear potential for efficacy against established prion infection, and poor tolerability of drug delivery by osmotic pumps. Here we test antisense oligonucleotides (ASOs) delivered by bolus intracerebroventricular injection to intracerebrally prion-infected wild-type mice. Prophylactic treatments given every 2-3 months extended survival times 61-98%, and a single injection at 120 days post-infection, near the onset of clinical signs, extended survival 55% (87 days). In contrast, a non-targeting control ASO was ineffective. Thus, PrP lowering is the mechanism of action of ASOs effective against prion disease in vivo, and infrequent, or even single, bolus injections of ASOs can slow prion neuropathogenesis and markedly extend survival, even when initiated near clinical signs. These findings should empower development of PrP-lowering therapy for prion disease.

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Huntington's disease is an autosomal-dominant neurodegenerative disease caused by CAG trinucleotide repeat expansion in HTT , resulting in a mutant huntingtin protein. IONIS-HTT Rx (hereafter, HTT Rx ) is an antisense oligonucleotide designed to inhibit HTT messenger RNA and thereby reduce concentrations of mutant huntingtin., Methods: We conducted a randomized, double-blind, multiple-ascending-dose, phase 1-2a trial involving adults with early Huntington's disease. Patients were randomly assigned in a 3:1 ratio to receive HTT Rx or placebo as a bolus intrathecal administration every 4 weeks for four doses. Dose selection was guided by a preclinical model in mice and nonhuman primates that related dose level to reduction in the concentration of huntingtin. The primary end point was safety. The secondary end point was HTT Rx pharmacokinetics in cerebrospinal fluid (CSF). Prespecified exploratory end points included the concentration of mutant huntingtin in CSF., Results: Of the 46 patients who were enrolled in the trial, 34 were randomly assigned to receive HTT Rx (at ascending dose levels of 10 to 120 mg) and 12 were randomly assigned to receive placebo. Each patient received all four doses and completed the trial. Adverse events, all of grade 1 or 2, were reported in 98% of the patients. No serious adverse events were seen in HTT Rx -treated patients. There were no clinically relevant adverse changes in laboratory variables. Predose (trough) concentrations of HTT Rx in CSF showed dose dependence up to doses of 60 mg. HTT Rx treatment resulted in a dose-dependent reduction in the concentration of mutant huntingtin in CSF (mean percentage change from baseline, 10% in the placebo group and -20%, -25%, -28%, -42%, and -38% in the HTT Rx 10-mg, 30-mg, 60-mg, 90-mg, and 120-mg dose groups, respectively)., Conclusions: Intrathecal administration of HTT Rx to patients with early Huntington's disease was not accompanied by serious adverse events. We observed dose-dependent reductions in concentrations of mutant huntingtin. (Funded by Ionis Pharmaceuticals and F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02519036.)., (Copyright © 2019 Massachusetts Medical Society.)

Background: Microglia are multifunctional cells that are key players in brain development and homeostasis. Recent years have seen tremendous growth in our understanding of the role microglia play in neurodegeneration, CNS injury, and developmental disorders. Given that microglia show diverse functional phenotypes, there is a need for more precise tools to characterize microglial states. Here, we experimentally define gene modules as the foundation for describing microglial functional states., Results: In an effort to develop a comprehensive classification scheme, we profiled transcriptomes of mouse microglia in a stimulus panel with 96 different conditions. Using the transcriptomic data, we generated fine-resolution gene modules that are robustly preserved across datasets. These modules served as the basis for a combinatorial code that we then used to characterize microglial activation under various inflammatory stimulus conditions., Conclusions: The microglial gene modules described here were robustly preserved, and could be applied to in vivo as well as in vitro conditions to dissociate the signaling pathways that distinguish acutely inflamed microglia from aged microglia. The microglial gene modules presented here are a novel resource for classifying and characterizing microglial states in health and disease.

Interactions of chemically modified nucleic acid therapeutics with plasma proteins play an important role in facilitating distribution from the injection site to peripheral tissues by reducing renal clearance. Despite the importance of these interactions, analytical methods that can characterize binding constants with individual plasma proteins in a reliable and high throughput manner are not easily available. We developed a fluorescence polarization (FP) based assay and measured binding constants for the 25 most abundant human plasma proteins with phosphorothioate (PS) modified antisense oligonucleotides (ASOs). We evaluated the influence of sequence, sugar modifications, and PS content on ASO interactions with several abundant human plasma proteins and determined the effect of salt and pH on these interactions. PS ASOs were found to associate predominantly with albumin and histidine-rich glycoprotein (HRG) in mouse and human plasma by size-exclusion chromatography. In contrast, PS ASOs associate predominantly with HRG in monkey plasma because of higher concentrations of this protein in monkeys. Finally, plasma proteins capable of binding PS ASOs in human plasma were confirmed by employing affinity chromatography and proteomics. Our results indicate distinct differences in contributions from the PS backbone, nucleobase composition and oligonucleotide flexibility to protein binding., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We recently reported that ( E )-5'-vinylphosphonate (5'-VP) is a metabolically-stable phosphate mimic for siRNA and demonstrated that 5'-VP improves the potency of the fully modified siRNAs in vivo. Here, we report an alternative synthesis of 5'-VP modified guide strand using S -pivaloyl-2-thioethyl ( t Bu-SATE) protecting group. The t Bu-SATE group is readily removed during the final cleavage of the oligonucleotide from the solid support and providing a more convenient route for the synthesis of siRNA guide strand carrying a 5'-vinylphosphonate.

The potency of antisense oligonucleotide (ASO) drugs has significantly improved in the clinic after exploiting asialoglycoprotein receptor (ASGR) mediated delivery to hepatocytes. To further this technology, we evaluated the structure-activity relationships of oligonucleotide chemistry on in vivo potency of GalNAc-conjugated Gapmer ASOs. GalNAc conjugation improved potency of ASOs containing 2'-O-methyl (2'-O-Me), 3'-fluoro hexitol nucleic acid (FHNA), locked nucleic acid (LNA), and constrained ethyl bicyclo nucleic acid (cEt BNA) 10-20-fold compared to unconjugated ASOs. We further demonstrate that GalNAc conjugation improves activity of 2'-O-(2-methoxyethyl) (2'-O-MOE) and Morpholino ASOs designed to correct splicing of survival motor neuron (SMN2) pre-mRNA in liver after subcutaneous administration. GalNAc modification thus represents a viable strategy for enhancing potency of ASO with diverse nucleic acid modifications and mechanisms of action for targets expressed in hepatocytes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited ataxia caused by expansion of a translated CAG repeat encoding a glutamine tract in the ataxin-1 (ATXN1) protein. Despite advances in understanding the pathogenesis of SCA1, there are still no therapies to alter its progressive fatal course. RNA-targeting approaches have improved disease symptoms in preclinical rodent models of several neurological diseases. Here, we investigated the therapeutic capability of an antisense oligonucleotide (ASO) targeting mouse Atxn1 in Atxn1154Q/2Q-knockin mice that manifest motor deficits and premature lethality. Following a single ASO treatment at 5 weeks of age, mice demonstrated rescue of these disease-associated phenotypes. RNA-sequencing analysis of genes with expression restored to WT levels in ASO-treated Atxn1154Q/2Q mice was used to demonstrate molecular differences between SCA1 pathogenesis in the cerebellum and disease in the medulla. Finally, select neurochemical abnormalities detected by magnetic resonance spectroscopy in vehicle-treated Atxn1154Q/2Q mice were reversed in the cerebellum and brainstem (a region containing the pons and the medulla) of ASO-treated Atxn1154Q/2Q mice. Together, these findings support the efficacy and therapeutic importance of directly targeting ATXN1 RNA expression as a strategy for treating both motor deficits and lethality in SCA1.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder characterized by cerebellar and retinal degeneration, and is caused by a CAG-polyglutamine repeat expansion in the ATAXIN-7 gene. Patients with SCA7 develop progressive cone-rod dystrophy, typically resulting in blindness. Antisense oligonucleotides (ASOs) are single-stranded chemically modified nucleic acids designed to mediate the destruction, prevent the translation, or modify the processing of targeted RNAs. Here, we evaluated ASOs as treatments for SCA7 retinal degeneration in representative mouse models of the disease after injection into the vitreous humor of the eye. Using Ataxin-7 aggregation, visual function, retinal histopathology, gene expression, and epigenetic dysregulation as outcome measures, we found that ASO-mediated Ataxin-7 knockdown yielded improvements in treated SCA7 mice. In SCA7 mice with retinal disease, intravitreal injection of Ataxin-7 ASOs also improved visual function despite initiating treatment after symptom onset. Using color fundus photography and autofluorescence imaging, we also determined the nature of retinal degeneration in human SCA7 patients. We observed variable disease severity and cataloged rapidly progressive retinal degeneration. Given the accessibility of neural retina, availability of objective, quantitative readouts for monitoring therapeutic response, and the rapid disease progression in SCA7, ASOs targeting ATAXIN-7 might represent a viable treatment for SCA7 retinal degeneration., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) protein, resulting in acquisition of toxic functions. Previous studies have shown that lowering mutant HTT has the potential to be broadly beneficial. We previously identified HTT single-nucleotide polymorphisms (SNPs) tightly linked to the HD mutation and developed antisense oligonucleotides (ASOs) targeting HD-SNPs that selectively suppress mutant HTT. We tested allele-specific ASOs in a mouse model of HD. Both early and late treatment reduced cognitive and behavioral impairments in mice. To determine the translational potential of the treatment, we examined the effect of ASO administration on HTT brain expression in nonhuman primates. The treatment induced robust HTT suppression throughout the cortex and limbic system, areas implicated in cognition and psychiatric function. The results suggest that ASOs specifically targeting mutated HTT might have therapeutic effects on HD-mediated cognitive impairments., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% of familial ALS. Given the gain of toxic function in this dominantly inherited disease, lowering SOD1 mRNA and protein is predicted to provide therapeutic benefit. An early generation antisense oligonucleotide (ASO) targeting SOD1 was identified and tested in a phase I human clinical trial, based on modest protection in animal models of SOD1 ALS. Although the clinical trial provided encouraging safety data, the drug was not advanced because there was progress in designing other, more potent ASOs for CNS application. We have developed next-generation SOD1 ASOs that more potently reduce SOD1 mRNA and protein and extend survival by more than 50 days in SOD1G93A rats and by almost 40 days in SOD1G93A mice. We demonstrated that the initial loss of compound muscle action potential in SOD1G93A mice is reversed after a single dose of SOD1 ASO. Furthermore, increases in serum phospho-neurofilament heavy chain levels, a promising biomarker for ALS, are stopped by SOD1 ASO therapy. These results define a highly potent, new SOD1 ASO ready for human clinical trial and suggest that at least some components of muscle response can be reversed by therapy.

Oligonucleotide therapeutics have emerged as a third distinct platform for drug discovery within the pharmaceutical industry. Five oligonucleotide-based drugs have been approved by the US FDA and over 100 oligonucleotides drugs are currently at different stages of human trials. Several of these oligonucleotide drugs are modified using the phosphorothioate (PS) backbone modification where one of the nonbridging oxygen atoms of the phosphodiester linkage is replaced with sulfur. In this review, we summarize our knowledge on receptor-mediated uptake of PS antisense oligonucleotides (ASOs) within different cell types of the liver-a privileged organ for the discovery of oligonucleotide-based therapeutics.

Charcot-Marie-Tooth disease type 1A (CMT1A) is caused by duplication of peripheral myelin protein 22 (PMP22) and is the most common hereditary peripheral neuropathy. CMT1A is characterized by demyelination and axonal loss, which underlie slowed motor nerve conduction velocity (MNCV) and reduced compound muscle action potentials (CMAP) in patients. There is currently no known treatment for this disease. Here, we show that antisense oligonucleotides (ASOs) effectively suppress PMP22 mRNA in affected nerves in 2 murine CMT1A models. Notably, initiation of ASO treatment after disease onset restored myelination, MNCV, and CMAP almost to levels seen in WT animals. In addition to disease-associated gene expression networks that were restored with ASO treatment, we also identified potential disease biomarkers through transcriptomic profiling. Furthermore, we demonstrated that reduction of PMP22 mRNA in skin biopsies from ASO-treated rats is a suitable biomarker for evaluating target engagement in response to ASO therapy. These results support the use of ASOs as a potential treatment for CMT1A and elucidate potential disease and target engagement biomarkers for use in future clinical trials.

There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several ASOs are currently being tested in clinical trials for various neurological disorders. Here, we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that activity of ASOs lasts up to 2 months after a single intrathecal bolus dose. Interestingly, comparison of subcutaneous, intracerebroventricular, and intrathecal administration shows that DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations, thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.

No treatments exist to slow or halt Parkinson's disease (PD) progression; however, inhibition of leucine-rich repeat kinase 2 (LRRK2) activity represents one of the most promising therapeutic strategies. Genetic ablation and pharmacological LRRK2 inhibition have demonstrated promise in blocking α-synuclein (α-syn) pathology. However, LRRK2 kinase inhibitors may reduce LRRK2 activity in several tissues and induce systemic phenotypes in the kidney and lung that are undesirable. Here, we test whether antisense oligonucleotides (ASOs) provide an alternative therapeutic strategy, as they can be restricted to the CNS and provide a stable, long-lasting reduction of protein throughout the brain. Administration of LRRK2 ASOs to the brain reduces LRRK2 protein levels and fibril-induced α-syn inclusions. Mice exposed to α-syn fibrils treated with LRRK2 ASOs show more tyrosine hydroxylase (TH)-positive neurons compared to control mice. Furthermore, intracerebral injection of LRRK2 ASOs avoids unwanted phenotypes associated with loss of LRRK2 expression in the periphery. This study further demonstrates that a reduction of endogenous levels of normal LRRK2 reduces the formation of α-syn inclusions. Importantly, this study points toward LRRK2 ASOs as a potential therapeutic strategy for preventing PD-associated pathology and phenotypes without causing potential adverse side effects in peripheral tissues associated with LRRK2 inhibition., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphorothioate (PS) modified antisense oligonucleotides (ASOs) have progressed rapidly in the clinic for treating a variety of disease indications. We previously demonstrated that the activity of PS ASOs in the liver can be enhanced by co-infusion of an excipient oligonucleotide (EON). It was posited that the EON saturates a nonproductive uptake pathway(s) thereby permitting accumulation of the PS ASO in a productive tissue compartment. In this report, we measured PS ASO activity following administration by bolus, infusion or co-fusion with EON within hepatocytes and nonparenchymal cells (NPCs), of the liver. This revealed that while ASOs accumulate preferentially in NPCs, they are intrinsically more active in hepatocytes. Furthermore, we show that the EON enhances ASO potency when infused up to 72 h before or after administration of the active ASO suggesting that the EON can saturate and displace the ASO from nonproductive to productive compartments. Physical presence of the EON in tissues was required for optimal potentiation suggesting that there is a dynamic distribution of the ASO and EON between the compartments. Lastly, using a candidate approach, we confirmed Stabilin-2 as a molecular pathway for ASO uptake in sinusoidal endothelial cells and the ASGR as a pathway for ASO uptake into hepatocytes in the liver.

Antisense oligonucleotides (ASOs) have the potential to discriminate between subtle RNA mismatches such as SNPs. Certain mismatches, however, allow ASOs to bind at physiological conditions and result in RNA cleavage mediated by RNase H. We showed that replacing DNA nucleotides in the gap region of an ASO with other chemical modification can improve allele selectivity. Herein, we systematically substitute every position in the gap region of an ASO targeting huntingtin gene (HTT) with fluorinated nucleotides. Potency is determined in cell culture against mutant HTT (mtHTT) and wild-type HTT (wtHTT) mRNA and RNase H cleavage intensities, and patterns are investigated. This study profiled five different fluorinated nucleotides and showed them to have predictable, site-specific effects on RNase H cleavage, and the cleavage patterns were rationalized from a published X-ray structure of human RNase H1. The results herein can be used as a guide for future projects where ASO discrimination of SNPs is important., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs ∼30-fold in the clinic (1). In order to fully characterize the effect of GalNAc valency, oligonucleotide length, flexibility and chemical composition on ASGR binding, we tested and validated a fluorescence polarization competition binding assay. The ASGR binding, and in vitro and in vivo activities of 1, 2 and 3 GalNAc conjugated single stranded and duplexed ASOs were studied. Two and three GalNAc conjugated single stranded ASOs bind the ASGR with the strongest affinity and display optimal in vitro and in vivo activities. 1 GalNAc conjugated ASOs showed 10-fold reduced ASGR binding affinity relative to three GalNAc ASOs but only 2-fold reduced activity in mice. An unexpected observation was that the ASGR also appears to play a role in the uptake of unconjugated phosphorothioate modified ASOs in the liver as evidenced by the loss of activity of GalNAc conjugated and unconjugated ASOs in ASGR knockout mice. Our results provide insights into how backbone charge and chemical composition assist in the binding and internalization of highly polar anionic single stranded oligonucleotides into cells and tissues., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global benefit. Thus there is a need for preclinical models of HD recapitulating human HTT genetics. We previously generated a humanized mouse model of HD, Hu97/18, by intercrossing BACHD and YAC18 mice with knockout of the endogenous mouse HD homolog (Hdh). Hu97/18 mice recapitulate the genetics of HD, having two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of Caucasian descent. We have now generated a companion model, Hu128/21, by intercrossing YAC128 and BAC21 mice on the Hdh-/- background. Hu128/21 mice have two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of East Asian descent and in a minority of patients from other ethnic groups. Hu128/21 mice display a wide variety of HD-like phenotypes that are similar to YAC128 mice. Additionally, both transgenes in Hu128/21 mice match the human HTT exon 1 reference sequence. Conversely, the BACHD transgene carries a floxed, synthetic exon 1 sequence. Hu128/21 mice will be useful for investigations of human HTT that cannot be addressed in Hu97/18 mice, for developing therapies targeted to exon 1, and for preclinical screening of personalized HTT lowering therapies in HD patients of East Asian descent., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Accumulation of hyperphosphorylated tau directly correlates with cognitive decline in Alzheimer's disease and other primary tauopathies. One therapeutic strategy may be to reduce total tau expression. We identified antisense oligonucleotides (ASOs) that selectively decreased human tau mRNA and protein in mice expressing mutant P301S human tau. After reduction of human tau in this mouse model of tauopathy, fewer tau inclusions developed, and preexisting phosphorylated tau and Thioflavin S pathology were reversed. The resolution of tau pathology was accompanied by the prevention of hippocampal volume loss, neuronal death, and nesting deficits. In addition, mouse survival was extended, and pathological tau seeding was reversed. In nonhuman primates, tau ASOs distributed throughout the brain and spinal cord and reduced tau mRNA and protein in the brain, spinal cord, and cerebrospinal fluid. These data support investigation of a tau-lowering therapy in human patients who have tau-positive inclusions even after pathological tau deposition has begun., (Copyright © 2017, American Association for the Advancement of Science.)

Triantennary N-acetyl galactosamine (GalNAc 3 )-conjugated antisense oligonucleotides (ASOs) have greatly improved potency due to receptor-mediated uptake into hepatocyte. The disposition and pharmacokinetics of ISIS 681257, a GalNAc 3 -conjugated ASO, were studied in monkeys. Following subcutaneous (SC) injection, ISIS 681257 was rapidly absorbed into the systemic circulation, with peak plasma levels observed within hours after dosing. After reaching C max , plasma concentrations rapidly declined in a multiexponential manner and were characterized by a dominant initial rapid distribution phase in which drug transferred to tissues from circulation, followed by a much slower terminal elimination phase (half-life of 4 weeks). Intact ISIS 681257 is the major full-length oligonucleotide species in plasma (≥70%). In tissues, the conjugated-GalNAc sugar moiety was rapidly metabolized, leaving the fully unconjugated form as the only full-length oligonucleotide detected at 48 h after dosing. Unconjugated ISIS 681257 cleared slowly from tissues with a half-life of 4 weeks. ISIS 681257 was highly bound to plasma proteins (>97% bound), which limited its urinary excretion. Disposition of ISIS 681257 in plasma and liver appeared nonlinear over the 1-40 mg/kg dose range studied. The plasma and liver tissue concentration data were well described by a population based mixed-effects modeling approach with Michaelis-Menten uptake from plasma to liver. Safety data from the study and the good exposure, as well as the extended half-life of the unconjugated ASO in the liver, support further development and less frequent dosing in Phase I clinical study.

Human haploinsufficiency of the transcription factor Tcf4 leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS), which is associated with severe language impairment and development delay. Here, we demonstrate that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4(+/-) mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, we found that small-molecule HDAC inhibitors normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified "memory-associated" genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, we observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4(+/-) mice., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)-a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5-10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

To investigate the pharmacokinetics (PKs) and pharmacodynamics (PDs) for ION-353382, an antisense oligonucleotide (ASO) targeting scavenger receptor class B type I (SRB1) mRNA, using alpha-2-macroglobulin (A2M), murinoglobulin double-knockout (DKO), and wild-type mice. Wild-type and DKO homozygous mice were administered a single subcutaneous injection of ION-353382 at 0, 5, 15, 30, and 60 mg/kg. Mice were sacrificed at 72 h with plasma and organs harvested. Both liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA) were used to determine ASO exposure with real-time PCR for SRB1 expression. Immunohistochemistry was evaluated to explore hepatic uptake of ASOs. The total plasma protein binding and profiling was assessed. Finally, two-dimensional gel electrophoresis identified protein expression differences. PK exposures were comparable between wild-type and DKO mice in plasma, liver, and kidney, yet a near twofold reduction in EC50 was revealed for DKO mice based on an inhibitory effect liver exposure response model. Total plasma protein binding and profiling revealed no major dissimilarities between both groups. Plasma proteome fingerprinting confirmed protein expression variations related to A2M. Histological examination revealed enhanced ASO distribution into hepatocytes and less nonparenchymal uptake for DKO mice compared to wild-type mice. Knocking out A2M showed improved PD activities without an effect on total plasma and tissue exposure kinetics. Binding to A2M could mediate ASOs to nonproductive compartments, and thus, decreased binding of ASOs to A2M could potentially improve ASO pharmacology.

Chemical modifications are essential to improve metabolic stability and pharmacokinetic properties of siRNA to enable their systemic delivery. We investigated the effect of combing the phosphorothioate (PS) modification with metabolically stable phosphate analog (E)-5'-vinylphosphonate and GalNAc cluster conjugation on the activity of fully 2'-modified siRNA in cell culture and mice. Our data suggest that integrating multiple chemical approaches in one siRNA molecule improved potency 5-10 fold and provide a roadmap for developing more efficient siRNA drugs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Triantennary N-acetyl galactosamine (GalNAc3) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of (3)H-radiolabeled ((3)H placed in THA) or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5'-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs.

Triantennary N-acetyl galactosamine (GalNAc3)-conjugated antisense oligonucleotides (ASOs) have greatly improved potency via receptor-mediated uptake. In the present study, the in vivo pharmacology of a 2'-O-(2-methoxyethyl)-modified ASO conjugated with GalNAc3 (ISIS 681257) together with its unmodified congener (ISIS 494372) targeting human apolipoprotein (a) (apo(a)), were studied in human LPA transgenic mice. Further, the disposition kinetics of ISIS 681257 was studied in CD-1 mice. ISIS 681257 demonstrated over 20-fold improvement in potency over ISIS 494372 as measured by liver apo(a) mRNA and plasma apo(a) protein levels. Following subcutaneous (SC) dosing, ISIS 681257 cleared rapidly from plasma and distributed to tissues. Intact ISIS 681257 was the major full-length oligonucleotide species in plasma. In tissues, however, GalNAc sugar moiety was rapidly metabolized and unconjugated ISIS 681257 accounted > 97% of the total exposure, which was then cleared slowly from tissues with a half-life of 7-8 days, similar to the half-life in plasma. ISIS 681257 is highly bound to plasma proteins (> 94% bound), which limited its urinary excretion. This study confirmed dose-dependent exposure to the parent drug ISIS 681257 in plasma and rapid conversion to unconjugated ASO in tissues. Safety data and the extended half-life support its further development and weekly dosing in phase 1 clinical studies.

A convenient method for the synthesis of several triantennary GalNAc clusters based on a nitromethanetrispropionic acid core was developed. The synthetic approach involves pentafluorophenolic ester intermediates which can be used in a one-pot, seven reaction procedure to quickly prepare a variety of triantennary GalNAc conjugated ASOs. The GalNAc clusters were conjugated to the 5'-end of an antisense oligonucleotide and evaluated for activity in primary mouse hepatocytes where they showed ∼10-fold improvement in activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)