RESUMEN
Endocytosis is a mechanism by which cells sense their environment and internalize various nutrients, growth factors and signaling molecules. This process initiates at the plasma membrane, converges with autophagy, and terminates at the lysosome. It is well-established that cellular uptake of antisense oligonucleotides (ASOs) proceeds through the endocytic pathway; however, only a small fraction escapes endosomal trafficking while the majority are rendered inactive in the lysosome. Since these pathways converge and share common molecular machinery, it is unclear if autophagy-related trafficking participates in ASO uptake or whether modulation of autophagy affects ASO activity and localization. To address these questions, we investigated the effects of autophagy modulation on ASO activity in cells and mice. We found that enhancing autophagy through small-molecule mTOR inhibition, serum-starvation/fasting, and ketogenic diet, increased ASO-mediated target reduction in vitro and in vivo. Additionally, autophagy activation enhanced the localization of ASOs into autophagosomes without altering intracellular concentrations or trafficking to other compartments. These results support a novel role for autophagy and the autophagosome as a previously unidentified compartment that participates in and contributes to enhanced ASO activity. Further, we demonstrate non-chemical methods to enhance autophagy and subsequent ASO activity using translatable approaches such as fasting or ketogenic diet.
Asunto(s)
Autofagia/fisiología , Oligonucleótidos Antisentido/metabolismo , Animales , Autofagosomas/metabolismo , Transporte Biológico/fisiología , Células Cultivadas , Endocitosis/fisiología , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/genética , Interferencia de ARN , Transducción de SeñalRESUMEN
Volanesorsen (previously known as ISIS 304801) is a 20-nucleotide partially 2'-O-(2-methoxyethyl) (2'-MOE)-modified antisense oligonucleotide (ASO) gapmer, which was recently approved in the European Union as a novel, first-in-class treatment in the reduction of triglyceride levels in patients with familial chylomicronemia syndrome. We characterized the absorption, distribution, metabolism, and excretion characteristics of volanesorsen in mice, rats, monkeys, and humans, in either radiolabeled or nonradiolabeled studies. This also included the characterization of all of the observed ASO metabolite species excreted in urine. Volanesorsen is highly bound to plasma proteins that are similar in mice, monkeys, and humans. In all species, plasma concentrations declined in a multiphasic fashion, characterized by a relatively fast initial distribution phase and then a much slower terminal elimination phase following subcutaneous bolus administration. The plasma metabolite profiles of volanesorsen are similar across species, with volanesorsen as the major component. Various shortened oligonucleotide metabolites (5-19 nucleotides long) were identified in tissues in the multiple-dose mouse and monkey studies, but fewer in the [3H]-volanesorsen rat study, likely due to a lower accumulation of metabolites following a single dose in rats. In urine, all metabolites identified in tissues were observed, consistent with both endo- and exonuclease-mediated metabolism and urinary excretion being the major elimination pathway for volanesorsen and its metabolites. SIGNIFICANCE STATEMENT: We characterized the absorption, distribution, metabolism, and excretion (ADME) of volanesorsen, a partially 2'-MOE-modified antisense oligonucleotide, from mouse to man utilizing novel extraction and quantitation techniques in samples collected from preclinical toxicology studies, a 3H rat ADME study, and a phase 1 clinical trial.
Asunto(s)
Apolipoproteína C-III/antagonistas & inhibidores , Proteínas Sanguíneas/metabolismo , Oligonucleótidos/farmacocinética , Adulto , Animales , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Femenino , Voluntarios Sanos , Humanos , Hiperlipoproteinemia Tipo I/sangre , Hiperlipoproteinemia Tipo I/tratamiento farmacológico , Hiperlipoproteinemia Tipo I/genética , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Tasa de Depuración Metabólica , Ratones , Persona de Mediana Edad , Mutación , Oligonucleótidos/administración & dosificación , Ratas , Eliminación Renal , Especificidad de la Especie , Distribución Tisular , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-ß (IFN-ß) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-ß in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-ß or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-ß promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-ß and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.
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ARN Helicasas DEAD-box/inmunología , ADN/inmunología , Inmunidad Innata/inmunología , Interferón Tipo I/inmunología , Oligonucleótidos Antisentido/inmunología , Ribosa/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Alanina Transaminasa/genética , Alanina Transaminasa/inmunología , Alanina Transaminasa/metabolismo , Animales , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN/genética , ADN/metabolismo , Hepatocitos/inmunología , Hepatocitos/metabolismo , Inmunidad Innata/genética , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Helicasa Inducida por Interferón IFIH1 , Interferón beta/genética , Interferón beta/inmunología , Interferón beta/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Oligonucleótidos Antisentido/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/inmunología , Receptores de Interleucina-1/metabolismo , Ribosa/genética , Ribosa/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismoRESUMEN
Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion in the huntingtin gene (HTT) that results in a toxic gain of function in the mutant huntingtin protein (mHTT). Reducing the expression of mHTT is therefore an attractive therapy for HD. However, wild-type HTT protein is essential for development and has critical roles in maintaining neuronal health. Therapies for HD that reduce wild-type HTT may therefore generate unintended negative consequences. We have identified single-nucleotide polymorphism (SNP) targets in the human HD population for the disease-specific targeting of the HTT gene. Using primary cells from patients with HD and the transgenic YAC18 and BACHD mouse lines, we developed antisense oligonucleotide (ASO) molecules that potently and selectively silence mHTT at both exonic and intronic SNP sites. Modification of these ASOs with S-constrained-ethyl (cET) motifs significantly improves potency while maintaining allele selectively in vitro. The developed ASO is potent and selective for mHTT in vivo after delivery to the mouse brain. We demonstrate that potent and selective allele-specific knockdown of the mHTT protein can be achieved at therapeutically relevant SNP sites using ASOs in vitro and in vivo.
Asunto(s)
Enfermedad de Huntington/terapia , Proteínas Mutantes/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Oligonucleótidos Antisentido/uso terapéutico , Polimorfismo de Nucleótido Simple/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Alelos , Animales , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Silenciador del Gen , Terapia Genética , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Masculino , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Linaje , ARN Mensajero/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
The molecular mechanisms of toxicity of chemically modified phosphorothioate antisense oligonucleotides (PS-ASOs) are not fully understood. Here, we report that toxic gapmer PS-ASOs containing modifications such as constrained ethyl (cEt), locked nucleic acid (LNA) and 2'-O-methoxyethyl (2'-MOE) bind many cellular proteins with high avidity, altering their function, localization and stability. We show that RNase H1-dependent delocalization of paraspeckle proteins to nucleoli is an early event in PS-ASO toxicity, followed by nucleolar stress, p53 activation and apoptotic cell death. Introduction of a single 2'-O-methyl (2'-OMe) modification at gap position 2 reduced protein-binding, substantially decreasing hepatotoxicity and improving the therapeutic index with minimal impairment of antisense activity. We validated the ability of this modification to generally mitigate PS-ASO toxicity with more than 300 sequences. Our findings will guide the design of PS-ASOs with optimal therapeutic profiles.
Asunto(s)
Oligonucleótidos Antisentido/química , Oligonucleótidos/química , Oligonucleótidos Fosforotioatos/química , Humanos , Hígado/efectos de los fármacos , Oligonucleótidos/uso terapéutico , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos Fosforotioatos/uso terapéutico , Unión Proteica/efectos de los fármacos , Ribonucleasa H/química , Ribonucleasa H/genética , Índice TerapéuticoRESUMEN
OBJECTIVE: C3H/HeJ (C3H) mice are extremely resistant to atherosclerosis. To identify the genetic factors involved in lesion initiation, we studied a cross between C3H and the susceptible strain C57BL/6J (B6) on a hyperlipidemic (apolipoprotein E-null) background. METHODS AND RESULTS: Whereas a previous cross in mice fed a Western diet for 16 weeks revealed a very complex inheritance pattern with many significant lesion QTLs, the present cross, on a chow diet, revealed a single major locus on chromosome 9 (lod=5.0, Ath29*), and a suggestive locus on chromosome 4 (lod=2.6, Ath8). QTLs for plasma HDL, total cholesterol, and triglyceride levels were found on chromosome 1 over the ApoA2 gene. Neither of the lesion QTLs were associated with differences in plasma lipid levels or other systemic risk factors, consistent with the concept that genetic factors affecting cellular functions of the vessel wall are important determinants of atherosclerosis susceptibility. We generated a congenic strain for Ath29 and confirmed its contribution to lesion development. Toll-like receptor 4 (Tlr4), the lipopolysaccharide (LPS) receptor, is located in the Ath8 region and is known to be defective in C3H/HeJ mice. We constructed a congenic strain carrying a normal Tlr4 gene on the C3H Apoe-null background and found that the defective Tlr4 does not contribute significantly to lesion resistance during early lesion development. CONCLUSIONS: We identified one major QTL on chromosome 9, Ath29, for early lesion development in the BXH ApoE(-/-) cross fed on a chow diet and confirmed its contribution in congenic mice. We have also determined that Tlr4 on the C3H ApoE(-/-) background does not contribute to early lesion development. *Ath29 is referred to as Ath22 in Su et al 2006.
Asunto(s)
Apolipoproteínas E/metabolismo , Aterosclerosis/genética , Mapeo Cromosómico , Ratones Endogámicos C3H/genética , Sitios de Carácter Cuantitativo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Cruzamientos Genéticos , Grasas de la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Lípidos/sangre , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Factores de Riesgo , Receptor Toll-Like 4/genéticaRESUMEN
To determine if the pharmacokinetics and pharmacodynamics of gapmer antisense oligonucleotides (ASOs), containing phosphorothioate backbones and 2'-O-methoxyethyl RNA modifications (2'-MOE ASOs), can be altered by renal disease, a series of experiments were performed in models of chronic kidney disease (CKD) and acute kidney injury (AKI). In an adenine diet model of CKD, 2'-MOE ASO activity in the whole kidney was preserved and the reduction in target RNA was sustained for 2-4 weeks postdose. Additionally, 2'-MOE ASO distribution within the kidney was altered in mice with CKD, in that ASO delivery to cortical regions with tubular damage was reduced while distribution to the medulla was increased. Finally, the concentration of 2'-MOE ASO in liver of mice with CKD was elevated relative to mice without CKD, indicating a reduction in renal function and ASO excretion can potentially alter the systemic delivery of 2'-MOE ASOs. These data were generally reproduced in an aristolochic acid model of AKI, with the exception that 2'-MOE ASO activity in the whole kidney was slightly reduced with acute injury. The results from these studies have important implications for the development of 2'-MOE ASO therapeutics as both renal and extrarenal 2'-MOE ASO pharmacokinetics and pharmacodynamics may be altered in patients with renal disease. Importantly, the underlying mechanisms that alter 2'-MOE ASO distribution in the context of kidney disease warrant further examination.
Asunto(s)
Lesión Renal Aguda/terapia , Hígado/efectos de los fármacos , Oligonucleótidos Antisentido/administración & dosificación , Insuficiencia Renal Crónica/terapia , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Adenina/metabolismo , Animales , Línea Celular , Humanos , Hígado/patología , Ratones , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacocinética , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Distribución TisularRESUMEN
ISIS 104838, a 2'-O-methoxyethyl (2'-MOE)-modified antisense oligonucleotide (ASO), causes a moderate, reproducible, dose-dependent, but selflimiting decrease in platelet (PLT) counts in monkeys and humans. To determine the etiology of PLT decrease in cynomolgus monkeys, a 12-week repeat dose toxicology study in 5 cynomolgus monkeys given subcutaneous injections of ISIS 104838 (30-60 mg/kg/week). Monkeys were also injected intravenously with 111Indium(In)-oxine-labeled PLTs to investigate PLT sequestration. In response to continued dosing, PLT counts were decreased by 50%-90% by day 30 in all monkeys. PLT decreases were accompanied by 2- to 4.5-fold increases in immunoglobulin M(IgM), which were typified by a 2- to 5-fold increase in antiplatelet factor 4 (antiPF4) IgM and antiPLT IgM, respectively. Monocyte chemotactic protein 1 increased upon dosing of ISIS 104838, concomitant with a 2- to 6-fold increase in monocyte-derived extracellular vesicles (EVs), indicating monocyte activation but not PLT activation. Despite a 2- to 3-fold increase in von Willebrand factor antigen in all monkeys following ASO administration, only 2 monkeys showed a 2- to 4-fold increase in endothelial EVs. Additionally, a â¼60 - 80%% increase in PLT sequestration in liver and spleen was also observed. Collectively, these results suggest the overall increase in total IgM, antiPLT IgM and/or antiPF4 IgM, in concert with monocyte activation contributed to increased PLT sequestration in spleen and liver, leading to decreased PLTs in peripheral blood.
Asunto(s)
Plaquetas/efectos de los fármacos , Macaca fascicularis/sangre , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Fosforotioatos/farmacología , Animales , Plaquetas/citología , Quimiocina CCL2/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/metabolismo , Inmunoglobulina M/sangre , Inmunoglobulina M/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Selectina-P/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Oligonucleótidos Fosforotioatos/farmacocinética , Recuento de Plaquetas , Bazo/efectos de los fármacos , Bazo/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. METHODS: We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. RESULTS: The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. CONCLUSIONS: Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung.
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Fibrosis Quística , Canales Epiteliales de Sodio/metabolismo , Oligonucleótidos Antisentido/farmacología , Mucosa Respiratoria , Administración por Inhalación , Animales , Fibrosis Quística/metabolismo , Fibrosis Quística/fisiopatología , Modelos Animales de Enfermedad , Bloqueadores del Canal de Sodio Epitelial/farmacología , Ratones , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/fisiopatología , Fármacos del Sistema Respiratorio/farmacología , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Excipientes/farmacocinética , Hígado/metabolismo , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Fosforotioatos/farmacocinética , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Excipientes/administración & dosificación , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/citología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Fosforotioatos/administración & dosificación , Distribución TisularRESUMEN
Centronuclear myopathies (CNM) are non-dystrophic muscle diseases for which no effective therapy is currently available. The most severe form, X-linked CNM, is caused by myotubularin 1 (MTM1) loss-of-function mutations, while the main autosomal dominant form is due to dynamin2 (DNM2) mutations. We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. Here we show that systemic delivery of Dnm2 antisense oligonucleotides (ASOs) into Mtm1KO mice efficiently reduces DNM2 protein level in muscle and prevents the myopathy from developing. Moreover, systemic ASO injection into severely affected mice leads to reversal of muscle pathology within 2 weeks. Thus, ASO-mediated DNM2 knockdown can efficiently correct muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease.
Asunto(s)
Dinamina II/genética , Miopatías Estructurales Congénitas/genética , Oligonucleótidos Antisentido/genética , Proteínas Tirosina Fosfatasas no Receptoras/genética , Animales , Modelos Animales de Enfermedad , Dinamina II/metabolismo , Femenino , Riñón/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Contracción Muscular , Músculo Esquelético/metabolismo , Mutación , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/terapia , Fenotipo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Recombinación GenéticaRESUMEN
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 ß-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining ß-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-ß-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.