RESUMEN
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.
Asunto(s)
Ácidos Nucleicos , Ácidos Nucleicos/química , ARN/química , Fósforo , Cinética , ADN/química , Oligonucleótidos/química , Conformación de Ácido NucleicoRESUMEN
Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'-O-methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2'-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5' end of the gap or in the 3'-wing of a Gap 2'-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2'-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.
Asunto(s)
Oligonucleótidos Antisentido , Oligonucleótidos Fosforotioatos , Animales , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/química , Oligonucleótidos Fosforotioatos/genética , Oligonucleótidos Fosforotioatos/farmacología , Oligonucleótidos Fosforotioatos/química , Nucleótidos , Unión Proteica , ARN/metabolismoRESUMEN
The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association (ka) and dissociation (kd). 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.
Asunto(s)
Hidrocarburos/química , ARN/química , Cinética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Organofosfonatos/química , ARN/metabolismoRESUMEN
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.
Asunto(s)
Oligonucleótidos Antisentido , Oligonucleótidos Fosforotioatos , Animales , Endosomas/metabolismo , Ratones , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos Fosforotioatos/genética , Fósforo , Índice TerapéuticoRESUMEN
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.
Asunto(s)
Oligonucleótidos Antisentido/síntesis química , Índice Terapéutico de los Medicamentos , Animales , Células HEK293 , Células HeLa , Humanos , Hígado/metabolismo , Masculino , Mesilatos/química , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/toxicidad , Fosforamidas/química , Unión Proteica , Distribución TisularRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the main causes of cancer-related death. Sorafenib, which is the first-line therapy for this disease, is associated with reduced therapeutic efficacy that could potentially be overcome by combination with selumetinib. In this context, the main goal of this work was to develop a new nanosystem, composed of a polymeric core coated by a lipid bilayer containing the targeting ligand GalNAc, to specifically and efficiently co-deliver both drugs into HCC cells, in order to significantly increase their therapeutic efficacy. METHODS: The physicochemical characterization of hybrid nanosystems (HNP) and their components was performed by dynamic light scattering, zeta potential, matrix-assisted laser desorption ionization - time of flight mass spectroscopy, and transmission electron microscopy. Cellular binding, uptake and specificity of HNP were evaluated through flow cytometry and confocal microscopy. The therapeutic activity was evaluated namely through: cell viability by the Alamar Blue assay; cell death by flow cytometry using FITC-Annexin V; caspases activity by luminescence; mitochondrial membrane potential by flow cytometry; and molecular target levels by Western blot. RESULTS: The obtained data show that these hybrid nanosystems present high stability and loading capacity of both drugs, and suitable physicochemical properties, namely in terms of size and surface charge. Moreover, the generated formulation allows to circumvent drug resistance and presents high specificity, promoting great cell death levels in HCC cells, but not in non-tumor cells. This potentiation of the antitumor effect of co-loaded drugs was carried out by an increased programmed cell death, being associated with a strong reduction in the mitochondrial membrane potential, a significant increase in the activity of caspases 3/7 and caspase 9, and much greater number of annexin V-positive cells. CONCLUSION: The developed formulation resulted in a high and synergistic antitumor effect, revealing a translational potential to improve therapeutic approaches against HCC.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Terapia Molecular Dirigida/métodos , Nanomedicina/métodos , Animales , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/patología , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Neoplasias Hepáticas/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Sorafenib/farmacología , Sorafenib/uso terapéuticoRESUMEN
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.
Asunto(s)
ADN/química , Oligonucleótidos Antisentido/química , Animales , Glucosa/análogos & derivados , Glucosa/química , Células HeLa , Humanos , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos Antisentido/toxicidad , Compuestos Organofosforados/síntesis química , Ribonucleasa HRESUMEN
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.
Asunto(s)
Oligonucleótidos Antisentido/análisis , Oligonucleótidos Fosforotioatos/análisis , Espectrometría de Masa de Ion Secundario/métodos , Células 3T3-L1 , Acetilgalactosamina/administración & dosificación , Acetilgalactosamina/análisis , Animales , Receptor de Asialoglicoproteína/análisis , Cesio , Células HEK293 , Células HeLa , Humanos , Riñón/química , Riñón/ultraestructura , Hígado/química , Hígado/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Miocardio/química , Miocardio/ultraestructura , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Fosforotioatos/farmacocinética , Seudópodos/química , Seudópodos/ultraestructura , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/biosíntesis , ARN Largo no Codificante/genética , Fracciones Subcelulares/química , Azufre/análisis , Isótopos de Azufre/análisis , Distribución TisularRESUMEN
The activity of PS-ASOs is strongly influenced by association with both inter- and intracellular proteins. The sequence, chemical nature, and structure of the ASO can have profound influences on the interaction of PS-ASOs with specific proteins. A more thorough understanding of how these pharmacological agents interact with various proteins and how chemical modifications, sequence, and structure influence interactions with proteins is needed to inform future ASO design efforts. To better understand the chemistry of PS-ASO interactions, we have focused on human positive cofactor 4 (PC4). Although several studies have investigated the in vitro binding properties of PC4 with endogenous nucleic acids, little is known about the chemistry of interaction of PS-ASOs with this protein. Here we examine in detail the impact of ASO backbone chemistry, 2'-modifications, and buffer environment on the binding affinity of PC4. In addition, using site-directed mutagenesis, we identify those amino acids that are specifically required for ASO binding interactions, and by substitution of abasic nucleotides we identify the positions on the ASO that most strongly influence affinity for PC4. Finally, to confirm that the interactions observed in vitro are biologically relevant, we use a recently developed complementation reporter system to evaluate the kinetics and subcellular localization of the interaction of ASO and PC4 in live cells.
Asunto(s)
Proteínas de Unión al ADN/química , Oligonucleótidos Antisentido/química , Factores de Transcripción/química , Células HEK293 , Células HeLa , Humanos , CinéticaRESUMEN
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.
Asunto(s)
ADN/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Fosforotioatos/genética , Ribonucleasa H/genética , Animales , ADN/química , Ratones , Oligonucleótidos Antisentido/química , Oligonucleótidos Fosforotioatos/química , Unión Proteica/genética , Ribonucleasa H/químicaRESUMEN
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
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.
Asunto(s)
Membrana Celular/metabolismo , Citoplasma/metabolismo , Oligonucleótidos Antisentido/química , Organofosfonatos/química , Oligonucleótidos Fosforotioatos/química , Células 3T3-L1 , Animales , Caspasas/metabolismo , Línea Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Proteínas de Unión al ADN , Células HeLa , Hepatocitos/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción de Octámeros/genética , Factores de Transcripción de Octámeros/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Fosforotioatos/administración & dosificación , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismoRESUMEN
A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.
Asunto(s)
Regiones no Traducidas 5' , Oligonucleótidos Antisentido/farmacología , Proteínas Tirosina Fosfatasas/genética , Proteínas Proto-Oncogénicas/genética , Receptores de LDL/genética , Ribonucleasa H/genética , Animales , Humanos , Lipoproteínas LDL/farmacocinética , Masculino , Ratones Endogámicos BALB C , Oligonucleótidos Antisentido/química , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Mensajero/química , Receptores de LDL/metabolismo , Ribonucleasa H/metabolismoRESUMEN
Increasing the levels of therapeutic proteins in vivo remains challenging. Antisense oligonucleotides (ASOs) are often used to downregulate gene expression or to modify RNA splicing, but antisense technology has not previously been used to directly increase the production of selected proteins. Here we used a class of modified ASOs that bind to mRNA sequences in upstream open reading frames (uORFs) to specifically increase the amounts of protein translated from a downstream primary ORF (pORF). Using ASO treatment, we increased the amount of proteins expressed from four genes by 30-150% in a dose-dependent manner in both human and mouse cells. Notably, systemic treatment of mice with ASO resulted in an â¼80% protein increase of LRPPRC. The ASO-mediated increase in protein expression was sequence-specific, occurred at the level of translation and was dependent on helicase activity. We also found that the type of RNA modification and the position of modified nucleotides in ASOs affected translation of a pORF. ASOs are a useful class of therapeutic agents with broad utility.
Asunto(s)
Mejoramiento Genético/métodos , Oligonucleótidos Antisentido/genética , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas/genética , Ingeniería de Proteínas/métodos , Regulación de la Expresión Génica/genética , Marcación de Gen/métodosRESUMEN
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.
RESUMEN
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 â¼10-fold improvement in activity.
Asunto(s)
Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/síntesis química , Nitrocompuestos/síntesis química , Oligonucleótidos Antisentido/síntesis química , Propionatos/síntesis química , Acetilgalactosamina/farmacología , Animales , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Indicadores y Reactivos , Ratones , Nitrocompuestos/farmacología , Oligonucleótidos Antisentido/farmacología , Propionatos/farmacología , Receptores Depuradores de Clase B/metabolismoRESUMEN
The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.
Asunto(s)
Acetilgalactosamina/síntesis química , Acetilgalactosamina/farmacología , Hepatocitos/efectos de los fármacos , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/farmacología , Animales , Apolipoproteína C-III/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Factor XI/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Depuradores de Clase B/biosíntesis , Receptores Depuradores de Clase B/genética , Relación Estructura-ActividadRESUMEN
A convenient solid-phase synthetic method was developed for assembling a triantennary N-acetylgalactosamine (GalNAc) cluster on the 5'-end of antisense oligonucleotide using phosphoramidite chemistry. Conjugation of the 5'-triantennary GalNAc cluster improved potency of the 14 mer ASO 7-fold in mice and more than 50 fold in hepatocytes. The synthetic approach described in this Letter simplifies the synthesis of 5'-triantennary GalNAc cluster conjugated ASOs and helps understand the structure-activity relationship for targeting hepatocytes with oligonucleotide therapeutics.
Asunto(s)
Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/química , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/síntesis química , Compuestos Organofosforados/química , Receptores Depuradores de Clase B/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Hígado/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Depuradores de Clase B/metabolismo , Relación Estructura-ActividadRESUMEN
Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. â¼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. â¼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.
Asunto(s)
Acetilgalactosamina/química , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/farmacología , Animales , Células Cultivadas , Hepatocitos/citología , Hígado/citología , Ratones , Ratones Endogámicos C57BLRESUMEN
Approaches to the synthesis of the constrained 5-methyluracil nucleoside (S)-cEt-BNA, a key "gapmer" unit in a number of biologically relevant antisense oligonucleotides, are described using 5-methyluridine as starting material. In the shorter synthesis, a nine-step linear sequence afforded a O-protected (S)-cEt-BNA consisting of a [2.2.1]dioxabicycloheptane core in 7% overall yield. A competing reaction in an intramolecular cyclization of a tosylate led to a bicyclic oxetane.