RESUMEN
Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.
Asunto(s)
Barrera Hematoencefálica , Oligonucleótidos Antisentido , ARN Largo no Codificante , Receptores de Transferrina , Animales , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/administración & dosificación , Barrera Hematoencefálica/metabolismo , Receptores de Transferrina/metabolismo , Humanos , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Ratones , Transporte Biológico , Macaca fascicularis , Técnicas de Silenciamiento del Gen , Distribución TisularRESUMEN
We evaluated the potential of AGTR1, the principal receptor for angiotensin II (Ang II) and a member of the G protein-coupled receptor family, for targeted delivery of antisense oligonucleotides (ASOs) in cells and tissues with abundant AGTR1 expression. Ang II peptide ASO conjugates maintained robust AGTR1 signaling and receptor internalization when ASO was placed at the N-terminus of the peptide, but not at C-terminus. Conjugation of Ang II peptide improved ASO potency up to 12- to 17-fold in AGTR1-expressing cells. Additionally, evaluation of Ang II conjugates in cells lacking AGTR1 revealed no enhancement of ASO potency. Ang II peptide conjugation improves potency of ASO in mouse heart, adrenal, and adipose tissues. The data presented in this report add to a growing list of approaches for improving ASO potency in extrahepatic tissues.
Asunto(s)
Oligonucleótidos Antisentido , Receptor de Angiotensina Tipo 1 , Animales , Ratones , Oligonucleótidos Antisentido/farmacología , Receptor de Angiotensina Tipo 1/genética , Transducción de SeñalRESUMEN
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 (â¼2-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-/-).
Asunto(s)
Oligonucleótidos Antisentido/farmacocinética , Ácido Palmítico , Albúminas/genética , Albúminas/metabolismo , Animales , Proteínas Sanguíneas/metabolismo , Caveolina 1/genética , Femenino , Corazón , Células Hep G2 , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Sistema Linfático/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Oligonucleótidos Antisentido/química , Músculo Cuádriceps/metabolismo , Receptores Fc/genética , Distribución TisularRESUMEN
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.
Asunto(s)
Músculo Esquelético , Miocardio , Oligonucleótidos Antisentido/química , Células 3T3-L1 , Albúminas/metabolismo , Animales , Colesterol/química , Interacciones Hidrofóbicas e Hidrofílicas , Lipoproteínas/metabolismo , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/toxicidad , Palmitatos/química , Ratas Sprague-Dawley , Tocoferoles/químicaRESUMEN
Reactive oxygen species such as H2O2 are believed to play a prominent role in the injury and loss of transport function that affect the intestinal epithelium in inflammatory conditions such as inflammatory bowel diseases. Defects in intestinal epithelial ion transport regulation contribute to dysbiosis and inflammatory phenotypes. We previously showed that H2O2 inhibits Ca2+-dependent Cl- secretion across intestinal epithelial cells (IECs) via a phosphatidylinositol 3-kinase (PI3K)- and extracellular signal-regulated kinase (ERK)-dependent mechanism that occurs, at least in part, through inhibition of the basolateral Na+-K+-2Cl- cotransporter NKCC1. NKCC1 governs Cl- entry into crypt IECs and thus plays a critical role in maintaining the driving force for Cl- secretion. Electrolyte transport consumes large amounts of cellular energy, and direct pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) has been shown to inhibit a number of ion transport proteins. Here, we show that H2O2 activates AMPK in human IEC lines and ex vivo human colon. Moreover, we demonstrate that the inhibitory effect of H2O2 on Ca2+-dependent Cl- secretion and NKCC1 activity is AMPK-dependent. This inhibitory effect is associated with a physical interaction between AMPK and NKCC1, as well as increased phosphorylation (Thr212,217) of NKCC1, without causing NKCC1 internalization. These data identify a key role for AMPK-NKCC1 interaction as a point of convergence for suppression of colonic epithelial ion transport by inflammatory reactive oxygen species.NEW & NOTEWORTHY H2O2 inhibition of intestinal epithelial Ca2+-dependent Cl- secretion involves recruitment of AMP-activated protein kinase (AMPK) downstream of ERK and phosphatidylinositol 3-kinase signaling pathways, physical interaction of AMPK with the Na+-K+-2Cl- cotransporter NKCC1, and AMPK-dependent suppression of NKCC1-mediated electrolyte influx without causing NKCC1 internalization. It is intriguing that, in human intestinal epithelial cell lines and human colon, H2O2 activation of AMPK increased phosphorylation of NKCC1 residues required for promoting, not inhibiting, NKCC1 activity. These data identify an elevated complexity of AMPK regulation of NKCC1 in the setting of an inflammatory stimulus.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Enfermedades Inflamatorias del Intestino , Mucosa Intestinal/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Proteínas Quinasas Activadas por AMP , Proteínas Portadoras , Células Cultivadas , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Transporte Iónico/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Músculo Esquelético/metabolismo , Miocardio/metabolismo , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacocinética , Ácido Palmítico/química , Animales , Proteínas Sanguíneas/metabolismo , Antígenos CD36/genética , Caveolina 3/genética , Ácidos Grasos/química , Ácidos Grasos Insaturados/química , Masculino , Ratones Endogámicos C57BL , Proteína Quinasa de Distrofia Miotónica/genética , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/metabolismo , ARN Largo no Codificante/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Polarización de Fluorescencia , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , Animales , Carbocianinas , Colorantes Fluorescentes , Humanos , Concentración de Iones de Hidrógeno , Ratones , Oligonucleótidos Fosforotioatos/metabolismo , Unión Proteica , Ratas , Albúmina Sérica/metabolismo , Cloruro de SodioRESUMEN
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.
Asunto(s)
Acetilgalactosamina/farmacología , Receptor de Asialoglicoproteína/metabolismo , Hepatocitos/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Acetilgalactosamina/administración & dosificación , Acetilgalactosamina/química , Animales , Relación Dosis-Respuesta a Droga , Hepatocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Conformación Molecular , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Receptores Depuradores de Clase B/antagonistas & inhibidores , Receptores Depuradores de Clase B/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Acetilgalactosamina/metabolismo , Organofosfonatos/metabolismo , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfatos/metabolismo , ARN Interferente Pequeño/farmacología , Compuestos de Vinilo/metabolismo , Acetilgalactosamina/química , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Transgénicos , Estructura Molecular , Organofosfonatos/química , Fosfohidrolasa PTEN/metabolismo , Fosfatos/química , ARN Interferente Pequeño/metabolismo , Relación Estructura-Actividad , Compuestos de Vinilo/químicaRESUMEN
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
The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog. In this report we used crystal structure of the 5'-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs containing various 5'-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity. Chemically modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.
Asunto(s)
ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Animales , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Secuencia de Bases , Sitios de Unión , LDL-Colesterol/sangre , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , Fosfatos/química , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triglicéridos/sangreRESUMEN
Differences in sensitivity of monkeys and humans to antisense oligonucleotide (ASO)-induced complement alternative pathway (AP) activation were evaluated in monkeys, humans, and in serum using biochemical assays. Transient AP activation was evident in monkeys at higher doses of two 2'-O-methoxyethyl (2'-MOE) ASOs (ISIS 426115 and ISIS 183750). No evidence of AP activation was observed in humans for either ASO, even with plasma ASO concentrations that reached the threshold for activation in monkeys. The absence of complement activation in humans is consistent with a query of the Isis Clinical Safety Database containing 767 subjects. The in vivo difference in sensitivity was confirmed in vitro, as monkey and human serum exposed to increasing concentrations of ASO indicated that monkeys were more sensitive to AP activation with this class of compounds. The mechanistic basis for the greater sensitivity of monkeys to AP activation by 2'-MOE ASO was evaluated using purified human or monkey factor H protein. The binding affinities between a representative 2'-MOE ASO and either purified protein are similar. However, the IC50 of fluid-phase complement inhibition for monkey factor H is about 3-fold greater than that for human protein using either monkey serum or factor H-depleted human serum. Interestingly, there is a sequence variant in the monkey complement factor H gene similar to a single nucleotide polymorphism in humans that is correlated with decreased factor H protein function. These findings show that monkeys are more sensitive to 2'-MOE ASO-mediated complement activation than humans likely because of differences in factor H inhibitory capacity.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Activación de Complemento/fisiología , Factor H de Complemento/genética , Comprensión , Oligonucleótidos Antisentido/farmacología , Oligorribonucleótidos/farmacología , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Humanos , Macaca fascicularis , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Oligonucleótidos , Oligonucleótidos Antisentido/genética , Oligorribonucleótidos/genética , Adulto JovenRESUMEN
Triantennary N-acetyl galactosamine (GalNAc, GN3: ), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6-10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2'-O-Et-2',4'-bridged nucleic acid) gapmer ASOs, â¼ 60-fold enhancement in potency relative to the parent MOE (2'-O-methoxyethyl RNA) ASO was observed. GN3: -conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3: -ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3: -ASO conjugate was detected in plasma suggesting that GN3: acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.
Asunto(s)
Galactosamina/análogos & derivados , Glucolípidos/química , Hepatocitos/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Animales , Apolipoproteína C-III/genética , Receptor de Asialoglicoproteína/metabolismo , Factor XI/antagonistas & inhibidores , Galactosamina/química , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligonucleótidos Antisentido/metabolismo , Prealbúmina/antagonistas & inhibidores , alfa 1-AntitripsinaRESUMEN
We evaluated the abilities of an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), and a single-stranded siRNA (ss-siRNA) to inhibit expression from the PTEN gene in mice when formulated identically with lipid nanoparticles (LNPs). Significantly greater reductions in levels of PTEN mRNA were observed for LNP-formulated agents compared to unformulated drugs when gene silencing was evaluated after a single dose in the livers of mice. An unformulated ss-siRNA modified with a metabolically stable phosphate mimic 5'-(E)-vinylphosphonate showed dose-dependent reduction of PTEN mRNA in mice, albeit at doses significantly higher than those observed for formulated ss-siRNA. These results demonstrate that LNPs can be used to deliver functional antisense and ss-siRNA therapeutics to the liver, indicating that progress in the field of siRNA delivery is transferable to other classes of nucleic acid-based drugs.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Lípidos/química , Nanopartículas/química , Oligonucleótidos Antisentido , Fosfohidrolasa PTEN/genética , ARN Interferente Pequeño , Animales , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Células HeLa , Humanos , Concentración 50 Inhibidora , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacología , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacologíaRESUMEN
The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5' phosphate to achieve activity in vivo, and we developed a metabolically stable 5'-(E)-vinylphosphonate (5'-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.
Asunto(s)
Proteínas Argonautas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Células HeLa , Hepatocitos/metabolismo , Humanos , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Organofosfonatos/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Complejo Silenciador Inducido por ARN/metabolismo , Compuestos de Vinilo/metabolismoRESUMEN
We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in T(m), cell culture and animal experiments and compare them to their gap-matched 20-mer 2'-O-methoxyethyl (MOE) and 14-mer 2',4'-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar T(m) and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED(50) 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower T(m) and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animals.
Asunto(s)
Oligodesoxirribonucleótidos Antisentido/química , Receptores Depuradores de Clase B/genética , Animales , Línea Celular , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Oligodesoxirribonucleótidos Antisentido/farmacocinética , ARN Mensajero/metabolismo , Receptores Depuradores de Clase B/metabolismo , Distribución TisularRESUMEN
Single-stranded antisense oligonucleotides (SSOs) are used to modulate the expression of genes in animal models and are being investigated as potential therapeutics. To better understand why synthetic SSOs accumulate in the same intracellular location as the target RNA, we have isolated a novel mouse hepatocellular SV40 large T-antigen carcinoma cell line, MHT that maintains the ability to efficiently take up SSOs over several years in culture. Sequence-specific antisense effects are demonstrated at low nanomolar concentrations. SSO accumulation into cells is both time and concentration dependent. At least two distinct cellular pathways are responsible for SSO accumulation in cells: a non-productive pathway resulting in accumulation in lysosomes, and a functional uptake pathway in which the SSO gains access to the targeted RNA. We demonstrate that functional uptake, as defined by a sequence-specific reduction in target mRNA, is inhibited by brefeldin A and chloroquine. Functional uptake is blocked by siRNA inhibitors of the adaptor protein AP2M1, but not by clathrin or caveolin. Furthermore, we document that treatment of mice with an AP2M1 siRNA blocks functional uptake into liver tissue. Functional uptake of SSO appears to be mediated by a novel clathrin- and caveolin-independent endocytotic process.
Asunto(s)
Hepatocitos/metabolismo , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Animales , Línea Celular Tumoral , Endocitosis , Cinética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Oligonucleótidos Antisentido/análisis , Oligonucleótidos Antisentido/química , Oligonucleótidos Fosforotioatos/análisis , Oligonucleótidos Fosforotioatos/químicaRESUMEN
Reactive oxygen species (ROS) are key mediators in a number of inflammatory conditions, including inflammatory bowel disease (IBD). ROS, including hydrogen peroxide (H(2)O(2)), modulate intestinal epithelial ion transport and are believed to contribute to IBD-associated diarrhea. Intestinal crypt fluid secretion, driven by electrogenic Cl(-) secretion, hydrates and sterilizes the crypt, thus reducing bacterial adherence. Here, we show that pathophysiological concentrations of H(2)O(2) inhibit Ca(2+)-dependent Cl(-) secretion across T(84) colonic epithelial cells by elevating cytosolic Ca(2+), which contributes to activation of two distinct signaling pathways. One involves recruitment of the Ca(2+)-responsive kinases, Src and Pyk-2, as well as extracellular signal-regulated kinase (ERK). A separate pathway recruits p38 MAP kinase and phosphoinositide 3-kinase (PI3-K) signaling. The ion transport response to Ca(2+)-dependent stimuli is mediated in part by K(+) efflux through basolateral K(+) channels and Cl(-) uptake by the Na(+)-K(+)-2Cl(-) cotransporter, NKCC1. We demonstrate that H(2)O(2) inhibits Ca(2+)-dependent basolateral K(+) efflux and also inhibits NKCC1 activity independently of inhibitory effects on apical Cl(-) conductance. Thus, we have demonstrated that H(2)O(2) inhibits Ca(2+)-dependent Cl(-) secretion through multiple negative regulatory signaling pathways and inhibition of specific ion transporters. These findings increase our understanding of mechanisms by which inflammation disturbs intestinal epithelial function and contributes to intestinal pathophysiology.