RESUMEN
Selective and controlled expansion of endogenous ß-cells has been pursued as a potential therapy for diabetes. Ideally, such therapies would preserve feedback control of ß-cell proliferation to avoid excessive ß-cell expansion and an increased risk of hypoglycemia. Here, we identified a regulator of ß-cell proliferation whose inactivation results in controlled ß-cell expansion: the protein deacetylase Sirtuin 2 (SIRT2). Sirt2 deletion in ß-cells of mice increased ß-cell proliferation during hyperglycemia with little effect in homeostatic conditions, indicating preservation of feedback control of ß-cell mass. SIRT2 restrains proliferation of human islet ß-cells cultured in glucose concentrations above the glycemic set point, demonstrating conserved SIRT2 function. Analysis of acetylated proteins in islets treated with a SIRT2 inhibitor revealed that SIRT2 deacetylates enzymes involved in oxidative phosphorylation, dampening the adaptive increase in oxygen consumption during hyperglycemia. At the transcriptomic level, Sirt2 inactivation has context-dependent effects on ß-cells, with Sirt2 controlling how ß-cells interpret hyperglycemia as a stress. Finally, we provide proof-of-principle that systemic administration of a GLP1-coupled Sirt2-targeting antisense oligonucleotide achieves ß-cell selective Sirt2 inactivation and stimulates ß-cell proliferation under hyperglycemic conditions. Overall, these studies identify a therapeutic strategy for increasing ß-cell mass in diabetes without circumventing feedback control of ß-cell proliferation.
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Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). Current therapies such as chemotherapy and radiation have limited efficacy, and new treatment options are needed urgently. We have previously shown that FLC tumors are dependent on the fusion kinase DNAJB1::PRKACA, making the oncokinase an ideal drug target. mRNA degrading modalities such as antisense oligonucleotides or small interfering RNAs (siRNAs) provide an opportunity to specifically target the fusion junction. Here, we identify a potent and specific siRNA that inhibits DNAJB1::PRKACA expression. We found expression of the asialoglycoprotein receptor in FLC to be maintained at sufficient levels to effectively deliver siRNA conjugated to the GalNAc ligand. We observe productive uptake and siRNA activity in FLC patient-derived xenografts (PDX) models in vitro and in vivo. Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/metabolismo , ARN Interferente Pequeño/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ARN Bicatenario , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismoRESUMEN
Insulin resistance is the major risk factor for Type 2 diabetes (T2D). In vulnerable individuals, insulin resistance induces a progressive loss of insulin secretion with islet pathology revealing a partial deficit of beta cells and islet amyloid derived from islet amyloid polypeptide (IAPP). IAPP is co-expressed and secreted with insulin by beta cells, expression of both proteins being upregulated in response to insulin resistance. If IAPP expression exceeds the threshold for clearance of misfolded proteins, beta cell failure occurs exacerbated by the action of IAPP toxicity to compromise the autophagy lysosomal pathway. We postulated that suppression of IAPP expression by an IAPP antisense oligonucleotide delivered to beta cells by the GLP-1 agonist exenatide (eGLP1-IAPP-ASO) is a potential disease modifying therapy for T2D. While eGLP1-IAPP-ASO suppressed mouse IAPP and transgenic human IAPP expression in mouse islets, it had no discernable effects on IAPP expression in human islets under the conditions studied. Suppression of transgenic human IAPP expression in mouse islets attenuated disruption of the autophagy lysosomal pathway in beta cells, supporting the potential of this strategy.
RESUMEN
Frataxin deficiency in Friedreich's ataxia results from transcriptional downregulation of the FXN gene caused by expansion of the intronic trinucleotide guanine-adenine-adenine (GAA) repeats. We used multiple transcriptomic approaches to determine the molecular mechanism of transcription inhibition caused by long GAAs. We uncovered that transcription of FXN in patient cells is prematurely terminated upstream of the expanded repeats leading to the formation of a novel, truncated and stable RNA. This FXN early terminated transcript (FXN-ett) undergoes alternative, non-productive splicing and does not contribute to the synthesis of functional frataxin. The level the FXN-ett RNA directly correlates with the length of the longer of the two expanded GAA tracts. Targeting GAAs with antisense oligonucleotides or excision of the repeats eliminates the transcription impediment, diminishes expression of the aberrant FXN-ett, while increasing levels of FXN mRNA and frataxin. Non-productive transcription may represent a common phenomenon and attractive therapeutic target in diseases caused by repeat-mediated transcription aberrations.
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Ataxia de Friedreich , Adenina , Arsenicales , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Galio , Guanina , Humanos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Oligonucleótidos Antisentido , Poliadenilación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Expansión de Repetición de Trinucleótido/genética , FrataxinaRESUMEN
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.
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Oligonucleótidos Antisentido , Receptor de Angiotensina Tipo 1 , Animales , Ratones , Oligonucleótidos Antisentido/farmacología , Receptor de Angiotensina Tipo 1/genética , Transducción de SeñalRESUMEN
Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, hyperinsulinemia, and insulin resistance (IR). During the early phase of T2D, insulin synthesis and secretion by pancreatic ß cells is enhanced, which can lead to proinsulin misfolding that aggravates endoplasmic reticulum (ER) protein homeostasis in ß cells. Moreover, increased circulating insulin may contribute to fatty liver disease. Medical interventions aimed at alleviating ER stress in ß cells while maintaining optimal insulin secretion are therefore an attractive therapeutic strategy for T2D. Previously, we demonstrated that germline Chop gene deletion preserved ß cells in high-fat diet (HFD)-fed mice and in leptin receptor-deficient db/db mice. In the current study, we further investigated whether targeting Chop/Ddit3 specifically in murine ß cells conferred therapeutic benefits. First, we showed that Chop deletion in ß cells alleviated ß cell ER stress and delayed glucose-stimulated insulin secretion (GSIS) in HFD-fed mice. Second, ß cell-specific Chop deletion prevented liver steatosis and hepatomegaly in aged HFD-fed mice without affecting basal glucose homeostasis. Third, we provide mechanistic evidence that Chop depletion reduces ER Ca2+ buffering capacity and modulates glucose-induced islet Ca2+ oscillations, leading to transcriptional changes of ER chaperone profile ("ER remodeling"). Last, we demonstrated that a GLP1-conjugated Chop antisense oligonucleotide strategy recapitulated the reduction in liver triglycerides and pancreatic insulin content. In summary, our results demonstrate that Chop depletion in ß cells provides a therapeutic strategy to alleviate dysregulated insulin secretion and consequent fatty liver disease in T2D.
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Diabetes Mellitus Tipo 2 , Hígado Graso , Células Secretoras de Insulina , Animales , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa/efectos adversos , Estrés del Retículo Endoplásmico , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Site-specific incorporation of 2'-modifications and neutral linkages in the deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined the effect of introducing 2',5'-linked RNA in the deoxynucleotide gap region on toxicity and potency of PS ASOs. Our results demonstrate that incorporation of 2',5'-linked RNA in the gap region dramatically improved hepatotoxicity profile of PS-ASOs without compromising potency and provide a novel alternate chemical approach for improving therapeutic index of ASO drugs.
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Synthetic therapeutic oligonucleotides (STO) represent the third bonafide platform for drug discovery in the pharmaceutical industry after small molecule and protein therapeutics. So far, thirteen STOs have been approved by regulatory agencies and over one hundred of them are in different stages of clinical trials. STOs hybridize to their target RNA or DNA in cells via Watson-Crick base pairing to exert their pharmacological effects. This unique class of therapeutic agents has the potential to target genes and gene products that are considered undruggable by other therapeutic platforms. However, STOs must overcome several extracellular and intracellular obstacles to interact with their biological RNA targets inside cells. These obstacles include degradation by extracellular nucleases, scavenging by the reticuloendothelial system, filtration by the kidney, traversing the capillary endothelium to access the tissue interstitium, cell-surface receptor-mediated endocytic uptake, and escape from endolysosomal compartments to access the nuclear and/or cytoplasmic compartments where their targets reside. In this review, we present the recent advances in this field with a specific focus on antisense oligonucleotides (ASOs) and siRNA therapeutics.
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Oligonucleótidos Antisentido , Oligonucleótidos , Descubrimiento de Drogas , ARN Interferente PequeñoRESUMEN
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.
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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
The extra hepatic delivery of antisense oligonucleotides (ASOs) remains a challenge and hampers the widespread application of this powerful class of therapeutic agents. In that regard, pancreatic beta cells are a particularly attractive but challenging cell type because of their pivotal role in diabetes and the fact that they are refractory to uptake of unconjugated ASOs. To circumvent this, we have expanded our understanding of the structure activity relationship of ASOs conjugated to Glucagon Like Peptide 1 Receptor (GLP1R) agonist peptide ligands. We demonstrate the key role of the linker chemistry and its optimization to design maleimide based conjugates with improved in vivo efficacy. In addition, truncation studies and scoping of a diverse set of GLP1R agonists proved fruitful to identify additional targeting ligands efficacious in vivo including native hGLP1(7-36)NH2. Variation of the carrier peptide also shed some light on the dramatic impact of subtle sequence differences on the corresponding ASO conjugate performance in vivo, an area which clearly warrant further investigations. We have confirmed the remarkable potential of GLP1R agonist conjugation for the delivery of ASOs to pancreatic beta cell by effectively knocking down islet amyloid polypeptide (IAPP) mRNA, a potential proapoptotic target, in mice.
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Portadores de Fármacos/química , Receptor del Péptido 1 Similar al Glucagón/química , Células Secretoras de Insulina/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Péptidos/química , Secuencia de Aminoácidos , Animales , Receptor del Péptido 1 Similar al Glucagón/agonistas , Células HEK293 , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Ratones Endogámicos C57BL , Estructura Molecular , ARN Mensajero/metabolismo , Relación Estructura-ActividadRESUMEN
Despite recent advances, targeted delivery of therapeutic oligonucleotide to extra-hepatic tissues continues to be a challenging endeavor and efficient ligand-receptor systems need to be identified. To determine the feasibility of using neurotensin to improve the productive uptake of antisense oligonucleotides (ASO), we synthesized neurotensin-ASO conjugates and evaluated their cellular uptake and activity in cells and in mice. We performed a comprehensive structure-activity relationship study of the conjugates and determined the influence of ASO charge, ASO length, peptide charge, linker chemistry and ligand identity on receptor binding and internalization. We identified a modified neurotensin peptide capable of improving the cellular uptake and activity of gapmer ASOs in sortilin expressing cells (sixfold) and in spinal cord in mice (twofold). Neurotensin conjugation also improved the potency of morpholino ASO designed to correct splicing of survival motor neuron pre-mRNA in the cortex and striatum after intracerebroventricular injection. Neurotensin-mediated targeted delivery represents a possible approach for enhancing the potency of ASOs with diverse nucleic acid modifications.
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Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Neurotensina/química , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacocinética , Animales , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Morfolinos/administración & dosificación , Morfolinos/química , Morfolinos/farmacocinética , Oligonucleótidos Antisentido/químicaRESUMEN
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-/-).
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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
The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein-protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO-protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed.
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Proteínas de Unión al ADN/metabolismo , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa H/metabolismo , Dominio Catalítico , Nucléolo Celular/metabolismo , Supervivencia Celular , Proteínas de Unión al ADN/química , Células HEK293 , Células HeLa , Humanos , Cinética , Unión Proteica , Proteínas de Unión al ARN/química , Ribonucleasa H/químicaRESUMEN
Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (-) cells. Both human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC.
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Acetilgalactosamina/química , Técnicas de Transferencia de Gen , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Animales , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular , Células Cultivadas , Expresión Génica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , RatonesRESUMEN
Antisense oligonucleotides modified with phosphorothioate linkages (PS-ASOs) can enter cells via endocytic pathways and must escape from membraned organelles to reach target RNAs. We recently found that membrane destabilization induced by different lipid species contributes to PS-ASO release from late endosomes (LEs). In this study, we characterized intracellular uptake, trafficking, and activities of PS-ASOs conjugated with different lipid species. We found that palmitic acid-, tocopherol-, and cholesterol-conjugated PS-ASOs have increased protein binding and enhanced intracellular uptake compared to unconjugated PS-ASOs. Similar to the parental PS-ASO, the lipid-conjugated PS-ASOs traffic from early to LEs without incorporation into lipid droplets. Unlike parental PS-ASOs, the lipid-conjugated PS-ASOs tend to remain associated with plasma or endosomal membranes, and this appears to influence their release from endosomes. The lipid-conjugated PS-ASOs were released more rapidly than parental PS-ASO. These results suggest that lipid conjugation enhances the interactions of PS-ASOs with proteins or membranes, in turn facilitating intracellular trafficking and endosomal release.
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Endocitosis/efectos de los fármacos , Lípidos/farmacología , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Fosforotioatos/farmacología , Animales , Transporte Biológico/genética , Endocitosis/genética , Endosomas/efectos de los fármacos , Endosomas/genética , Células HeLa , Humanos , Lípidos/genética , Oligonucleótidos Antisentido/genética , Unión ProteicaRESUMEN
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.
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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
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.
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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
We recently reported that (E)-5'-vinylphosphonate (5'-VP) is a metabolically-stable phosphate mimic for siRNA and demonstrated that 5'-VP improves the potency of the fully modified siRNAs in vivo. Here, we report an alternative synthesis of 5'-VP modified guide strand using S-pivaloyl-2-thioethyl (tBu-SATE) protecting group. The tBu-SATE group is readily removed during the final cleavage of the oligonucleotide from the solid support and providing a more convenient route for the synthesis of siRNA guide strand carrying a 5'-vinylphosphonate.
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Organofosfonatos/química , ARN Interferente Pequeño/síntesis química , Compuestos de Vinilo/química , Estructura Molecular , ARN Interferente Pequeño/químicaRESUMEN
The potency of antisense oligonucleotide (ASO) drugs has significantly improved in the clinic after exploiting asialoglycoprotein receptor (ASGR) mediated delivery to hepatocytes. To further this technology, we evaluated the structure-activity relationships of oligonucleotide chemistry on in vivo potency of GalNAc-conjugated Gapmer ASOs. GalNAc conjugation improved potency of ASOs containing 2'-O-methyl (2'-O-Me), 3'-fluoro hexitol nucleic acid (FHNA), locked nucleic acid (LNA), and constrained ethyl bicyclo nucleic acid (cEt BNA) 10-20-fold compared to unconjugated ASOs. We further demonstrate that GalNAc conjugation improves activity of 2'-O-(2-methoxyethyl) (2'-O-MOE) and Morpholino ASOs designed to correct splicing of survival motor neuron (SMN2) pre-mRNA in liver after subcutaneous administration. GalNAc modification thus represents a viable strategy for enhancing potency of ASO with diverse nucleic acid modifications and mechanisms of action for targets expressed in hepatocytes.
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Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/farmacología , Morfolinos/química , Morfolinos/farmacología , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacología , Animales , Receptor de Asialoglicoproteína/metabolismo , Halogenación , Hepatocitos/metabolismo , Metilación , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Oligonucleótidos/química , Oligonucleótidos/farmacología , Alcoholes del Azúcar/química , Alcoholes del Azúcar/farmacología , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by reduced expression of the mitochondrial protein frataxin (FXN). The genetic cause of the disease is an expanded GAA repeat within the FXN gene. Agents that increase expression of FXN protein are a potential approach to therapy. We previously described anti-trinucleotide GAA duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) that activate FXN protein expression in multiple patient derived cell lines. Here we test two distinct series of compounds for their ability to increase FXN expression. ASOs with butane linkers showed low potency, which is consistent with the low Tm values and suggesting that flexible conformation impairs activity. By contrast, single-stranded siRNAs (ss-siRNAs) that combine the strengths of dsRNA and ASO approaches had nanomolar potencies. ss-siRNAs provide an additional option for developing nucleic acid therapeutics to treat FRDA.