RESUMEN
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
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 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.
Asunto(s)
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
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
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
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
Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global benefit. Thus there is a need for preclinical models of HD recapitulating human HTT genetics. We previously generated a humanized mouse model of HD, Hu97/18, by intercrossing BACHD and YAC18 mice with knockout of the endogenous mouse HD homolog (Hdh). Hu97/18 mice recapitulate the genetics of HD, having two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of Caucasian descent. We have now generated a companion model, Hu128/21, by intercrossing YAC128 and BAC21 mice on the Hdh-/- background. Hu128/21 mice have two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of East Asian descent and in a minority of patients from other ethnic groups. Hu128/21 mice display a wide variety of HD-like phenotypes that are similar to YAC128 mice. Additionally, both transgenes in Hu128/21 mice match the human HTT exon 1 reference sequence. Conversely, the BACHD transgene carries a floxed, synthetic exon 1 sequence. Hu128/21 mice will be useful for investigations of human HTT that cannot be addressed in Hu97/18 mice, for developing therapies targeted to exon 1, and for preclinical screening of personalized HTT lowering therapies in HD patients of East Asian descent.
Asunto(s)
Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Mutación/genética , Alelos , Animales , Modelos Animales de Enfermedad , Exones/genética , Heterocigoto , Humanos , Enfermedad de Huntington/patología , Ratones , Ratones Transgénicos , FenotipoRESUMEN
Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs â¼30-fold in the clinic (1). In order to fully characterize the effect of GalNAc valency, oligonucleotide length, flexibility and chemical composition on ASGR binding, we tested and validated a fluorescence polarization competition binding assay. The ASGR binding, and in vitro and in vivo activities of 1, 2 and 3 GalNAc conjugated single stranded and duplexed ASOs were studied. Two and three GalNAc conjugated single stranded ASOs bind the ASGR with the strongest affinity and display optimal in vitro and in vivo activities. 1 GalNAc conjugated ASOs showed 10-fold reduced ASGR binding affinity relative to three GalNAc ASOs but only 2-fold reduced activity in mice. An unexpected observation was that the ASGR also appears to play a role in the uptake of unconjugated phosphorothioate modified ASOs in the liver as evidenced by the loss of activity of GalNAc conjugated and unconjugated ASOs in ASGR knockout mice. Our results provide insights into how backbone charge and chemical composition assist in the binding and internalization of highly polar anionic single stranded oligonucleotides into cells and tissues.
Asunto(s)
Acetilgalactosamina/química , Receptor de Asialoglicoproteína/metabolismo , Bioensayo , ADN de Cadena Simple/química , ADN/química , Oligonucleótidos Antisentido/química , Oligonucleótidos Fosforotioatos/química , Animales , Receptor de Asialoglicoproteína/genética , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Transporte Biológico , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Polarización de Fluorescencia , Glicoconjugados/química , Glicoconjugados/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Cinética , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Noqueados , Microsomas Hepáticos/metabolismo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Cultivo Primario de Células , Unión Proteica , Electricidad EstáticaRESUMEN
The anticancer nucleoside 5-fluoro 2'-deoxyuridine-5'-phosphate (5-FdU-P) was attached via an amide chain linker to a triantennary GalNAc cluster as a means to deliver the drug to hepatic cells that recognize the amino sugar units.
Asunto(s)
Antineoplásicos/administración & dosificación , Receptor de Asialoglicoproteína/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Desoxiuridina/análogos & derivados , Neoplasias Hepáticas/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Desoxiuridina/administración & dosificación , Desoxiuridina/uso terapéutico , Sistemas de Liberación de Medicamentos , Células Hep G2 , Humanos , Fosforilación , Profármacos/administración & dosificaciónRESUMEN
Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro™, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform of Stabilin-2 (315-HARE and 190-HARE), we have determined that PS ASOs bind with high affinity and these receptors are responsible for bulk, clathrin-mediated endocytosis within the cell. Binding is primarily dependent on salt-bridge formation and correct folding of the intact protein receptor. Increased internalization rates also enhanced ASO potency for reducing expression of the non-coding RNA Malat-1, in Stabilin-expressing cell lines. A more thorough understanding of mechanisms by which ASOs are internalized in cells and their intracellular trafficking pathways will aid in the design of next generation antisense agents with improved therapeutic properties.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Células Endoteliales/metabolismo , Hígado/metabolismo , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/genética , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Expresión Génica , Células HEK293 , Humanos , Cinética , Hígado/citología , Hígado/efectos de los fármacos , Ratones , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Fosforotioatos/síntesis química , Oligonucleótidos Fosforotioatos/farmacocinética , Cultivo Primario de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Huntington's disease (HD) is a fatal neurodegenerative disease, caused by expansion of polyglutamine repeats in the Huntingtin gene, with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however, the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines, which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity, as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed, blocking glutamate signaling, not just through the NMDA but also mGlu and AMPA/Kainate receptors, completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of 'persistent' neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together, these results provide important insight into HD mechanisms at early developmental time points, which may suggest novel approaches to HD therapeutics.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Ácido Glutámico/fisiología , Enfermedad de Huntington/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/fisiología , Edad de Inicio , Animales , Apoptosis , Supervivencia Celular , Células Cultivadas , Humanos , Enfermedad de Huntington/patología , RatonesRESUMEN
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 design, synthesis and biophysical evaluation of two highly-constrained tricyclic analogues of locked nucleic acid (LNA), which restrict rotation around the C4'-C5'-exocyclic bond (torsion angle γ) and enhance hydrophobicity in the minor groove and along the major groove, are reported. A structural model that provides insights into the sugar-phosphate backbone conformations required for efficient hybridization to complementary nucleic acids is also presented.
Asunto(s)
Diseño de Fármacos , Oligonucleótidos/química , Conformación Molecular , Oligonucleótidos/síntesis químicaRESUMEN
Antisense oligonucleotides (ASOs) modified with ligands which target cell surface receptors have the potential to significantly improve potency in the target tissue. This has recently been demonstrated using triantennary N-acetyl d-galactosamine conjugated ASOs. CD22 is a cell surface receptor expressed exclusively on B cells thus presenting an attractive target for B cell specific delivery of drugs. Herein, we reported the synthesis of monovalent and trivalent ASO conjugates with biphenylcarbonyl (BPC) modified sialic acids and their study as ASO delivery agents into B cells. CD22 positive cells exhibited reduced potency when treated with ligand modified ASOs and mechanistic examination suggested reduced uptake into cells potentially as a result of sequestration of ASO by other cell-surface proteins.
Asunto(s)
Linfocitos B/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Ácidos Nucleicos/metabolismo , Oligonucleótidos/farmacología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Ácidos Siálicos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Ácidos Siálicos/síntesis química , Ácidos Siálicos/química , 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
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
Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD.
Asunto(s)
Encéfalo/patología , Enfermedad de Huntington/terapia , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/genética , Oligonucleótidos Antisentido/administración & dosificación , Tionucleótidos/administración & dosificación , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Silenciador del Gen , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Inyecciones , Ratones , Ratones Endogámicos C57BL , Terapia Molecular Dirigida , Proteínas del Tejido Nervioso/metabolismo , Oligonucleótidos Antisentido/farmacología , Polimorfismo de Nucleótido Simple , Ratas , Ratas Sprague-Dawley , Tionucleótidos/farmacologíaRESUMEN
Autosomal dominant diseases such as Huntington's disease (HD) are caused by a gain of function mutant protein and/or RNA. An ideal treatment for these diseases is to selectively suppress expression of the mutant allele while preserving expression of the wild-type variant. RNase H active antisense oligonucleotides (ASOs) or small interfering RNAs can achieve allele selective suppression of gene expression by targeting single nucleotide polymorphisms (SNPs) associated with the repeat expansion. ASOs have been previously shown to discriminate single nucleotide changes in targeted RNAs with â¼5-fold selectivity. Based on RNase H enzymology, we enhanced single nucleotide discrimination by positional incorporation of chemical modifications within the oligonucleotide to limit RNase H cleavage of the non-targeted transcript. The resulting oligonucleotides demonstrate >100-fold discrimination for a single nucleotide change at an SNP site in the disease causing huntingtin mRNA, in patient cells and in a completely humanized mouse model of HD. The modified ASOs were also well tolerated after injection into the central nervous system of wild-type animals, suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings lay the foundation for efficient allele-selective downregulation of gene expression using ASOs-an outcome with broad application to HD and other dominant genetic disorders.
Asunto(s)
Alelos , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Oligonucleótidos Antisentido/química , Polimorfismo de Nucleótido Simple , Animales , Emparejamiento Base , Encéfalo/metabolismo , Células Cultivadas , Regulación hacia Abajo , Flúor/química , Humanos , Proteína Huntingtina , Enfermedad de Huntington/metabolismo , Ratones , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Ratas , Ratas Sprague-Dawley , Ribonucleasa H/metabolismoRESUMEN
We compare the duplex stabilizing properties of 2'-fluorinated nucleic acid analogues with furanose and non-furanose ring systems and dissect the relative contributions of hydration, sugar conformation, and fluorine configuration toward the overall T(m) value. We find that the stabilization imparted by fluorine substitution is additive over that obtained by restricting the conformation of the sugar ring itself. Our studies support further evaluation of fluorinated nucleic acid analogues with non-furanose sugar rings as surrogates of 2'-F RNA for therapeutic antisense applications.
Asunto(s)
Carbohidratos/química , Ácidos Nucleicos/química , ARN/química , Ribosa/química , Emparejamiento Base , Conformación de Carbohidratos , Conformación Molecular , Estructura Molecular , Conformación de Ácido NucleicoRESUMEN
Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers.