RESUMO
Therapeutic oligonucleotides are chemically modified to enhance their drug-like properties - including binding affinity for target RNA. Many nucleic acid analogs that enhance RNA binding affinity constrain the furanose sugar in an RNA-like sugar pucker. The improvements in binding affinity result primarily from increased off-rates with minimal effects on on-rates for hybridization. To identify alternate chemical modification strategies that can modulate on- and off-rates for oligonucleotide hybridization, we hypothesized that extending conformational restraint across multiple nucleotides could modulate hybridization kinetics by restricting rotational freedom of the sugar-phosphate backbone. As part of that effort, we recently reported that using hydrocarbon tethers to bridge adjacent phosphodiester linkages as phosphonate tethered bridges can pre-organize nucleic acids in conformations conducive for Watson-Crick base-pairing and modulate hybridization kinetics. In this report, we describe the synthesis of locked nucleic acid (LNA) trimers linked through alkylphosphonate tethers which restrict conformation of the furanose sugar in addition to restricting conformational mobility of the sugar-phosphate backbone across three nucleotide units.
RESUMO
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-/-).
Assuntos
Oligonucleotídeos Antissenso/farmacocinética , Ácido Palmítico , Albuminas/genética , Albuminas/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Caveolina 1/genética , Feminino , Coração , Células Hep G2 , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Sistema Linfático/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Oligonucleotídeos Antissenso/química , Músculo Quadríceps/metabolismo , Receptores Fc/genética , Distribuição TecidualRESUMO
Neuroblastoma (NB) is the most common extracranial solid tumor in children. The outcomes for aggressive forms of NB remain poor. The aim of this study was to develop a new molecular-targeted therapy for NB using an antisense oligonucleotide (ASO) and superparamagnetic iron oxide (SPIO) nanoparticles (NPs), as a delivery vehicle, targeting the transcription regulator MAX dimerization protein 3 (MXD3). We previously discovered that MXD3 was highly expressed in high-risk NB, acting as an anti-apoptotic factor; therefore, it can be a good therapeutic target. In this study, we developed two ASO-NP complexes using electrostatic conjugation to polyethylenimine-coated SPIO NPs and chemical conjugation to amphiphilic polymers on amine-functionalized SPIO NPs. Both ASO-NP complexes demonstrated MXD3 knockdown, which resulted in apoptosis in NB cells. ASO chemically-conjugated NP complexes have the potential to be used in the clinic as they showed great efficacy with minimum NP-associated cytotoxicity.
Assuntos
Compostos Férricos/química , Compostos Férricos/farmacologia , Nanopartículas de Magnetita/química , Nanopartículas Metálicas/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Inativação Gênica/fisiologia , Humanos , Immunoblotting , Imuno-Histoquímica , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Repressoras/genética , Eletricidade EstáticaRESUMO
Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.
Assuntos
Encéfalo/metabolismo , Antagonistas de Receptores de GABA-A/farmacocinética , Atrofia Muscular Espinal/tratamento farmacológico , Oligonucleotídeos Antissenso/farmacocinética , Animais , Artérias/diagnóstico por imagem , Artérias/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Flumazenil/administração & dosagem , Flumazenil/análogos & derivados , Antagonistas de Receptores de GABA-A/administração & dosagem , Técnicas de Silenciamento de Genes , Humanos , Injeções Espinhais , Microscopia Intravital , Masculino , Terapia de Alvo Molecular/métodos , Neuroglia/metabolismo , Neurônios/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Pia-Máter/diagnóstico por imagem , Pia-Máter/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Receptores de AMPA/análise , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/genética , Receptores de GABA-A/análise , Receptores de GABA-A/genética , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Análise Espaço-Temporal , Tionucleotídeos/administração & dosagem , Tionucleotídeos/farmacocinética , Distribuição TecidualRESUMO
Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.
Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Oligonucleotídeos Antissenso/química , Organofosfonatos/química , Oligonucleotídeos Fosforotioatos/química , Células 3T3-L1 , Animais , Caspases/metabolismo , Linhagem Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Proteínas de Ligação a DNA , Células HeLa , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Fosforotioatos/administração & dosagem , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismoRESUMO
The exponential rise in molecular and genomic data has generated a vast array of therapeutic targets. Oligonucleotide-based technologies to down regulate these molecular targets have promising therapeutic efficacy. However, there is relatively limited success in translating this into effective in vivo cancer therapeutics. The primary challenge is the lack of effective cancer cell-targeted delivery methods, particularly for a systemic disease such as leukemia. We developed a novel leukemia-targeting compound composed of a monoclonal antibody directly conjugated to an antisense oligonucleotide (ASO). Our compound uses an ASO that specifically targets the transcription factor MAX dimerization protein 3 (MXD3), which was previously identified to be critical for precursor B cell (preB) acute lymphoblastic leukemia (ALL) cell survival. The MXD3 ASO was conjugated to an anti-CD22 antibody (αCD22 Ab) that specifically targets most preB ALL. We demonstrated that the αCD22 Ab-ASO conjugate treatment showed MXD3 protein knockdown and leukemia cell apoptosis in vitro. We also demonstrated that the conjugate treatment showed cytotoxicity in normal B cells, but not in other hematopoietic cells, including hematopoietic stem cells. Furthermore, the conjugate treatment at the lowest dose tested (0.2mg/kg Ab for 6 doses - twice a week for 3 weeks) more than doubled the mouse survival time in both Reh (median survival time 20.5 vs. 42.5 days, p<0.001) and primary preB ALL (median survival time 29.3 vs. 63 days, p<0.001) xenograft models. Our conjugate that uses αCD22 Ab to target the novel molecule MXD3, which is highly expressed in preB ALL cells, appears to be a promising novel therapeutic approach.