RESUMO
Antisense-oligonucleotides (ASOs) are a promising drug modality for the treatment of neurological disorders, but the currently established route of administration via intrathecal delivery is a major limitation to its broader clinical application. An attractive alternative is the conjugation of the ASO to an antibody that facilitates access to the central nervous system (CNS) after peripheral application and target engagement at the blood-brain barrier, followed by transcytosis. Here, we show that the diligent conjugate design of Brainshuttle-ASO conjugates is the key to generating promising delivery vehicles and thereby establishing design principles to create optimized molecules with drug-like properties. An innovative site-specific transglutaminase-based conjugation technology was chosen and optimized in a stepwise process to identify the best-suited conjugation site, tags, reaction conditions, and linker design. The overall conjugation performance was found to be specifically governed by the choice of buffer conditions and the structure of the linker. The combination of the peptide tags YRYRQ and RYESK was chosen, showing high conjugation fidelity. Elaborate conjugate analysis revealed that one leading differentiating factor was hydrophobicity. The increase of hydrophobicity by the ASO payload could be mitigated by the appropriate choice of conjugation site and the heavy chain position 297 proved to be the most optimal. Evaluating the properties of the linker suggested a short bicyclo[6.1.0]nonyne (BCN) unit as best suited with regards to conjugation performance and potency. Promising in vitro activity and in vivo pharmacokinetic behavior of optimized Brainshuttle-ASO conjugates, based on a microtubule-associated protein tau (MAPT) targeting oligonucleotide, suggest that such designs have the potential to serve as a blueprint for peripherally delivered ASO-based drugs for the CNS in the future.
Assuntos
Anticorpos , Oligonucleotídeos Antissenso , Oligonucleotídeos Antissenso/química , Oligonucleotídeos , PeptídeosRESUMO
The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.
Assuntos
Apolipoproteína B-100/genética , Oligonucleotídeos/genética , Fosfatos/química , Oligonucleotídeos Fosforotioatos/genética , RNA Longo não Codificante/genética , Animais , Apolipoproteína B-100/antagonistas & inibidores , Apolipoproteína B-100/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Fosfatos/metabolismo , Oligonucleotídeos Fosforotioatos/síntese química , Oligonucleotídeos Fosforotioatos/metabolismo , Estabilidade de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo , EstereoisomerismoRESUMO
Antisense oligonucleotides (ASOs) are chemically modified nucleic acids with therapeutic potential, some of which have been approved for marketing. We performed a study in rats to investigate mechanisms of toxicity after administration of 3 tool locked nucleic acid (LNA)-containing ASOs with differing established safety profiles. Four male rats per group were dosed once, 3, or 6 times subcutaneously, with 7 days between dosing, and sacrificed 3 days after the last dose. These ASOs were either unconjugated (naked) or conjugated with N-acetylgalactosamine for hepatocyte-targeted delivery. The main readouts were in-life monitoring, clinical and anatomic pathology, exposure assessment and metabolite identification in liver and kidney by liquid chromatography coupled to tandem mass spectrometry, ASO detection in liver and kidney by immunohistochemistry, in situ hybridization, immune electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry imaging. The highly toxic compounds showed the greatest amount of metabolites and a low degree of tissue accumulation. This study reveals different patterns of cell death associated with toxicity in liver (apoptosis and necrosis) and kidney (necrosis only) and provides new ultrastructural insights on the tissue accumulation of ASOs. We observed that the immunostimulatory properties of ASOs can be either primary from sequence-dependent properties or secondary to cell necrosis.
Assuntos
Oligonucleotídeos Antissenso , Oligonucleotídeos , Acetilgalactosamina , Animais , Masculino , Oligonucleotídeos Antissenso/toxicidade , Ratos , Distribuição TecidualRESUMO
Antisense oligonucleotides linked by phosphorothioates are an important class of therapeutics under investigation in various pharmaceutical companies. Antisense oligonucleotides may be coupled to high-affinity ligands (triantennary N-acetyl galactosamine = GalNAc) for hepatocyte-specific asialoglycoprotein receptors (ASGPR) to enhance uptake to hepatocytes and to increase potency. Since disposition and biotransformation of GalNAc-conjugated oligonucleotides is different from unconjugated oligonucleotides, appropriate analytical methods are required to identify main cleavage sites and degradation products of GalNAc conjugated and unconjugated oligonucleotides in target cells. A highly sensitive method was developed to identify metabolites of oligonucleotides using capillary flow liquid chromatography with column switching coupled to a high resolution Orbitrap Fusion mass spectrometer. Detection of GalNAc-conjugated oligonucleotides and their metabolites was achieved by combining full scan MS with two parallel MS2 experiments, one data-dependent scan and an untargeted MS2 experiment (all ion fragmentation) applying high collision energy. In the all ion fragmentation scan, a diagnostic fragment originating from the phosphorothioate backbone (O2PS-: m/z 94.936) was formed efficiently upon collisional activation. Based on this fragment an accurate determination of metabolites of oligonucleotides was achieved, independent of their sequence or conjugation in an untargeted but highly selective manner. The method was effectively applied to investigate uptake and metabolism of GalNAc-conjugated oligonucleotides in incubations of primary rat hepatocytes; the elucidation of expected and unexpected degradation products was achieved in subnanomolar range.
Assuntos
Acetilgalactosamina/química , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Oligonucleotídeos Antissenso/metabolismo , Animais , Biotransformação , Células Cultivadas , Hepatócitos/citologia , Hepatócitos/metabolismo , Masculino , Oligonucleotídeos/análise , Oligonucleotídeos/isolamento & purificação , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/isolamento & purificação , Ratos , Ratos Wistar , Extração em Fase SólidaRESUMO
UNLABELLED: Identification of microRNAs (miRNAs) that regulate lipid metabolism is important to advance the understanding and treatment of some of the most common human diseases. In the liver, a few key miRNAs have been reported that regulate lipid metabolism, but since many genes contribute to hepatic lipid metabolism, we hypothesized that other such miRNAs exist. To identify genes repressed by miRNAs in mature hepatocytes in vivo, we injected adult mice carrying floxed Dicer1 alleles with an adenoassociated viral vector expressing Cre recombinase specifically in hepatocytes. By inactivating Dicer in adult quiescent hepatocytes we avoided the hepatocyte injury and regeneration observed in previous mouse models of global miRNA deficiency in hepatocytes. Next, we combined gene and miRNA expression profiling to identify candidate gene/miRNA interactions involved in hepatic lipid metabolism and validated their function in vivo using antisense oligonucleotides. A candidate gene that emerged from our screen was lipoprotein lipase (Lpl), which encodes an enzyme that facilitates cellular uptake of lipids from the circulation. Unlike in energy-dependent cells like myocytes, LPL is normally repressed in adult hepatocytes. We identified miR-29a as the miRNA responsible for repressing LPL in hepatocytes, and found that decreasing hepatic miR-29a levels causes lipids to accumulate in mouse livers. CONCLUSION: Our screen suggests several new miRNAs are regulators of hepatic lipid metabolism. We show that one of these, miR-29a, contributes to physiological lipid distribution away from the liver and protects hepatocytes from steatosis. Our results, together with miR-29a's known antifibrotic effect, suggest miR-29a is a therapeutic target in fatty liver disease.
Assuntos
Metabolismo dos Lipídeos , Lipase Lipoproteica/biossíntese , Fígado/metabolismo , MicroRNAs/metabolismo , Animais , Repressão Enzimática , Fígado Gorduroso/etiologia , Hepatócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Oligodesoxirribonucleotídeos Antissenso/química , Receptores Depuradores Classe B/genética , Animais , Linhagem Celular , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/farmacocinética , RNA Mensageiro/metabolismo , Receptores Depuradores Classe B/metabolismo , Distribuição TecidualRESUMO
Regulators of apoptosis in acute myeloid leukemia (AML) have been extensively studied and are considered excellent therapeutic targets. Apoptosis repressor with caspase recruitment domain (ARC), an antiapoptotic protein originally found to be involved in apoptosis of cardiac cells, was recently demonstrated to be overexpressed in several solid tumors. To assess its importance in AML, we profiled ARC expression in 511 newly diagnosed AML patients using a validated robust reverse-phase protein array and correlated ARC levels with clinical outcomes. ARC was variably expressed in samples from patients with AML. ARC level was not associated with cytogenetic groups or with FLT-3 mutation status. However, patients with low or medium ARC protein levels had significantly better outcomes than those with high ARC levels: longer overall survival (median, 53.9 or 61.6 vs 38.9 weeks, P = .0015) and longer remission duration (median, 97.6 or 44.7 vs 31.1 weeks, P = .0007). Multivariate analysis indicated that ARC was a statistically significant independent predictor of survival in AML (P = .00013). Inhibition of ARC promoted apoptosis and sensitized cytosine arabinoside-induced apoptosis in OCI-AML3 cells. These results suggest that ARC expression levels are highly prognostic in AML and that ARC is a potential therapeutic target in AML.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Leucemia Mieloide/metabolismo , Proteínas Musculares/metabolismo , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citarabina/farmacologia , Feminino , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Proteínas Musculares/genética , Mutação , Oligonucleotídeos Antissenso/genética , Prognóstico , Análise de Sobrevida , Adulto Jovem , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
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.
Assuntos
Hepatócitos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Fosforotioatos/metabolismo , Animais , Linhagem Celular Tumoral , Endocitose , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Oligonucleotídeos Antissenso/análise , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/análise , Oligonucleotídeos Fosforotioatos/químicaRESUMO
Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a-induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic.
Assuntos
Apoptose , Crise Blástica/metabolismo , Leucemia Mieloide Aguda/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Crise Blástica/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 6/biossíntese , Caspase 6/genética , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/genética , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Células Estromais/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
Many in vitro and in vivo models are used in pharmacological research to evaluate the role of targeted proteins in a disease. Understanding the translational relevance and limitation of these models for analyzing a drug's disposition, pharmacokinetic/pharmacodynamic (PK/PD) profile, mechanism, and efficacy, is essential when selecting the most appropriate model of the disease of interest and predicting clinically efficacious doses of the investigational drug. Selected animal models used in ophthalmology, infectious diseases, oncology, autoimmune diseases, and neuroscience are reviewed here. Each area has specific challenges around translatability and determination of an efficacious dose: new patient-specific dosing methods may help overcome these limitations.
Assuntos
Drogas em Investigação , Oncologia , Animais , Modelos BiológicosRESUMO
The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS2) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS2 linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS2 and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.
RESUMO
The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed.
Assuntos
Portadores de Fármacos/química , Vesículas Extracelulares/química , Leite/citologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Administração Oral , Animais , Composição de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Neurônios , Oligonucleotídeos/farmacocinética , Oligonucleotídeos Antissenso/farmacocinética , Células-Tronco Pluripotentes , Cultura Primária de Células , Distribuição TecidualRESUMO
Across the pharmaceutical industry, there is increasing interest and need to investigate the biotransformation of oligonucleotide drugs. The method of choice is high-resolution mass spectrometry due to its unmet sensitivity and specificity.Here, we describe a method developed and applied in our laboratory studying the biotransformation of phosphorothioate-containing oligonucleotide drugs. This method is based on capillary flow liquid chromatography with column switching coupled to high-resolution mass spectrometry.
Assuntos
Cromatografia Líquida , Oligonucleotídeos Fosforotioatos/química , Oligonucleotídeos Fosforotioatos/metabolismo , Espectrometria de Massas em Tandem , Biotransformação , Análise de Dados , Hepatócitos/metabolismo , Oligonucleotídeos Fosforotioatos/análise , Oligonucleotídeos Fosforotioatos/isolamento & purificaçãoRESUMO
OBJECTIVES: Microtubular inhibitors, including docetaxel, are active cytotoxics in many cancers, including prostate cancer (CaP). The Eg5 gene, a member of the kinesin-5 family, plays critical roles in proper mitotic spindle function, and is a potential microtubule-related target for proliferating cancer cells. To investigate the functional activities of Eg5 in CaP, we used an antisense oligonucleotide (ASO) targeting Eg5 to assess the potency and anti-cancer activity of Eg5 ASO treatment for androgen-independent CaP cells in vitro and in vivo. RESULTS: PC3 cells express higher Eg5 protein and mRNA levels compared to LNCaP cells. In both cell lines, Eg5 ASO treatment reduced mRNA and protein levels in a dose-dependent manner and a complete reduction of Eg5 protein levels was observed at 100 nM. Dose-dependent inhibition in cell growth, potent G2/M phase arrest, and increases in apoptotic sub-G1 fraction were also observed using Eg5 ASO. Surprisingly, low dose Eg5 ASO significantly antagonized cytotoxic effects of paclitaxel. In vivo, Eg5 ASO monotherapy significantly reduced both LNCaP and PC-3 tumor growth but combination treatment with paclitaxel did not yield additive benefits. CONCLUSIONS: These findings suggest that while Eg5 is a potential target to delay androgen-independent CaP growth, combination treatment with paclitaxel may not be desirable.
Assuntos
Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Cinesinas/genética , Oligonucleotídeos Antissenso/farmacologia , Neoplasias da Próstata/patologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Humanos , Cinesinas/metabolismo , Masculino , Oligonucleotídeos/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Paclitaxel/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , RNA Mensageiro/metabolismo , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.
Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/metabolismo , Ligação Competitiva , Linhagem Celular Tumoral , Humanos , Cinesinas/genética , PTEN Fosfo-Hidrolase/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismoRESUMO
A library of 2'-methoxyethyl-modified antisense oligonucleotides (2'MOE ASO) targeting 1,510 different genes has been developed, validated, and used to identify cell cycle regulatory genes. The most effective molecular target identified was Eg5 (kinesin-like-1), which when inhibited gave the largest increase in 4N DNA in various tumor cells. The Eg5 ASO reduced Eg5 levels, inhibited proliferation, increased apoptosis, and altered the expression of other cell cycle proteins, including survivin and Aurora-A. To examine the therapeutic utility of the Eg5 ASO, the compound was also evaluated in xenograft models. Treatment with Eg5 ASO produced a statistically significant reduction of tumor growth, reduction in Eg5 expression in the tumors, and changes in histone phosphorylation, consistent with a loss of Eg5 protein expression. These data show, for the first time, the utility of a 2'MOE ASO library for high-throughput cell culture-based functional assays and suggest that an Eg5 ASO also has potential in a therapeutic strategy.
Assuntos
Cinesinas/antagonistas & inibidores , Cinesinas/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Biblioteca Gênica , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Células HeLa , Humanos , Cinesinas/biossíntese , Camundongos , Camundongos Nus , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Malignant gliomas are almost uniformly fatal and display exquisite radiation resistance. Glioma cells lacking wild-type (WT) p53 function are more susceptible to radiation-induced apoptosis than their isogenic counterparts expressing WT p53. We explored the mechanisms of such apoptosis and found that, in the absence of WT p53, radiation increases caspase-8 expression and activity. Inhibition of caspase-8 expression using caspase-8 antisense or small interfering RNA (siRNA) oligonucleotides partially blocks radiation-induced apoptosis. In contrast, inhibition of the mitochondrial death pathway by expression of Bcl-2 has no effect on radiation-induced caspase-8 activity or apoptosis. Our data indicate that, in contrast to commonly accepted models of p53-dependent radiation-induced apoptosis, in our cell system, radiation relies on caspase-8 activity to help mediate p53-independent cell death. In a system of inducible E2F1 activity, E2F1 activated caspase-8 and, accordingly, decreased cellular viability, effects that were abolished by caspase-8 siRNA. In this model, in the absence of WT p53, p21Cip1 is not induced, and E2F1 activity is sustained and allows transcription and activation of caspase-8. This model may explain why p53 mutations in adult gliomas paradoxically correlate with improved survival and enhanced response to radiation.
Assuntos
Apoptose/efeitos da radiação , Caspases/fisiologia , Glioma/enzimologia , Apoptose/fisiologia , Caspase 8 , Caspases/biossíntese , Caspases/genética , Caspases/efeitos da radiação , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fator de Transcrição E2F1/metabolismo , Indução Enzimática/efeitos da radiação , Glioma/patologia , Glioma/radioterapia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Transcrição Gênica/efeitos da radiação , Proteína Supressora de Tumor p53/fisiologiaRESUMO
High-content imaging (HCI) provides researchers with a powerful tool for understanding cellular processes. Although phenotypic analysis generated through HCI is a potent technique to determine the overall cellular effects of a given treatment, it frequently produces complex data sets requiring extensive interpretation. The authors developed statistical analyses to decrease the time spent to determine the outcome of each HCI assay and to better understand complex phenotypic changes. To test these tools, the authors performed a comparison experiment between 2 types of oligonucleotide-mediated gene silencing (OMGS), antisense oligonucleotides (ASOs), and short, double-stranded RNAs (siRNAs). Although similar in chemical structure, these 2 methods differ in cellular mechanism of action and off-target effects. Using a library of 50 validated ASOs and siRNAs to the same targets, the authors characterized the differential effects of these 2 technologies using a HeLa cell G2-M cell cycle assay. Although knockdown of a variety of targets by ASOs or siRNAs affected the cell cycle profile, few of those targets were affected by both ASOs and siRNAs. Distribution analysis of population changes induced through target knockdown led to the identification of targets that, when inhibited, could affect the G2-M transition in the cell cycle in a statistically significant manner. The distinctly different mechanisms of action of these 2 forms of gene silencing may help define the use of these treatments in both clinical and research environments.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Oligonucleotídeos Antissenso/farmacologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Perfilação da Expressão Gênica , Células HeLa , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Organismos Geneticamente ModificadosRESUMO
To address the increased need to understand the similarities and differences in targeting Aurora A or Aurora B for the treatment of cancer, we systematically evaluated the relative importance of Aurora A and/or Aurora B as molecular targets using antisense oligonucleotides. It was found that perturbations in Aurora A and Aurora B signaling result in growth arrest and apoptosis preferentially in cancer cells. The biological fingerprints of Aurora A and Aurora B inhibition were compared and contrasted in efforts to identify the superior therapeutic target. Due to the different biological responses, we conclude that each Aurora kinase should be treated as autonomous drug targets, which can be targeted independently or in combination. We observed no advantages to targeting both kinases simultaneously and feel that an Aurora A-targeted therapy may have some beneficial consequences over an Aurora B-targeted therapy, such as mitotic arrest and the rapid induction of apoptosis.
Assuntos
Neoplasias Pancreáticas/patologia , Proteínas Serina-Treonina Quinases/biossíntese , Apoptose , Aurora Quinase B , Aurora Quinases , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ativação Enzimática , Perfilação da Expressão Gênica , Humanos , Oligonucleotídeos Antissenso/farmacologia , Neoplasias Pancreáticas/enzimologia , Poliploidia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ensaio Tumoral de Célula-TroncoRESUMO
Single-stranded oligonucleotides (ON) comprise a promising therapeutic platform that enables selective modulation of currently undruggable targets. The development of novel ON drug candidates has demonstrated excellent efficacy, but in certain cases also some safety liabilities were reported. Among them are events of thrombocytopenia, which have recently been evident in late stage trials with ON drugs. The underlying mechanisms are poorly understood and the risk for ON candidates causing such events cannot be sufficiently assessed pre-clinically. We investigated potential thrombocytopenia risk factors of ONs and implemented a set of in vitro assays to assess these risks. Our findings support previous observations that phosphorothioate (PS)-ONs can bind to platelet proteins such as platelet collagen receptor glycoprotein VI (GPVI) and activate human platelets in vitro to various extents. We also show that these PS-ONs can bind to platelet factor 4 (PF4). Binding to platelet proteins and subsequent activation correlates with ON length and connected to this, the number of PS in the backbone of the molecule. Moreover, we demonstrate that locked nucleic acid (LNA) ribosyl modifications in the wings of the PS-ONs strongly suppress binding to GPVI and PF4, paralleled by markedly reduced platelet activation. In addition, we provide evidence that PS-ONs do not directly affect hematopoietic cell differentiation in culture but at higher concentrations show a pro-inflammatory potential, which might contribute to platelet activation. Overall, our data confirm that certain molecular attributes of ONs are associated with a higher risk for thrombocytopenia. We propose that applying the in vitro assays discussed here during the lead optimization phase may aid in deprioritizing ONs with a potential to induce thrombocytopenia.