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1.
Nucleic Acids Res ; 46(11): 5366-5380, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29790953

RESUMO

Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.


Assuntos
Terapia Genética/métodos , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA Complementar/metabolismo , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Animais , Apolipoproteínas B/genética , Sítios de Ligação/genética , Células Cultivadas , Feminino , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/genética
2.
Nucleic Acids Res ; 45(5): 2262-2282, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28426096

RESUMO

All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery.


Assuntos
Descoberta de Drogas/métodos , Oligonucleotídeos Antissenso/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Análise de Sequência de RNA/estatística & dados numéricos , Animais , Pareamento de Bases , Avaliação Pré-Clínica de Medicamentos , Humanos , Terapia de Alvo Molecular , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Sensibilidade e Especificidade , Termodinâmica
3.
Nucleic Acids Res ; 45(22): 12932-12944, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29126318

RESUMO

RNase H cleaves RNA in RNA-DNA duplexes. It is present in all domains of life as well as in multiple viruses and is essential for mammalian development and for human immunodeficiency virus replication. Here, we developed a sequencing-based method to measure the cleavage of thousands of different RNA-DNA duplexes and thereby comprehensively characterized the sequence preferences of HIV-1, human and Escherichia coli RNase H enzymes. We find that the catalytic domains of E. coli and human RNase H have nearly identical sequence preferences, which correlate with the efficiency of RNase H-recruiting antisense oligonucleotides. The sequences preferred by HIV-1 RNase H are distributed in the HIV genome in a way suggesting selection for efficient RNA cleavage during replication. Our findings can be used to improve the design of RNase H-recruiting antisense oligonucleotides and show that sequence preferences of HIV-1 RNase H may have shaped evolution of the viral genome and contributed to the use of tRNA-Lys3 as primer during viral replication.


Assuntos
Oligonucleotídeos Antissenso/metabolismo , Clivagem do RNA , RNA/metabolismo , Ribonuclease H/metabolismo , Sequência de Bases , Domínio Catalítico , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , RNA/química , RNA/genética , Ribonuclease H/química , Especificidade por Substrato , Replicação Viral
4.
Nucleic Acids Res ; 43(17): 8476-87, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26220183

RESUMO

Processing and post-transcriptional regulation of RNA often depend on binding of regulatory molecules to short motifs in RNA. The effects of such interactions are difficult to study, because most regulatory molecules recognize partially degenerate RNA motifs, embedded in a sequence context specific for each RNA. Here, we describe Library Sequencing (LibSeq), an accurate massively parallel reporter method for completely characterizing the regulatory potential of thousands of short RNA sequences in a specific context. By sequencing cDNA derived from a plasmid library expressing identical reporter genes except for a degenerate 7mer subsequence in the 3'UTR, the regulatory effects of each 7mer can be determined. We show that LibSeq identifies regulatory motifs used by RNA-binding proteins and microRNAs. We furthermore apply the method to cells transfected with RNase H recruiting oligonucleotides to obtain quantitative information for >15000 potential target sequences in parallel. These comprehensive datasets provide insights into the specificity requirements of RNase H and allow a specificity measure to be calculated for each tested oligonucleotide. Moreover, we show that inclusion of chemical modifications in the central part of an RNase H recruiting oligonucleotide can increase its sequence-specificity.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Oligonucleotídeos/química , Sequências Reguladoras de Ácido Ribonucleico , Ribonuclease H/metabolismo , Análise de Sequência de RNA/métodos , Regiões 3' não Traduzidas , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Células HeLa , Humanos , MicroRNAs , Motivos de Nucleotídeos , Oligonucleotídeos/metabolismo , Plasmídeos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Termodinâmica
6.
Blood ; 118(22): 5891-900, 2011 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21865341

RESUMO

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.


Assuntos
Perfilação da Expressão Gênica , Linfoma Cutâneo de Células T/diagnóstico , Linfoma Cutâneo de Células T/genética , MicroRNAs/genética , Animais , Células Cultivadas , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Análise em Microsséries , Prognóstico , Psoríase/patologia , Transplante Heterólogo
7.
Exp Dermatol ; 21(4): 299-301, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22417307

RESUMO

MicroRNAs are non-coding RNA molecules modulating gene expression post-transcriptionally. Formalin-fixed, paraffin-embedding (FFPE) is a standard preservation method often used in clinical practices, but induces RNA degradation. Extracting high-quality RNA from human skin can be challenging as skin contains high levels of RNases. As microRNAs are 19-23 nucleotides long and lack a poly-A tail, they may be less prone to RNA degradation than mRNAs. We investigated whether microRNAs in psoriatic (FFPE) samples reliably reflect microRNA expression in samples less prone to RNA degradation such as fresh-frozen (FS) and Tissue-Tek-embedding (OCT). We found a strong correlation of the microRNA expression levels between all preservation methods of matched psoriatic skin samples (r(s) ranging from 0.91 to 0.95 (P < 0.001)). These observations were further confirmed with qRT-PCR. Our results demonstrate that microRNA detection in human skin is robust irrespective of preservation method; thus, microRNAs offer an appropriate and flexible approach in clinical practices and for diagnostic purposes in skin disorders.


Assuntos
Técnicas de Preparação Histocitológica/métodos , MicroRNAs/genética , Psoríase/genética , Pele/metabolismo , Formaldeído , Congelamento , Expressão Gênica , Humanos , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Inclusão em Parafina , Psoríase/diagnóstico , Psoríase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fixação de Tecidos
8.
Contact Dermatitis ; 67(5): 298-305, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22594804

RESUMO

BACKGROUND: MicroRNAs are short, endogenous RNA molecules that can bind to parts of target mRNAs, thus inhibiting their translation and causing accelerated turnover or degradation of transcripts, thereby regulating gene expression. Several microRNAs have been found to be upregulated in atopic dermatitis and psoriasis, indicating a role in inflammatory skin diseases. However, there have been no studies on the expression of microRNAs in allergic contact dermatitis. OBJECTIVES: To investigate expression of microRNAs in allergic contact dermatitis. Methods. Lesional and non-lesional skin biopsies were collected from subjects with allergic responses to diphenylcyclopropenone (DPCP). Additional samples for profiling were collected from an experimental mouse model by use of the strong allergen dinitrofluorobenzene. RNA was purified from all samples, and locked nucleic acid microarray analysis was performed, followed by validation with quantitative polymerase chain reaction (PCR). RESULTS: In humans sensitized with DPCP, we found significant upregulation of miR-21, miR-142-3p, miR-142-5p and miR-223 in challenged skin. The same microRNAs were significantly upregulated in the skin of mice in a mouse model of contact allergy. The upregulation of microRNA was confirmed by quantitative PCR. CONCLUSION: These are the first results indicating that microRNAs may be involved in the pathogenesis of allergic contact dermatitis, and they show that mouse models are valuable tools for further study of the involvement of microRNAs in allergic contact dermatitis.


Assuntos
Dermatite Alérgica de Contato/metabolismo , MicroRNAs/metabolismo , Pele/metabolismo , Regulação para Cima , Adulto , Animais , Ciclopropanos/efeitos adversos , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Pessoa de Meia-Idade , Pele/imunologia
9.
Nucleic Acid Ther ; 32(3): 151-162, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35166597

RESUMO

Antisense oligonucleotides are a relatively new therapeutic modality and safety evaluation is still a developing area of research. We have observed that some oligonucleotides can produce acute, nonhybridization dependent, neurobehavioral side effects after intracerebroventricular (ICV) dosing in mice. In this study, we use a combination of in vitro, in vivo, and bioinformatics approaches to identify a sequence design algorithm, which can reduce the number of acutely toxic molecules synthesized and tested in mice. We find a cellular assay measuring spontaneous calcium oscillations in neuronal cells can predict the behavioral side effects after ICV dosing, and may provide a mechanistic explanation for these observations. We identify sequence features that are overrepresented or underrepresented among oligonucleotides causing these reductions in calcium oscillations. A weighted linear combination of the five most informative sequence features predicts the outcome of ICV dosing with >80% accuracy. From this, we develop a bioinformatics tool that allows oligonucleotide designs with acceptable acute neurotoxic potential to be identified, thereby reducing the number of toxic molecules entering drug discovery pipelines. The informative sequence features we identified also suggest areas in which to focus future medicinal chemistry efforts.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Oligonucleotídeos Antissenso , Animais , Encéfalo , Camundongos , Oligonucleotídeos Antissenso/farmacologia
10.
Mol Ther Nucleic Acids ; 29: 625-642, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36090761

RESUMO

Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neurodegenerative disorders characterized by accumulation of hyperphosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we performed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 3' UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.

11.
BMC Genomics ; 12: 97, 2011 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-21294859

RESUMO

BACKGROUND: Several approaches have been developed for miRNA target prediction, including methods that incorporate expression profiling. However the methods are still in need of improvements due to a high false discovery rate. So far, none of the methods have used independent component analysis (ICA). Here, we developed a novel target prediction method based on ICA that incorporates both seed matching and expression profiling of miRNA and mRNA expressions. The method was applied on a cellular model of type 1 diabetes. RESULTS: Microarray profiling identified eight miRNAs (miR-124/128/192/194/204/375/672/708) with differential expression. Applying ICA on the mRNA profiling data revealed five significant independent components (ICs) correlating to the experimental conditions. The five ICs also captured the miRNA expressions by explaining > 97% of their variance. By using ICA, seven of the eight miRNAs showed significant enrichment of sequence predicted targets, compared to only four miRNAs when using simple negative correlation. The ICs were enriched for miRNA targets that function in diabetes-relevant pathways e.g. type 1 and type 2 diabetes and maturity onset diabetes of the young (MODY). CONCLUSIONS: In this study, ICA was applied as an attempt to separate the various factors that influence the mRNA expression in order to identify miRNA targets. The results suggest that ICA is better at identifying miRNA targets than negative correlation. Additionally, combining ICA and pathway analysis constitutes a means for prioritizing between the predicted miRNA targets. Applying the method on a model of type 1 diabetes resulted in identification of eight miRNAs that appear to affect pathways of relevance to disease mechanisms in diabetes.


Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , MicroRNAs/genética , Western Blotting , Linhagem Celular , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Immunology ; 132(3): 394-400, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21070236

RESUMO

Up to one in four lung-transplanted patients develop pulmonary infiltrates and impaired oxygenation within the first days after lung transplantation. Known as primary graft dysfunction (PGD), this condition increases mortality significantly. Complex interactions between donor lung and recipient immune system are the suspected cause. We took an integrative, systems-level approach by first exploring whether the recipient's immune response to PGD includes the development of long-lasting autoreactivity. We next explored whether proteins displaying such differential autoreactivity also display differential gene expression in donor lungs that later develop PGD compared with those that did not. We evaluated 39 patients from whom autoantibody profiles were already available for PGD based on chest radiographs and oxygenation data. An additional nine patients were evaluated for PGD based on their medical records and set aside for validation. From two recent donor lung gene expression studies, we reanalysed and paired gene profiles with autoantibody profiles. Primary graft dysfunction can be distinguished by a profile of differentially reactive autoantibodies binding to 17 proteins. Functional analysis showed that 12 of these proteins are part of a protein-protein interaction network (P=3 x 10⁻6) involved in proliferative processes. A nearest centroid classifier assigned correct PGD grades to eight out of the nine patients in the validation cohort (P=0·048). We observed significant positive correlation (r=0·63, P=0·011) between differences in IgM reactivity and differences in gene expression levels. This connection between donor lung gene expression and long-lasting recipient IgM autoantibodies towards a specific set of proteins suggests a mechanism for the development of autoimmunity in PGD.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Perfilação da Expressão Gênica , Transplante de Pulmão/imunologia , Disfunção Primária do Enxerto/imunologia , Adulto , Idoso , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Disfunção Primária do Enxerto/genética , Doadores de Tecidos
13.
Nucleic Acid Ther ; 31(6): 383-391, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34619060

RESUMO

Phosphorothioate (PS) modification of antisense oligonucleotides (ASOs) is a critical factor enabling their therapeutic use. Standard chemical synthesis incorporates this group in a stereorandom manner; however, significant effort was made over the years to establish and characterize the impact of chiral control. In this work, we present our in-depth characterization of interactions between Escherichia coli RNase H and RNA-DNA heteroduplexes carrying chirally defined PS groups. First, using a massive parallel assay, we showed that at least a single Rp-PS group is necessary for efficient RNase H-mediated cleavage. We followed by demonstrating that this group needs to be aligned to the phosphate-binding pocket of RNase H, and that chiral status of other PS groups in close proximity to RNase H does not affect cleavage efficiency. We have shown that RNase H's PS chiral preference can be utilized to guide cleavage to a specific chemical bond. Finally, we present a strategy for ASO optimization by mapping preferred RNase H cleavage sites of a non-thioated compound, followed by introduction of Rp-PS in a strategic position. This results in a cleaner cleavage profile and higher knockdown activity compared with a compound carrying an Sp-PS at the same location.


Assuntos
Escherichia coli , Ribonuclease H , DNA , Escherichia coli/genética , Fosfatos , Oligonucleotídeos Fosforotioatos , Ribonuclease H/genética , Estereoisomerismo
14.
Immunology ; 130(3): 427-35, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20201985

RESUMO

SUMMARY: Obliterative bronchiolitis (OB) continues to be the major limitation to long-term survival after lung transplantation. The specific aetiology and pathogenesis of OB are not well understood. To explore the role of autoreactivity in OB, we spotted 751 different self molecules onto glass slides, and used these antigen microarrays to profile 48 human serum samples for immunoglobulin G (IgG) and IgM autoantibodies; 27 patients showed no or mild bronchiolitis obliterans syndrome (BOS; a clinical correlate of OB) and 15 patients showed medium to severe BOS. We now report that these BOS grades could be differentiated by a profile of autoantibodies binding to 28 proteins or their peptides. The informative autoantibody profile included down-regulation as well as up-regulation of both IgM and IgG specific reactivities. This profile was evaluated for robustness using a panel of six independent test patients. Analysis of the functions of the 28 informative self antigens showed that eight of them are connected in an interaction network involved in apoptosis and protein metabolism. Thus, a profile of autoantibodies may reflect pathological processes in the lung allograft, suggesting a role for autoimmunity in chronic rejection leading to OB.


Assuntos
Autoanticorpos/imunologia , Bronquiolite Obliterante/diagnóstico , Bronquiolite Obliterante/imunologia , Rejeição de Enxerto/imunologia , Transplante de Pulmão/imunologia , Adulto , Apoptose/fisiologia , Autoanticorpos/sangue , Autoantígenos/imunologia , Pressão Sanguínea/fisiologia , Bronquiolite Obliterante/etiologia , Quimiotaxia/fisiologia , Progressão da Doença , Feminino , Rejeição de Enxerto/complicações , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Redes e Vias Metabólicas/fisiologia , Análise em Microsséries , Pessoa de Meia-Idade , Análise de Componente Principal , Proteínas/imunologia , Proteínas/metabolismo
15.
Mol Ther Nucleic Acids ; 19: 706-717, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-31951854

RESUMO

The identification of molecules that can modulate RNA or protein function and the subsequent chemical and structural optimization to refine such molecules into drugs is a key activity in drug discovery. Here, we explored the extent to which chemical and structural differences in antisense oligonucleotides, designed as gapmers and capable of recruiting RNase H for target RNA cleavage, can affect their functional properties. To facilitate structure-activity learning, we analyzed two sets of iso-sequential locked nucleic acid (LNA)-modified gapmers, where we systematically varied the number and positions of LNA modifications in the flanks. In total, we evaluated 768 different and architecturally diverse gapmers in HeLa cells for target knockdown activity and cytotoxic potential and found widespread differences in both of these properties. Binding affinity between gapmer and RNA target, as well as the presence of certain short sequence motifs in the gap region, can explain these differences, and we propose statistical and machine-learning models that can be used to predict region-specific, optimal LNA-modification architectures. Once accessible regions in the target of interest have been identified, our results show how to refine and optimize LNA gapmers with improved pharmacological profiles targeting such regions.

16.
Mol Ther Nucleic Acids ; 19: 1290-1298, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32092825

RESUMO

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

17.
Plant J ; 56(6): 867-80, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18694460

RESUMO

ATAF1 is a member of a largely uncharacterized plant-specific gene family encoding NAC transcription factors, and is induced in response to various abiotic and biotic stimuli in Arabidopsis thaliana. Previously, we showed that a mutant allele of ATAF1 compromises penetration resistance in Arabidopsis with respect to the non-host biotrophic pathogen Blumeria graminis f. sp. hordei (Bgh). In this study, we have used genome-wide transcript profiling to characterize signalling perturbations in ataf1 plants following Bgh inoculation. Comparative transcriptomic analyses identified an over-representation of abscisic acid (ABA)-responsive genes, including the ABA biosynthesis gene AAO3, which is significantly induced in ataf1 plants compared to wild-type plants following inoculation with Bgh. Additionally, we show that Bgh inoculation results in decreased endogenous ABA levels in an ATAF1-dependent manner, and that the ABA biosynthetic mutant aao3 showed increased penetration resistance to Bgh compared to wild-type plants. Furthermore, we show that ataf1 plants show ABA-hyposensitive phenotypes during seedling development and germination. Our data support a negative correlation between ABA levels and penetration resistance, and identify ATAF1 as a new stimuli-dependent attenuator of ABA signalling for the mediation of efficient penetration resistance in Arabidopsis upon Bgh attack.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Ascomicetos/patogenicidade , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Aldeído Oxidase/genética , Aldeído Oxidase/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Estômatos de Plantas/metabolismo , RNA de Plantas/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética
18.
Methods Mol Biol ; 2036: 261-282, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31410803

RESUMO

Antisense oligonucleotides (AONs) that promote degradation of complementary RNA are being developed as therapeutics. Here, we describe a simple computational workflow for identification of the regions on an RNA that are suitable for targeting with such AONs. The workflow is based on the statistical programming language R, and the calculations and data processing can be carried out on a desktop computer. Our workflow integrates well-established data resources and RNA structure-prediction tools and can be modified easily and expanded as new resources become available.


Assuntos
Biologia Computacional , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos/química , Oligonucleotídeos/genética , Software , Pareamento de Bases , Biologia Computacional/métodos , Humanos , Conformação de Ácido Nucleico , Polimorfismo Genético , Precursores de RNA/química , Precursores de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética
19.
Mol Ther Nucleic Acids ; 10: 45-54, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29499955

RESUMO

The successful development of high-affinity gapmer antisense oligonucleotide (ASO) therapeutics containing locked nucleic acid (LNA) or constrained ethyl (cEt) substitutions has been hampered by the risk of hepatotoxicity. Here, we present an in vitro approach using transfected mouse fibroblasts to predict the potential hepatic liabilities of LNA-modified ASOs (LNA-ASOs), validated by assessing 236 different LNA-ASOs with known hepatotoxic potential. This in vitro assay accurately reflects in vivo findings and relates hepatotoxicity to RNase H1 activity, off-target RNA downregulation, and LNA-ASO-binding affinity. We further demonstrate that the hybridization-dependent toxic potential of LNA-ASOs is also evident in different cell types from different species, which indicates probable translatability of the in vitro results to humans. Additionally, we show that the melting temperature (Tm) of LNA-ASOs maintained below a threshold level of about 55°C greatly diminished the hepatotoxic potential. In summary, we have established a sensitive in vitro screening approach for assessing the hybridization-dependent toxic potential of LNA-ASOs, enabling prioritization of candidate molecules in drug discovery and early development.

20.
Drug Discov Today ; 23(1): 101-114, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28988994

RESUMO

Over the past 20 years, the field of RNA-targeted therapeutics has advanced based on discoveries of modified oligonucleotide chemistries, and an ever-increasing understanding of how to apply cellular assays to identify oligonucleotides with improved pharmacological properties in vivo. Locked nucleic acid (LNA), which exhibits high binding affinity and potency, is widely used for this purpose. Our understanding of RNA biology has also expanded tremendously, resulting in new approaches to engage RNA as a therapeutic target. Recent observations indicate that each oligonucleotide is a unique entity, and small structural differences between oligonucleotides can often lead to substantial differences in their pharmacological properties. Here, we outline new principles for drug discovery exploiting oligonucleotide diversity to identify rare molecules with unique pharmacological properties.


Assuntos
Descoberta de Drogas , Oligonucleotídeos , Animais , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , RNA
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