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1.
Methods Mol Biol ; 2383: 491-513, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34766309

RESUMO

Oligonucleotides (ONs) are therapeutic macromolecules with great potential for the treatment of neurological conditions, including spinal muscular atrophy (SMA), a neurodegenerative disease. However, the neurovascular unit severely limits their distribution to the neural parenchyma of the brain and the spinal cord. Cell-penetrating peptides (CPPs) can be conjugated to oligonucleotides to increase their delivery across biological barriers. In this chapter, we describe the synthesis and conjugation of CPPs to oligonucleotides, and the use of a severe SMA mouse model to test in vivo the efficacy of CPP-delivered oligonucleotides, using ELISA, western blot, and TaqMan™ RT-qPCR assays.


Assuntos
Atrofia Muscular Espinal , Animais , Peptídeos Penetradores de Células , Modelos Animais de Doenças , Camundongos , Atrofia Muscular Espinal/tratamento farmacológico , Oligonucleotídeos , Oligonucleotídeos Antissenso
2.
Methods Mol Biol ; 2404: 207-218, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694611

RESUMO

microRNA capture affinity technology (miR-CATCH) uses affinity capture biotinylated antisense oligonucleotides to co-purify a target transcript together with all its endogenously bound miRNAs. The miR-CATCH assay is performed to investigate miRNAs bound to a specific mRNA. This method allows to have a total vision of miRNAs bound not only to the 3'UTR but also to the 5'UTR and Coding Region of target messenger RNAs (mRNAs).


Assuntos
MicroRNAs/genética , Regiões 3' não Traduzidas , Oligonucleotídeos Antissenso/genética , RNA Mensageiro , Tecnologia
3.
Methods Mol Biol ; 2383: 197-210, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34766291

RESUMO

Cationic cell-penetrating peptides spontaneously associate with negatively charged oligonucleotides to form submicron nanoparticles, so-called polyplexes. Contact with cells leads to endosomal uptake of these nanoparticles. Oligonucleotide activity critically depends on endosomal release and finally dissociation of polyplexes. Fluorescence provides a highly powerful means to follow the spatial dynamics of oligonucleotide uptake, trafficking and decomplexation, in particular when combined with markers of subcellular compartments that enable a quantitative analysis of colocalization and thereby mapping of trafficking routes. In this chapter, we describe protocols for a highly defined formation of polyplexes. We then point out the use of fluorescent fusion proteins to identify subcellular trafficking compartments and image analysis protocols to obtain quantitative information on trafficking routes and endosomal release.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Endossomos , Oligonucleotídeos , Oligonucleotídeos Antissenso , Pirazinas
4.
Drugs Today (Barc) ; 57(12): 707-717, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34909800

RESUMO

Duchenne muscular dystrophy (DMD) is a genetic disorder affecting 1 in 5,000 males which causes progressive muscle deterioration, loss of mobility and eventual death, with an average lifespan of around 25 years. While no cure currently exists for DMD, a novel treatment known as antisense-mediated exon skipping therapy has shown great promise. Exon skipping therapy induces the skipping of mutated exons, restoring the reading frame in dystrophin transcripts and resulting in a truncated but partially functional protein product. In February 2021, Sarepta Therapeutics received accelerated Food and Drug Administration (FDA) approval for their new antisense oligonucleotide, casimersen (brand name Amondys 45). Casimersen targets exon 45 of the dystrophin gene and is expected to treat ~8% of the DMD patient population. The continued approval of this drug will be dependent on satisfactory clinical results from an ongoing phase III trial. This article summarizes the preclinical and clinical data currently available for casimersen, emphasizing pharmacokinetics and safety.


Assuntos
Distrofia Muscular de Duchenne , Éxons , Humanos , Masculino , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos , Oligonucleotídeos Antissenso/uso terapêutico
5.
Nat Med ; 27(10): 1725-1734, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34642494

RESUMO

Chronic infection with hepatitis B virus (HBV) leads to an increased risk of death from cirrhosis and hepatocellular carcinoma. Functional cure rates are low with current treatment options (nucleos(t)ide analogs (NAs) and pegylated interferons). Bepirovirsen is an antisense oligonucleotide targeting all HBV messenger RNAs; in cell culture and animal models, bepirovirsen leads to reductions in HBV-derived RNAs, HBV DNA and viral proteins. This phase 2 double-blinded, randomized, placebo-controlled trial is the first evaluation of the safety and activity of an antisense oligonucleotide targeting HBV RNA in both treatment-naïve and virally suppressed individuals with chronic HBV infection. The primary objective was to assess the safety and tolerability of bepirovirsen in individuals with chronic hepatitis B (CHB) (NCT02981602). The secondary objective was to assess antiviral activity, including the change from baseline to day 29 in serum hepatitis B surface antigen (HBsAg) concentration. Participants with CHB infection ≥6 months and serum HBsAg ≥50 IU ml-1 were enrolled from seven centers across Hong Kong and the Republic of Korea and randomized (3:1 within each dose cohort) to receive bepirovirsen or placebo via subcutaneous injection twice weekly during weeks 1 and 2 (days 1, 4, 8 and 11) and once weekly during weeks 3 and 4 (days 15 and 22). Participants were then followed for 26 weeks. Twenty-four participants were treatment-naïve and seven were receiving stable NA therapy. Treatment-emergent adverse events were mostly mild/moderate (most commonly injection site reactions). Eleven (61.1%) and three (50.0%) treatment-naïve participants experienced one or more treatment-emergent adverse event in the bepirovirsen and placebo groups, respectively. In participants receiving NA therapy, the corresponding numbers were three (60.0%) and one (50.0%). Transient, self-resolving alanine aminotransferase flares (≥2× upper limit of normal) were observed in eight treatment-naïve participants and three participants on stable NA regimens in the bepirovirsen treatment arms. HBsAg reductions were observed and were significant versus placebo for treatment-naïve participants receiving bepirovirsen 300 mg (P = 0.001), but not for the bepirovirsen 150 mg group (P = 0.245) or participants receiving stable NA therapy (P = 0.762). Two participants in each of the 300 mg dose groups achieved HBsAg levels below the lower limit of quantitation by day 29 (n = 3) or day 36 (n = 1). Bepirovirsen had a favorable safety profile. These preliminary observations warrant further investigation of the safety and activity of bepirovirsen in a larger CHB patient population.


Assuntos
Antivirais/administração & dosagem , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Oligonucleotídeos Antissenso/administração & dosagem , Adolescente , Adulto , Antivirais/efeitos adversos , Quimioterapia Combinada , Feminino , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/sangue , Hepatite B Crônica/genética , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Oligonucleotídeos Antissenso/efeitos adversos , Placebos , Polietilenoglicóis/química , República da Coreia/epidemiologia , Adulto Jovem
6.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638554

RESUMO

Vascular smooth muscle cells (VSMCs) display extraordinary phenotypic plasticity. This allows them to differentiate or dedifferentiate, depending on environmental cues. The ability to 'switch' between a quiescent contractile phenotype to a highly proliferative synthetic state renders VSMCs as primary mediators of vascular repair and remodelling. When their plasticity is pathological, it can lead to cardiovascular diseases such as atherosclerosis and restenosis. Coinciding with significant technological and conceptual innovations in RNA biology, there has been a growing focus on the role of alternative splicing in VSMC gene expression regulation. Herein, we review how alternative splicing and its regulatory factors are involved in generating protein diversity and altering gene expression levels in VSMC plasticity. Moreover, we explore how recent advancements in the development of splicing-modulating therapies may be applied to VSMC-related pathologies.


Assuntos
Processamento Alternativo/fisiologia , Plasticidade Celular/genética , Músculo Liso Vascular/metabolismo , Processamento Alternativo/efeitos dos fármacos , Animais , Aterosclerose/etiologia , Aterosclerose/genética , Reestenose Coronária/etiologia , Reestenose Coronária/genética , Humanos , Músculo Liso Vascular/citologia , Oligonucleotídeos Antissenso/uso terapêutico , Fenótipo
8.
PLoS One ; 16(9): e0256938, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34492050

RESUMO

The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5'-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5'-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5' terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.


Assuntos
Oligonucleotídeos Antissenso/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/genética , Proteína Supressora de Tumor p53/genética , Sobrevivência Celular/efeitos dos fármacos , Códon de Iniciação/antagonistas & inibidores , Células HCT116 , Humanos , Íntrons/genética , Isoformas de Proteínas/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , Sítio de Iniciação de Transcrição/efeitos dos fármacos , Proteína Supressora de Tumor p53/antagonistas & inibidores
9.
Adv Drug Deliv Rev ; 178: 113834, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34492233

RESUMO

Recent medical advances have exploited the ability to address a given disease at the underlying level of transcription and translation. These treatment paradigms utilize nucleic acids - including short interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), and messenger RNA (mRNA) - to achieve a desired outcome ranging from gene knockdown to induced expression of a selected target protein. Towards this end, numerous strategies for encapsulation or stabilization of various nucleic acid structures have been developed in order to achieve intracellular delivery. In this review, we discuss several therapeutic applications of nucleic acids directed towards specific diseases and tissues of interest, in particular highlighting recent technologies which have reached late-stage clinical trials and received FDA approval.


Assuntos
Sistemas de Liberação de Medicamentos/tendências , Técnicas de Transferência de Genes/tendências , Ácidos Nucleicos/administração & dosagem , Ácidos Nucleicos/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Animais , COVID-19/genética , COVID-19/metabolismo , COVID-19/terapia , Ensaios Clínicos como Assunto/métodos , Aprovação de Drogas , Sistemas de Liberação de Medicamentos/métodos , Hepatite/genética , Hepatite/metabolismo , Hepatite/terapia , Humanos , MicroRNAs/administração & dosagem , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Ácidos Nucleicos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
10.
J Pharm Biomed Anal ; 206: 114368, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34571322

RESUMO

Therapeutic nucleic acids are various chemically modified RNA or DNA with different functions, which mainly play roles at the gene level. Owing to its accurately targeting at pathogenic genes, nucleic acid based therapeutics have a wide range of application prospects. Recently, the improvement on chemical synthesis and delivery materials accelerated the development of therapeutic nucleic acids rapidly. Up to now, 17 nucleic acid based therapeutics approved by Food and Drug Administration (FDA) or European Medicines Agency (EMA). The development of therapeutics raised higher requirements for analytical methods, both in quality control and in clinical research. The first part of this review introduces different classes of therapeutic nucleic acids, including antisense oligonucleotide (ASO), RNA interference (RNAi) therapy, mRNA, aptamer and other classes which are under research. The second part reviews the therapeutic nucleic acids commercialized from 2019 to now. The third part discusses the analytical methods for nucleic acid based therapeutics, including liquid chromatography-based methods, capillary gel electrophoresis (CGE), hybridization enzyme-linked immunosorbent assay (ELISA) and other infrequently used methods. Finally, the advantages and shortcomings of these methods are summarized, and the future development of analysis methods are prospected.


Assuntos
Ácidos Nucleicos , DNA , Oligonucleotídeos , Oligonucleotídeos Antissenso , RNA/genética
11.
Arterioscler Thromb Vasc Biol ; 41(10): 2538-2550, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34407634

RESUMO

Objective: A cardinal feature of Marfan syndrome is thoracic aortic aneurysm. The contribution of the renin-angiotensin system via AT1aR (Ang II [angiotensin II] receptor type 1a) to thoracic aortic aneurysm progression remains controversial because the beneficial effects of angiotensin receptor blockers have been ascribed to off-target effects. This study used genetic and pharmacological modes of attenuating angiotensin receptor and ligand, respectively, to determine their roles on thoracic aortic aneurysm in mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+). Approach and Results: Thoracic aortic aneurysm in Fbn1C1041G/+ mice was found to be strikingly sexual dimorphic. Males displayed aortic dilation over 12 months while aortic dilation in Fbn1C1041G/+ females did not differ significantly from wild-type mice. To determine the role of AT1aR, Fbn1C1041G/+ mice that were either +/+ or -/- for AT1aR were generated. AT1aR deletion reduced expansion of ascending aorta and aortic root diameter from 1 to 12 months of age in males. Medial thickening and elastin fragmentation were attenuated. An antisense oligonucleotide against angiotensinogen was administered to male Fbn1C1041G/+ mice to determine the effects of Ang II depletion. Antisense oligonucleotide against angiotensinogen administration attenuated dilation of the ascending aorta and aortic root and reduced extracellular remodeling. Aortic transcriptome analyses identified potential targets by which inhibition of the renin-angiotensin system reduced aortic dilation in Fbn1C1041G/+ mice. Conclusions: Deletion of AT1aR or inhibition of Ang II production exerted similar effects in attenuating pathologies in the proximal thoracic aorta of male Fbn1C1041G/+ mice. Inhibition of the renin-angiotensin system attenuated dysregulation of genes within the aorta related to pathology of Fbn1C1041G/+ mice.


Assuntos
Angiotensinogênio/metabolismo , Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/prevenção & controle , Fibrilina-1/genética , Deleção de Genes , Síndrome de Marfan/genética , Receptor Tipo 1 de Angiotensina/genética , Sistema Renina-Angiotensina , Angiotensinogênio/genética , Animais , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Modelos Animais de Doenças , Feminino , Fibrilina-1/metabolismo , Predisposição Genética para Doença , Haploinsuficiência , Masculino , Síndrome de Marfan/metabolismo , Síndrome de Marfan/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Receptor Tipo 1 de Angiotensina/deficiência , Sistema Renina-Angiotensina/genética , Caracteres Sexuais , Fatores Sexuais , Transcriptoma
12.
Nat Commun ; 12(1): 5180, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34462437

RESUMO

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp-/-), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Cardiomiopatias/genética , Cardiomiopatias/terapia , Terapia Genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Oligonucleotídeos Antissenso/genética , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatias/metabolismo , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Ratos , Ratos Endogâmicos Lew
13.
Nucleic Acids Res ; 49(16): 9026-9041, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417625

RESUMO

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.


Assuntos
Oligonucleotídeos Antissenso/síntese química , Índice Terapêutico do Medicamento , Animais , Células HEK293 , Células HeLa , Humanos , Fígado/metabolismo , Masculino , Mesilatos/química , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/toxicidade , Fosforamidas/química , Ligação Proteica , Distribuição Tecidual
14.
Biochem Biophys Res Commun ; 573: 140-144, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34411896

RESUMO

We have been developing a gene silencing technology by harnessing a tRNA 3' processing endoribonuclease, tRNase ZL, with antisense oligonucleotides. Here, to further improve this technology, we investigated how the length and the modifications of naked oligonucleotides affect the efficiency of their uptake by HeLa, HEK293, and HL60 cells by flow cytometry and fluorescence microscopy. 7-30-nt Alexa-Fluor-568-labeled DNAs with phosphorothioate linkages and 7-30-nt Alexa-Fluor-568-labeled, 2'-O-methylated RNAs without phosphorothioate linkages were examined, and, on the whole, longer oligonucleotides were shown to be intracellularly taken up more efficiently. In addition, a 2'-O-methoxyethylated RNA without phosphorothioate linkages, a 2'-fluoriated RNA without phosphorothioate linkages, a 2'-O-methylated RNA with phosphorothioate linkages, and a 2'-O-methylated RNA with phosphorothioate linkages and LNA modifications of 5'-/3'-terminal nucleotides were examined. The oligonucleotides with phosphorothioate linkages were taken up by the cells more efficiently than those without the linkages. Furthermore, we examined how the phosphorothioate linkages of oligonucleotides affect their antisense effects using 22-nt anti-miR16 oligonucleotides with and without phosphorothioate linkages. The latter oligonucleotide decreased the miR16 level much more intensively than the former, although the latter was intracellularly taken up much less efficiently. These observations may be not generalized and differ depending on features of oligonucleotides and cell types. Taken together these results suggest that the productive uptake efficiency for an antisense oligonucleotide needs to be considered to select its length and modifications.


Assuntos
Oligonucleotídeos Antissenso/metabolismo , Fosfatos/metabolismo , Células Cultivadas , Humanos , Oligonucleotídeos Antissenso/química , Fosfatos/química
15.
Nat Struct Mol Biol ; 28(9): 747-754, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34426697

RESUMO

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.


Assuntos
COVID-19/prevenção & controle , Microscopia Crioeletrônica/métodos , Mutação da Fase de Leitura/genética , Oligonucleotídeos Antissenso/genética , RNA Viral/genética , Elementos de Resposta/genética , SARS-CoV-2/genética , Células A549 , Animais , Sequência de Bases , COVID-19/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Genoma Viral/genética , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/farmacologia , RNA Viral/química , RNA Viral/ultraestrutura , SARS-CoV-2/fisiologia , SARS-CoV-2/ultraestrutura , Células Vero , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
16.
Appl Microbiol Biotechnol ; 105(18): 6669-6677, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34427763

RESUMO

The peptide nucleic acid (PNA) is a chimeric molecule with the nucleobases connected by peptide bonds. This chimeric nature gives the PNA certain therapeutic advantages over natural antisense nucleic acid molecules. The PNA probes are known for its better and stronger complementation with target nucleic acids. However, cellular delivery of PNA is a major hurdle due to the charge-neutral nature of the PNA. For cellular delivery of PNA, peptide-PNA conjugates are used. This approach may face some practical limitation in terms of PNA antisense activity. In this study, we propose a novel RATH-2 peptide-based non-covalent PNA delivery mechanism. We observed RATH-2 shows a favorable molecular interaction with PNA at 16:1 (peptide:PNA) molar ratio resulting in co-centric nanoparticle formation. With this combination, we could achieve as high as 93% cellular delivery of the PNA. The proposed non-covalent RATH:PNA delivery model showed endocytic entrapment free delivery of PNA. The study further demonstrated the therapeutic application of PNA with in vitro antiviral intervention model. Using RATH-2 non-covalent PNA delivery system, we could inhibit 69.5% viral load. The present study demonstrates a cell-penetrating peptide:PNA interaction can lead to nanoparticle formations that facilitated cellular delivery of PNA.Key points• A novel cell-penetrating peptide (RATH-2) was identified for non-covalent delivery of PNA.• RATH-2 and PNA formed co-centric nanoparticles at appropriate molar combination.• PNA delivered through the RATH-2 inhibited the viral gene expression and reduced the viral load.


Assuntos
Peptídeos Penetradores de Células , Nanopartículas , Ácidos Nucleicos Peptídicos , Antivirais , Oligonucleotídeos Antissenso
17.
Cells ; 10(8)2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34440661

RESUMO

Pulmonary arterial hypertension (PAH) is a debilitating condition of the pulmonary circulatory system that occurs in patients of all ages and if untreated, eventually leads to right heart failure and death. Despite existing medical treatment options that improve survival and quality of life, the disease remains incurable. Thus, there is an urgent need to develop novel therapies to treat this disease. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play critical roles in pulmonary vascular remodeling and PAH. LncRNAs are implicated in pulmonary arterial endothelial dysfunction by modulating endothelial cell proliferation, angiogenesis, endothelial mesenchymal transition, and metabolism. LncRNAs are also involved in inducing different pulmonary arterial vascular smooth muscle cell phenotypes, such as cell proliferation, apoptosis, migration, regulation of the phenotypic switching, and cell cycle. LncRNAs are essential regulators of gene expression that affect various diseases at the chromatin, transcriptional, post-translational, and even post-translational levels. Here, we focus on the role of LncRNAs and their molecular mechanisms in the pathogenesis of PAH. We also discuss the current research challenge and potential biomarker and therapeutic potentials of lncRNAs in PAH.


Assuntos
Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , RNA Longo não Codificante/metabolismo , Remodelação Vascular , Animais , Biomarcadores/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Humanos , Oligonucleotídeos Antissenso/uso terapêutico , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/fisiopatologia , Hipertensão Arterial Pulmonar/terapia , Artéria Pulmonar/fisiopatologia , RNA Longo não Codificante/genética , Terapêutica com RNAi , Transdução de Sinais , Disfunção Ventricular Direita/metabolismo , Disfunção Ventricular Direita/fisiopatologia , Função Ventricular Direita , Remodelação Ventricular
18.
Int J Mol Sci ; 22(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34445083

RESUMO

Intronic splicing silencer N1 (ISS-N1) located within Survival Motor Neuron 2 (SMN2) intron 7 is the target of a therapeutic antisense oligonucleotide (ASO), nusinersen (Spinraza), which is currently being used for the treatment of spinal muscular atrophy (SMA), a leading genetic disease associated with infant mortality. The discovery of ISS-N1 as a promising therapeutic target was enabled in part by Anti-N1, a 20-mer ASO that restored SMN2 exon 7 inclusion by annealing to ISS-N1. Here, we analyzed the transcriptome of SMA patient cells treated with 100 nM of Anti-N1 for 30 h. Such concentrations are routinely used to demonstrate the efficacy of an ASO. While 100 nM of Anti-N1 substantially stimulated SMN2 exon 7 inclusion, it also caused massive perturbations in the transcriptome and triggered widespread aberrant splicing, affecting expression of essential genes associated with multiple cellular processes such as transcription, splicing, translation, cell signaling, cell cycle, macromolecular trafficking, cytoskeletal dynamics, and innate immunity. We validated our findings with quantitative and semiquantitative PCR of 39 candidate genes associated with diverse pathways. We also showed a substantial reduction in off-target effects with shorter ISS-N1-targeting ASOs. Our findings are significant for implementing better ASO design and dosing regimens of ASO-based drugs.


Assuntos
Atrofia Muscular Espinal/terapia , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Transcriptoma , Células Cultivadas , Terapia Genética , Humanos , Íntrons/efeitos dos fármacos , Atrofia Muscular Espinal/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Transcriptoma/efeitos dos fármacos
19.
Nucleic Acids Res ; 49(14): 8277-8293, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34244781

RESUMO

Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs can trigger RNase H1 cleavage of cellular target RNAs to modulate gene expression. Internalized PS-ASOs must be released from membraned endosomal organelles, a rate limiting step that is not well understood. Recently we found that M6PR transport between Golgi and late endosomes facilitates productive release of PS-ASOs, raising the possibility that Golgi-mediated transport may play important roles in PS-ASO activity. Here we further evaluated the involvement of Golgi in PS-ASO activity by examining additional Golgi proteins. Reduction of certain Golgi proteins, including Golgi-58K, GCC1 and TGN46, decreased PS-ASO activity, without substantial effects on Golgi integrity. Upon PS-ASO cellular uptake, Golgi-58K was recruited to late endosomes where it colocalized with PS-ASOs. Reduction of Golgi-58K caused slower PS-ASO release from late endosomes, decreased GCC2 late endosome relocalization, and led to slower retrograde transport of M6PR from late endosomes to trans-Golgi. Late endosome relocalization of Golgi-58K requires Hsc70, and is most likely mediated by PS-ASO-protein interactions. Together, these results suggest a novel function of Golgi-58K in mediating Golgi-endosome transport and indicate that the Golgi apparatus plays an important role in endosomal release of PS-ASO, ensuring antisense activity.


Assuntos
Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi/genética , Glicoproteínas de Membrana/genética , Receptor IGF Tipo 2/genética , Transporte Biológico/genética , Endocitose/genética , Endossomos/genética , Complexo de Golgi/efeitos dos fármacos , Células HeLa , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Fosforotioatos/genética , Ribonuclease H/genética
20.
Angew Chem Int Ed Engl ; 60(39): 21226-21230, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34296814

RESUMO

The combination of gene therapy and chemotherapy provides a We developed a simple and versatile approach to prepare a series of two-in-one nanodrugs through direct self-assembly of cyanine-labeled single-stranded DNA (Cys-DNA) and different types of drug molecules. Molecular dynamics simulation showed that the Cys introduced into the DNA could enhance the noncovalent interaction between Cys-DNA and drug molecules. More drug molecules were incorporated into Cys-DNA, tending to spontaneously form hybrid Cys-DNA/drug nanosphere. Such nanospheres serve as both carriers and cargoes, excluding the extra use of nontherapeutic excipients and showing ultrahigh drug loading capacity. Following this approach, an antisense oligonucleotides/doxorubicin nanodrug model was constructed, demonstrating the significant synergistic anti-tumor therapeutic effect. As a proof of the concept, our study establishes a simple and reproducible two-in-one nucleic acid-based drug formulation.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Carbocianinas/química , DNA/química , Doxorrubicina/farmacologia , Nanopartículas/química , Oligonucleotídeos Antissenso/farmacologia , Antibióticos Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Dinâmica Molecular , Oligonucleotídeos Antissenso/química , Tamanho da Partícula
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