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1.
Oncotarget ; 8(46): 80651-80665, 2017 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-29113333

RESUMO

Excessive skin scars due to elective operations or trauma represent a challenging clinical problem. Pathophysiology of hypertrophic scars entails a prolonged inflammatory and proliferative phase of wound healing. Over expression of TGF-ß1 and COX-2 play key regulatory roles of the aberrant fibrogenic responses and proinflammatory mediators. When we silenced TGF-ß1 and COX-2 expression simultaneously in primary human fibroblasts, a marked increase in the apoptotic cell population occurred in contrast to those only treated with either TGF-ß1 or COX-2 siRNA alone. Furthermore, using human hypertrophic scar and skin graft implant models in mice, we observed significant size reductions of the implanted tissues following intra-scar administration of TGF-ß1/COX-2 specific siRNA combination packaged with Histidine Lysine Polymer (HKP). Gene expression analyses of those treated tissues revealed silencing of the target gene along with down regulations of pro-fibrotic factors such as α-SMA, hydroxyproline acid, Collagen 1 and Collagen 3. Using TUNEL assay detection, we found that the human fibroblasts in the implanted tissues treated with the TGF-ß1/COX-2siRNAs combination exhibited significant apoptotic activity. Therefore we conclude that a synergistic effect of the TGF-ß1/COX-2siRNAs combination contributed to the size reductions of the hypertrophic scar implants, through activation of fibroblast apoptosis and re-balancing between scar tissue deposition and degradation.

2.
Am J Pathol ; 185(4): 1073-84, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25700796

RESUMO

Ocular infection with herpes simplex virus 1 can result in a chronic immunoinflammatory stromal keratitis (SK) lesion that is a significant cause of human blindness. A key to controlling SK lesion severity is to identify cellular and molecular events responsible for tissue damage and to manipulate them therapeutically. Potential targets for therapy are miRNAs, but these are minimally explored especially in responses to infection. Here, we demonstrated that Mir155 expression was up-regulated after ocular herpes simplex virus 1 infection, with the increased Mir155 expression occurring mainly in macrophages and CD4(+) T cells and to a lesser extent in neutrophils. In vivo studies indicated that Mir155 knockout mice were more resistant to herpes SK with marked suppression of T helper cells type 1 and 17 responses both in the ocular lesions and the lymphoid organs. The reduced SK lesion severity was reflected by increased phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 and interferon-γ receptor α-chain levels in activated CD4(+) T cells in the lymph nodes. Finally, in vivo silencing of miR-155 by the provision of antagomir-155 nanoparticles to herpes simplex virus 1-infected mice led to diminished SK lesions and corneal vascularization. In conclusion, our results indicate that miR-155 contributes to the pathogenesis of SK and represents a promising target to control SK severity.


Assuntos
Substância Própria/patologia , Substância Própria/virologia , Ceratite Herpética/genética , Ceratite Herpética/virologia , MicroRNAs/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Quimiocinas/metabolismo , Substância Própria/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Herpesvirus Humano 1/fisiologia , Humanos , Inflamação/patologia , Inositol Polifosfato 5-Fosfatases , Ceratite Herpética/imunologia , Ceratite Herpética/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Modelos Biológicos , Nanopartículas/química , Oligonucleotídeos/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Receptores de Interferon/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Regulação para Cima/efeitos dos fármacos
3.
Mol Ther Nucleic Acids ; 3: e182, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25093706

RESUMO

Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed "chemical antibodies." In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

4.
Am J Pathol ; 181(2): 525-34, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22659469

RESUMO

MicroRNAs (miRNAs) are small regulatory molecules that control diverse biological processes that include angiogenesis. Herpes simplex virus (HSV) causes a chronic immuno-inflammatory response in the eye that may result in corneal neovascularization during blinding immunopathological lesion stromal keratitis (SK). miR-132 is a highly conserved miRNA that is induced in endothelial cells in response to growth factors, such as vascular endothelial growth factor (VEGF). In this study, we show that miR-132 expression was up-regulated (10- to 20-fold) after ocular infection with HSV, an event that involved the production of both VEGF-A and IL-17. Consequently, blockade of VEGF-A activity using soluble VEGF receptor 1 resulted in significantly lower levels of corneal miR-132 after HSV infection. In addition, low levels of corneal miR-132 were detected in IL-17 receptor knockout mice after HSV infection. In vivo silencing of miR-132 by the provision of anti-miR-132 (antagomir-132) nanoparticles to HSV-infected mice led to reduced corneal neovascularization and diminished SK lesions. The anti-angiogenic effect of antagomir-132 was reflected by a reduction in angiogenic Ras activity in corneal CD31-enriched cells (presumably blood vessel endothelial cells) during SK. To our knowledge, this is one of the first reports of miRNA involvement in an infectious ocular disease. Manipulating miRNA expression holds promise as a therapeutic approach to control an ocular lesion that is an important cause of human blindness.


Assuntos
Infecções Oculares/genética , Infecções Oculares/virologia , Ceratite Herpética/genética , MicroRNAs/metabolismo , Neovascularização Patológica/complicações , Neovascularização Patológica/genética , Simplexvirus/fisiologia , Animais , Córnea/irrigação sanguínea , Córnea/metabolismo , Córnea/patologia , Córnea/virologia , Neovascularização da Córnea/complicações , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Neovascularização da Córnea/virologia , Infecções Oculares/complicações , Infecções Oculares/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Interleucina-17/metabolismo , Ceratite Herpética/complicações , Ceratite Herpética/patologia , Ceratite Herpética/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Modelos Biológicos , Nanopartículas , Neovascularização Patológica/patologia , Oligorribonucleotídeos/administração & dosagem , Oligorribonucleotídeos/farmacologia , Receptores de Interleucina-17/metabolismo , Simplexvirus/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas ras/metabolismo
5.
Future Med Chem ; 1(9): 1671-81, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21425985

RESUMO

While the discovery of RNA interference (RNAi) has been considered one of the most significant breakthroughs in biomedicine, its prospects for novel therapeutic applications are even more exciting. The high specificity, exquisite selectivity and chemical homogeneity of small interfering RNAs (siRNA; intermediates in RNAi activity), provide unique advantages for these moieties as multi-targeted inhibitory drugs. Many such applications have demonstrated significant benefit compared with single gene-targeted siRNA inhibitors. In this article, we will review the current status of using a multi-targeted siRNA cocktail for novel therapeutic development in the treatment of cancer and viral infections. We will also propose the characteristics of various types of siRNA cocktails and their design, while recognizing the potential future impact of and challenges facing this unique therapeutic modality.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Humanos , Neoplasias/genética , Neoplasias/terapia , Viroses/genética , Viroses/terapia
6.
Drugs Future ; 34(9): 721, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20161621

RESUMO

Sequence-specific gene silencing with small interfering RNA (siRNA) has transformed basic science research, and the efficacy of siRNA therapeutics toward a variety of diseases is now being evaluated in pre-clinical and clinical trials. Despite its potential value, the highly negatively charged siRNA has the classic delivery problem of requiring transport across cell membranes to the cytosol. Consequently, carrier development for siRNA delivery is one of the most important problems to solve before siRNA can achieve widespread clinical use. An assortment of non-viral carriers including liposomes, peptides, polymers, and aptamers are being evaluated for their ability to shepherd siRNA to the target tissue and cross the plasma membrane barrier into the cell. Several promising carriers with low toxicity and increased specificity for disease targets have emerged for siRNA-based therapeutics. This review will discuss non-viral approaches for siRNA therapeutics, with particular focus on synthetic carriers for in vivo systemic delivery of siRNA.

7.
Mol Cancer Ther ; 7(6): 1355-64, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18524845

RESUMO

The rhomboid family of genes carry out a wide range of important functions in a variety of organisms. Little is known, however, about the function of the human rhomboid family-1 gene (RHBDF1). We show here that RHBDF1 function is essential to epithelial cancer cell growth. RHBDF1 mRNA level is significantly elevated in clinical specimens of invasive ductal carcinoma of the breast, and the protein is readily detectable in human breast cancer or head and neck cancer cell lines. Silencing the RHBDF1 gene with short interfering RNA (siRNA) results in apoptosis in breast cancer MDA-MB-435 cells and autophagy in head and neck squamous cell cancer 1483 cells. The treatment also leads to significant down-modulation of activated AKT and extracellular signal-regulated kinase in the cells, suggesting that critically diminished strength of these growth signals may be the key attributes of the induction of cell death. Furthermore, silencing the RHBDF1 gene in MDA-MB-435 or 1483 xenograft tumors on athymic nude mice by using i.v. administered histidine-lysine polymer nanoparticle-encapsulated siRNA results in marked inhibition of tumor growth. Our findings indicate that RHBDF1 has a pivotal role in sustaining growth signals in epithelial cancer cells and thus may serve as a therapeutic target for treating epithelial cancers.


Assuntos
Apoptose , Autofagia , Células Epiteliais/patologia , Receptores ErbB/genética , Inativação Gênica , Neoplasias/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Proteínas de Membrana , Camundongos , Camundongos Nus , Nanopartículas , Neoplasias/genética , Neoplasias de Células Escamosas/genética , Neoplasias de Células Escamosas/patologia , Polímeros , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos
8.
Methods Mol Biol ; 442: 139-58, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18369784

RESUMO

Containment of the SARS coronavirus (SCV) outbreak was accompanied by the rapid characterization of this new pathogen's genome sequence in 2003, encouraging the development of anti-SCV therapeutics using short interfering RNA (siRNA) inhibitors. A pair of siRNA duplexes identified as potent SCV inhibitors in vitro was evaluated for in vivo efficacy and safety in a rhesus macaque SARS model using intranasal administration with clinical viable delivery carrier in three dosing regimens. Observations of SCV-induced SARS-like symptoms, measurements of SCV RNA presence in the respiratory tract, microscopic inspections of lung histopathology, and immunohistochemistry sections from 21 tested macaques consistently demonstrated siRNA-mediated anti-SCV activity. The prophylactic and therapeutic efficacies resulted in relief of animals from SCV infection-induced fever, diminished SCV in upper airway and lung alveoli, and milder acute diffuse alveoli damage (DAD). The dosages of siRNA used, 10 to 40 mg/kg, did not show any sign of siRNA-induced toxicity. These results support that a clinical investigation of this anti-SARS siRNA therapeutic agent is warranted. The study also illustrates the capability of siRNA to enable a massive reduction in development time for novel targeted therapeutic agents. We detail a representative example of large-mammal siRNA use.


Assuntos
RNA Interferente Pequeno , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/terapia , Animais , Sequência de Bases , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Humanos , Pulmão/citologia , Pulmão/patologia , Pulmão/virologia , Macaca mulatta , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Vírus da SARS/metabolismo , Síndrome Respiratória Aguda Grave/virologia
9.
Methods Mol Biol ; 437: 93-107, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18369963

RESUMO

The gene silencing capability of RNA interference (RNAi) is being used to study individual gene's biological function and role in biochemical pathways. However, the efficacy of RNAi depends upon efficient delivery of the intermediates of RNAi, small interfering RNA (siRNA) oligonucleotides. The delivery challenge is even greater when the aim is to inhibit the expression of target genes in disease tissues. In vivo delivery of siRNA is complicated and challenging, and recent works on various animal disease models and early successes in human clinical trials are enlightening the tremendous potential of RNAi therapeutics. In this chapter, the latest developments of in vivo delivery of siRNA and the critical issues related to this effort are addressed.


Assuntos
Sistemas de Liberação de Medicamentos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Animais , Humanos
10.
Drug Discov Today ; 11(1-2): 67-73, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16478693

RESUMO

The use of RNA interference (RNAi) is spreading rapidly to nearly every aspect of biomedical research. The gene silencing capability of RNAi is being used to study individual gene's biological function and role in biochemical pathways. However, the efficacy of RNAi depends upon efficient delivery of the intermediates of RNAi, short interfering RNA (siRNA) and short hairpin RNA (shRNA) oligonucleotides. The delivery challenge is even greater when the aim is to inhibit the expression of target genes in animal models. Although i n vivo delivery of siRNA is complicated and challenging, recent results are encouraging. In this review, the latest developments of in vivo delivery of siRNA and the crucial issues related to this effort are addressed.


Assuntos
Desenho de Fármacos , RNA Interferente Pequeno , Animais , Vias de Administração de Medicamentos , Sistemas de Liberação de Medicamentos , Marcação de Genes , Humanos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico
11.
Nat Med ; 11(9): 944-51, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16116432

RESUMO

Development of therapeutic agents for severe acute respiratory syndrome (SARS) viral infection using short interfering RNA (siRNA) inhibitors exemplifies a powerful new means to combat emerging infectious diseases. Potent siRNA inhibitors of SARS coronavirus (SCV) in vitro were further evaluated for efficacy and safety in a rhesus macaque (Macaca mulatta) SARS model using clinically viable delivery while comparing three dosing regimens. Observations of SARS-like symptoms, measurements of SCV RNA presence and lung histopathology and immunohistochemistry consistently showed siRNA-mediated anti-SARS efficacy by either prophylactic or therapeutic regimens. The siRNAs used provided relief from SCV infection-induced fever, diminished SCV viral levels and reduced acute diffuse alveoli damage. The 10-40 mg/kg accumulated dosages of siRNA did not show any sign of siRNA-induced toxicity. These results suggest that a clinical investigation is warranted and illustrate the prospects for siRNA to enable a massive reduction in development time for new targeted therapeutic agents.


Assuntos
Antivirais/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Vírus da SARS/efeitos dos fármacos , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/prevenção & controle , Animais , Relação Dose-Resposta a Droga , Feminino , Genoma Viral , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Macaca mulatta , Masculino , Camundongos , Dados de Sequência Molecular , Síndrome Respiratória Aguda Grave/patologia
12.
Adv Genet ; 54: 117-42, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16096010

RESUMO

RNAi has rapidly become a powerful tool for drug target discovery and validation in cell culture, and now has largely displaced efforts with antisense and ribozymes. Consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Studies on RNAi have resulted in two basic methods for its use for gene selective inhibition: 1) cytoplasmic delivery of short dsRNA oligonucleotides (siRNA), which mimics an active intermediate of an endogenous RNAi mechanism and 2) nuclear delivery of gene expression cassettes that express a short hairpin RNA (shRNA), which mimics the micro interfering RNA (miRNA) active intermediate of a different endogenous RNAi mechanism. Non-viral gene delivery systems are a diverse collection of technologies that are applicable to both of these forms of RNAi. Importantly, unlike antisense and ribozyme systems, a remarkable trait of siRNA is a lack of dependence on chemical modifications blocking enzymatic degradation, although chemical protection methods developed for the earlier systems are being incorporated into siRNA and are generally compatible with non-viral delivery systems. The use of siRNA is emerging more rapidly than for shRNA, in part due to the increased effort required to construct shRNA expression systems before selection of active sequences and verification of biological activity are obtained. In contrast, screens of many siRNA sequences can be accomplished rapidly using synthetic oligos. It is not surprising that the use of siRNA in vivo is also emerging first. Initial in vivo studies have been reported for both viral and non-viral delivery but viral delivery is limited to shRNA. This review describes the emerging in vivo application of non-viral delivery systems for RNAi for functional genomics, which will provide a foundation for further development of RNAi therapeutics. Of interest is the rapid adaptation of ligand-targeted plasmid-based nanoparticles for RNAi agents. These systems are growing in capabilities and beginning to pose a serious rival to viral vector based gene delivery. The activity of siRNA in the cytoplasm may lower the hurdle and thereby accelerate the successful development of therapeutics based on targeted non-viral delivery systems.


Assuntos
Interferência de RNA , Animais , Sistemas de Liberação de Medicamentos , Marcação de Genes , Terapia Genética , Genômica , Humanos , Técnicas In Vitro , Neoplasias/genética , Neoplasias/terapia , Especificidade de Órgãos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico
13.
J Control Release ; 109(1-3): 5-14, 2005 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-15979191

RESUMO

The recently developed siRNA oligonucleotides are an attractive alternative to antisense as a therapeutic modality because of their robust, gene selective silencing of drug target protein expression. To achieve therapeutic success, however, several hurdles must be overcome including rapid clearance, nuclease degradation, and inefficient intracellular localization. In this presentation, we discuss design strategies for development of self-assembling nanoscale carriers for neovasculature targeted delivery of siRNA inhibiting tumor or ocular angiogenesis.


Assuntos
Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/uso terapêutico , Neovascularização Patológica/terapia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Animais , Células Cultivadas , Sistemas de Liberação de Medicamentos , Citometria de Fluxo , Humanos , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Peptídeos/química , Fosfatidiletanolaminas/química , Polietilenoglicóis
14.
Trends Mol Med ; 11(3): 104-13, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15760768

RESUMO

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. This article describes the latest developments using small-interfering RNA (siRNA) inhibitors to downregulate various angiogenic and tumor-associated factors, both in cell-culture assays and in animal disease models. The majority of research efforts are currently focused on understanding gene function, as well as proof-of-concept for siRNA-mediated anti-angiogenesis. The prospects for siRNA therapeutics, both advantages and looming hurdles, are evaluated.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Neovascularização Patológica/terapia , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Transdução de Sinais/fisiologia , Moléculas de Adesão Celular/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , Neovascularização Patológica/fisiopatologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
Nucleic Acids Res ; 32(19): e149, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15520458

RESUMO

Potent sequence selective gene inhibition by siRNA 'targeted' therapeutics promises the ultimate level of specificity, but siRNA therapeutics is hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these problems, ligand-targeted, sterically stabilized nanoparticles have been adapted for siRNA. Self-assembling nanoparticles with siRNA were constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG), as a means to target tumor neovasculature expressing integrins and used to deliver siRNA inhibiting vascular endothelial growth factor receptor-2 (VEGF R2) expression and thereby tumor angiogenesis. Cell delivery and activity of PEGylated PEI was found to be siRNA sequence specific and depend on the presence of peptide ligand and could be competed by free peptide. Intravenous administration into tumor-bearing mice gave selective tumor uptake, siRNA sequence-specific inhibition of protein expression within the tumor and inhibition of both tumor angiogenesis and growth rate. The results suggest achievement of two levels of targeting: tumor tissue selective delivery via the nanoparticle ligand and gene pathway selectivity via the siRNA oligonucleotide. This opens the door for better targeted therapeutics with both tissue and gene selectivity, also to improve targeted therapies with less than ideal therapeutic targets.


Assuntos
Neoplasias Experimentais/terapia , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Células Cultivadas , Coloides , Feminino , Humanos , Ligantes , Camundongos , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Fenótipo , Polietilenoglicóis/química , Polietilenoimina/química , Polímeros/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética
16.
Antivir Ther ; 9(3): 365-74, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15259899

RESUMO

OBJECTIVES: To identify and characterize the siRNA duplexes that are effective for inhibition of SARS-CoV infection and replication in the non-human primate cells. This in vitro study will serve as the foundation for development of novel anti-SARS therapeutics. METHODS: 48 siRNA sequences were designed for targeting regions throughout entire SARS-CoV genome RNA including open-reading frames for several key proteins. Chemically synthesized siRNA duplexes were transfected into foetal rhesus kidney (FRhK-4) cells prior to or after SARS-CoV infection. The inhibitory effects of the siRNAs were evaluated for reductions of intracellular viral genome copy number and viral titres in the cell culture medium measured by Q-RT-PCR and CPE-based titration, respectively. Four siRNA duplexes were found to achieve potent inhibition of SARS-CoV infection and replication. A prolonged prophylactic effect of siRNA duplexes with up to 90% inhibition that lasted for at least 72 h was observed. Combination of active siRNA duplexes targeting different regions of the viral genome resulted in therapeutic activity of up to 80% inhibition. CONCLUSION: Chemically synthesized siRNA duplexes targeting SARS-CoV genomic RNA are potent agents for inhibition of the viral infection and replication. The location effects of siRNAs were revealed at both genome sequence and open-reading frame levels. The rapid development of siRNA-based SARS-CoV inhibitors marked a novel approach for combating newly emergent infectious diseases.


Assuntos
Antivirais , Efeito Citopatogênico Viral/fisiologia , RNA Interferente Pequeno/genética , RNA Viral/antagonistas & inibidores , Vírus da SARS/fisiologia , Animais , Antivirais/síntese química , Linhagem Celular , Meios de Cultura , Terapia Genética , Genoma Viral , Macaca mulatta , RNA Interferente Pequeno/síntese química , RNA Viral/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus da SARS/genética , Vírus da SARS/isolamento & purificação , Fatores de Tempo , Transfecção , Replicação Viral/genética
17.
Curr Opin Mol Ther ; 5(3): 225-34, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12870431

RESUMO

Application of siRNA to knockdown a specific gene requires target mRNA accessibility, effective intracellular delivery of siRNA into target cells and potent siRNA inhibition of target mRNA. Use of siRNA as a tool is advancing in almost every field of biomedical research, but some of the most dynamic and exciting applications of siRNA are in cancer research. This review summarizes the results obtained with siRNA in cancer, in particular functional validation of tumorigenic genes in cell culture and animal tumor models, effective siRNA delivery systems, efficiency of siRNA agents compared with antisense oligonucleotides and efforts for potential therapeutic development. Along with the rapidly growing literature on using siRNA as a functional genomic tool, there is emerging evidence that siRNA may represent a novel therapeutic modality for cancer treatment when optimized local and systemic delivery systems are available.


Assuntos
Inativação Gênica , Técnicas de Transferência de Genes , Terapia Genética/métodos , Neoplasias/terapia , RNA Interferente Pequeno , Animais , Ciclo Celular/fisiologia , Marcação de Genes , Substâncias de Crescimento/metabolismo , Neoplasias/fisiopatologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo
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