RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoo tonic, highly pathogenic virus. The new type of coronavirus with contagious nature spread from Wuhan (China) to the whole world in a very short time and caused the new coronavirus disease (COVID-19). COVID-19 has turned into a global public health crisis due to spreading by close person-to-person contact with high transmission capacity. Thus, research about the treatment of the damages caused by the virus or prevention from infection increases everyday. Besides, there is still no approved and definitive, standardized treatment for COVID-19. However, this disaster experienced by human beings has made us realize the significance of having a system ready for use to prevent humanity from viral attacks without wasting time. As is known, nanocarriers can be targeted to the desired cells in vitro and in vivo. The nano-carrier system targeting a specific protein, containing the enzyme inhibiting the action of the virus can be developed. The system can be used by simple modifications when we encounter another virus epidemic in the future. In this review, we present a potential treatment method consisting of a nanoparticle-ribozyme conjugate, targeting ACE-2 receptors by reviewing the virus-associated ribozymes, their structures, types and working mechanisms.
Assuntos
Tratamento Farmacológico da COVID-19 , Nanopartículas/administração & dosagem , RNA Catalítico/uso terapêutico , RNA Viral/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Ensaios Clínicos como Assunto , Portadores de Fármacos , Composição de Medicamentos , Desenho de Fármacos , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Interferência de RNA , RNA Catalítico/administração & dosagem , RNA Catalítico/química , RNA Catalítico/classificação , RNA não Traduzido/classificação , RNA não Traduzido/genética , RNA não Traduzido/uso terapêutico , Receptores de Coronavírus/antagonistas & inibidores , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
The vast majority of the human genome is transcribed into RNA molecules that do not code for proteins, which could be small ones approximately 20 nucleotide in length, known as microRNAs, or transcripts longer than 200 bp, defined as long noncoding RNAs. The prevalent deregulation of microRNAs in human cancers prompted immediate interest on the therapeutic value of microRNAs as drugs and drug targets. Many features of microRNAs such as well-defined mechanisms, and straightforward oligonucleotide design further make them attractive candidates for therapeutic development. The intensive efforts of exploring microRNA therapeutics are reflected by the large body of preclinical studies using oligonucleotide-based mimicking and blocking, culminated by the recent entry of microRNA therapeutics in clinical trial for several human diseases including cancer. Meanwhile, microRNA therapeutics faces the challenge of effective and safe delivery of nucleic acid therapeutics into the target site. Various chemical modifications of nucleic acids and delivery systems have been developed to increase targeting specificity and efficacy, and reduce the associated side effects including activation of immune response. Recently, long noncoding RNAs become attractive targets for therapeutic intervention because of their association with complex and delicate phenotypes, and their unconventional pharmaceutical activities such as capacity of increasing output of proteins. Here I discuss the general therapeutic strategies targeting noncoding RNAs, review delivery systems developed to maximize noncoding RNA therapeutic efficacy, and offer perspectives on the future development of noncoding RNA targeting agents for colorectal cancer.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , RNA não Traduzido/uso terapêutico , Neoplasias Colorretais/genética , Sistemas de Liberação de Medicamentos , Endocitose , Previsões , Humanos , Lipossomos , MicroRNAs/administração & dosagem , MicroRNAs/antagonistas & inibidores , MicroRNAs/uso terapêutico , Terapia de Alvo Molecular/métodos , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/uso terapêutico , Veículos Farmacêuticos , RNA Catalítico/administração & dosagem , RNA Catalítico/uso terapêutico , RNA Longo não Codificante/administração & dosagem , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/uso terapêutico , RNA Neoplásico/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , RNA não Traduzido/administração & dosagem , RNA não Traduzido/antagonistas & inibidoresRESUMO
Although activity has been reported in vivo, free nucleic acid-based drugs are rapidly degraded and cleared following systemic administration. To address these challenges and improve the potency and bioavailability of genetic drugs, significant efforts have been made to develop effective delivery systems of which lipid nanoparticles (LNP) represent the most advanced technology currently available. In this review, we will describe and discuss the improvements to the pharmacokinetic and pharmacodynamic properties of nucleic acid-based drugs mediated by LNP delivery. It is envisioned that the significant improvements in potency and safety, largely driven by the development of LNP encapsulated siRNA drugs, will be translatable to other types of genetic drugs and enable the rapid development of potent molecular tools and drugs.
Assuntos
Portadores de Fármacos/química , Lipídeos/química , Nanopartículas/química , Nucleotídeos/administração & dosagem , Nucleotídeos/farmacocinética , Animais , DNA Catalítico/administração & dosagem , DNA Catalítico/farmacocinética , DNA Catalítico/farmacologia , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos , Humanos , MicroRNAs/administração & dosagem , MicroRNAs/farmacocinética , MicroRNAs/farmacologia , Nucleotídeos/farmacologia , RNA Catalítico/administração & dosagem , RNA Catalítico/farmacocinética , RNA Catalítico/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/farmacologiaRESUMO
Nucleic acid therapies targeting HIV replication have the potential to be used in conjunction with or in place of the standard small-molecule therapies. Among the different classes of nucleic acid therapies, several ribozymes (Rzs, RNA enzymes) have been developed to target HIV RNA. The design of Rzs targeting HIV RNA is complicated by the sequence diversity of viral strains and the structural diversity of their target sites. Using the SOFA-HDV Rz as an example, this chapter describes methods that can be used to design Rzs for controlling HIV replication. We describe how to (1) identify highly conserved Rz target sites in HIV RNA; (2) generate a set of Rzs with the potential to be used as therapeutics; and (3) screen these Rzs for activity against HIV production.
Assuntos
Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , RNA Catalítico/administração & dosagem , Infecções por HIV/virologia , Humanos , Biologia Molecular/métodos , RNA Catalítico/genética , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Replicação Viral/efeitos dos fármacosRESUMO
The thickening of the vessel wall (intimal hyperplasia) is a pathological process which often follows revascularization approaches such as transluminal angioplasty and artery bypass graft, procedures used to re-vascularize stenotic artery. Despite the significant improvements in the treatment of intimal hyperplasia obtained in the last years, the problem has not completely solved. Nucleic acid based-drugs (NABDs) represent an emergent class of molecules with potential therapeutic value for the treatment of intimal hyperplasia. NABDs of interest in the field of intimal hyperplasia are: ribozymes, DNAzymes, antisense oligonucleotides, decoy oligonucleotides, small interfering RNAs and micro interfering RNAs. These molecules can recognize, in a sequencespecific fashion, a target which, depending on the different NABDs, can be represented by a nucleic acid or a protein. Upon binding, NABDs can down-modulate the functions of the target (mRNA/proteins) and thus they are used to impair the functions of disease-causing biological molecules.In spite of the great therapeutic potential demonstrated by NABDs in many experimental model of intima hyperplasia, their practical use is hindered by the necessity to identify optimal delivery systems to the vasculature. In the first part of this review a brief description of the clinical problem related to intima hyperplasia formation after revascularization procedures is reported. In the second part, the attention is focused on the experimental evidences of NABD therapeutic potential in the prevention of intimal hyperplasia. Finally, in the third part, we will describe the strategies developed to optimize NABD delivery to the diseased vessel.
Assuntos
Ácidos Nucleicos/administração & dosagem , Doenças Vasculares/tratamento farmacológico , DNA Catalítico/administração & dosagem , DNA Catalítico/química , Portadores de Fármacos/química , Endotélio Vascular/patologia , Humanos , Hiperplasia , Ácidos Nucleicos/química , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/química , RNA Catalítico/administração & dosagem , RNA Catalítico/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/químicaRESUMO
Safe, effective, and tissue-specific delivery is a central issue for the therapeutic application of nucleic-acid-based gene interfering agents, such as ribozymes and siRNAs. In this study, we constructed a functional RNase P-based ribozyme (M1GS RNA) that targets the overlapping mRNA region of M80.5 and protease, two murine cytomegalovirus (MCMV) proteins essential for viral replication. In addition, a novel attenuated strain of Salmonella, which exhibited efficient gene transfer activity and little cytotoxicity and pathogenicity in mice, was constructed and used for delivery of anti-MCMV ribozyme. In MCMV-infected macrophages treated with the constructed attenuated Salmonella strain carrying the functional M1GS RNA construct, we observed an 80-85% reduction in the expression of M80.5/protease and a 2,500-fold reduction in viral growth. Oral inoculation of the attenuated Salmonella strain in mice efficiently delivered antiviral M1GS RNA into spleens and livers, leading to substantial expression of the ribozyme without causing significant adverse effects in the animals. Furthermore, the MCMV-infected mice that were treated orally with Salmonella carrying the functional M1GS sequence displayed reduced viral gene expression, decreased viral titers, and improved survival compared to the untreated mice or mice treated with Salmonella containing control ribozyme sequences. Our results provide direct evidence that oral delivery of M1GS RNA by Salmonella-based vectors effectively inhibits viral gene expression and replication in mice. Moreover, this study demonstrates the utility of Salmonella-mediated oral delivery of RNase P ribozyme for gene-targeting applications in vivo.
Assuntos
Terapia Genética/métodos , RNA Catalítico/administração & dosagem , Ribonuclease P/antagonistas & inibidores , Salmonella/genética , Viroses/terapia , Administração Oral , Animais , Macrófagos/enzimologia , Macrófagos/virologia , Camundongos , RNA Catalítico/farmacologia , Carga Viral/efeitos dos fármacos , Viroses/prevenção & controle , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Inhibition of inappropriate tyrosine kinase activity by neoplasms is an attractive strategy for the treatment of malignancy. OBJECTIVE: We aimed to produce a concise review of the potential role of hepatocyte growth factor (HGF)/Mesenchymal-epithelial transition factor (MET) tyrosine kinase pathway inhibition in the treatment of cancer. METHODS: The current literature, abstracts and internet resources related to HGF/MET structure, function and inhibition are summarized. The potential of inhibiting this pathway as a therapy for cancer and remaining hurdles prior to routine clinical use of MET inhibition are discussed. RESULTS/CONCLUSIONS: Current knowledge suggests that the inhibition of the HGF/MET pathway has significant potential for the treatment of cancer. A number of MET inhibitor molecules are nearing completion of their development for clinical use.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Benzoquinonas/administração & dosagem , Benzoquinonas/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fator de Crescimento de Hepatócito/imunologia , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Lactamas Macrocíclicas/administração & dosagem , Lactamas Macrocíclicas/uso terapêutico , Proteínas de Neoplasias/fisiologia , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-met , RNA Catalítico/administração & dosagem , RNA Catalítico/uso terapêutico , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: To determine the safety and efficacy of VIT100 (Immusol, Inc, San Diego, California), a ribozyme to proliferating cell nuclear antigen, in preventing recurrent proliferative vitreoretinopathy (PVR) in patients with established PVR who undergo vitrectomy for retinal reattachment repair. METHODS: A multicenter, double-masked, placebo-controlled, randomized clinical trial. One hundred seventy-five eyes from 175 patients with grade C or worse PVR were randomly assigned to receive high-dose VIT100, low-dose VIT100, or placebo by intravitreal injection at the conclusion of retinal reattachment surgery. MAIN OUTCOME MEASURES: The primary efficacy end point was recurrent retinal detachment secondary to PVR. The secondary end point was recurrent retinal detachment due to any cause. RESULTS: One hundred fifty-four patients completed the study. Forty-one patients (27%) developed recurrent retinal detachment due to PVR by 24 weeks, including 18 patients (33%) in the group receiving 0.75 mg, 13 patients (24%) in the group receiving 0.15 mg, and 10 patients (22%) in the placebo group. There was no statistically significant difference in patients reaching this end point by 24 weeks (P = .37). Ancillary statistical analyses are reported. CONCLUSIONS: VIT100 was not effective in preventing PVR recurrence in patients with established grade C or worse PVR. APPLICATION TO CLINICAL PRACTICE: To our knowledge, this is the most recent, meticulously designed clinical trial in PVR.
Assuntos
Antígeno Nuclear de Célula em Proliferação/genética , RNA Catalítico/administração & dosagem , Vitreorretinopatia Proliferativa/prevenção & controle , Método Duplo-Cego , Feminino , Humanos , Injeções , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Catalítico/efeitos adversos , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/efeitos adversos , Descolamento Retiniano/prevenção & controle , Descolamento Retiniano/cirurgia , Recurvamento da Esclera , Prevenção Secundária , Resultado do Tratamento , Acuidade Visual , Vitrectomia , Corpo VítreoRESUMO
BACKGROUND: Encapsulating peritoneal sclerosis (EPS) is a rare and devastating fibrotic complication in patients treated with peritoneal dialysis. Transforming growth factor-beta1 (TGF-beta1) has been reported to be a pivotal factor in the induction of EPS. Ribozymes are RNA molecules that enzymatically cleave the target mRNAs and are expected to be utilized as a novel nucleic acid-based therapy. We examined the effects of the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta1 mRNA on a peritoneal sclerosis rat model to develop a possible gene therapy for EPS. METHODS: To create an animal model of peritoneal sclerosis, rats were given a daily intraperitoneal injection of chlorhexidine gluconate and ethanol dissolved in saline (CHX) for 14 days. On day 4, the chimeric ribozyme or mismatch ribozyme was intraperitoneally injected. On day 15, samples of peritoneum were obtained from the rats, and expression of TGF-beta1 mRNA and fibronectin mRNA in peritoneal tissues were evaluated by quantitative real-time PCR analysis. RESULTS: Injections of CHX significantly increased the submesothelial thickness, and increased the expression of TGF-beta1 and fibronectin mRNA in the rat peritoneum. Treatment with the chimeric ribozyme significantly reduced the CHX-induced peritoneal thickness, and expression of TGF-beta1, and fibronectin mRNA in peritoneal tissues. CONCLUSIONS: These results indicate that the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta1 mRNA has the potential for use as a gene therapy agent for EPS.
Assuntos
Terapia Genética , Peritônio/patologia , RNA Catalítico/administração & dosagem , Fator de Crescimento Transformador beta1/genética , Parede Abdominal , Animais , Modelos Animais de Doenças , Fibronectinas/biossíntese , Masculino , Diálise Peritoneal/efeitos adversos , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Esclerose/etiologia , Esclerose/patologia , Esclerose/terapia , Fator de Crescimento Transformador beta1/biossínteseRESUMO
Combinatorial therapies for the treatment of HIV infection have changed the course of the AIDS epidemic in developed nations where the antiviral drug combinations are readily available. Despite this progress, there are many problems associated with chemotherapy for AIDS including toxicities and emergence of viral mutants resistant to the drugs. Our goal has been the development of a hematopoietic gene therapy treatment for HIV infection. Like chemotherapy, gene therapy for treatment of HIV infection should be used combinatorially. We have thus combined three different inhibitory genes for treatment of HIV infection into a single lentiviral vector backbone. The inhibitory agents engage RNAi via a short hairpin RNA targeting HIV tat/rev mRNAs, a nucleolar localizing decoy that binds and sequesters the HIV Tat protein, and a ribozyme that cleaves and downregulates the CCR5 chemokine receptor used by HIV for cellular entry. This triple combination has proven to be highly effective for inhibiting HIV replication in primary hematopoietic cells, and is currently on track for human clinical application.
Assuntos
Terapia Genética/métodos , Infecções por HIV/terapia , Células-Tronco Hematopoéticas/metabolismo , Antagonistas dos Receptores CCR5 , Células Cultivadas , Quimioterapia Combinada , Produtos do Gene rev/antagonistas & inibidores , Produtos do Gene tat/antagonistas & inibidores , Vetores Genéticos , HIV/efeitos dos fármacos , Repetição Terminal Longa de HIV/genética , Humanos , RNA Catalítico/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Resultado do Tratamento , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
There are numerous examples in the literature of gene therapy applications for recessive disorders. There are precious few instances, however, of studies conducted to treat dominantly inherited pathologies. The reasons are simple: there are fewer cases of dominantly inherited diseases on one hand, but mostly it is far easier to correct recessive mutations than dominant ones. Typically recessive mutations cause a loss of (or reduced) gene function which can be compensated for by introduction of a replacement allele into the cell. In contrast, dominant negative mutations not only display impaired function, but also exhibit a novel one that is pathologic to the cell. Treating these conditions by gene therapy implies silencing the dominant allele without altering the expression of the wild-type gene. We describe here different strategies aimed at silencing dominant mutations through mRNA destruction and provide examples of their application to known autosomal dominant diseases. An overview of the most common molecular tools (antisense DNA and RNA, ribozymes and RNA interference) suitable to utilize these strategies is also presented and we discuss the relevant aspects involved in the choice of a particular approach in a gene therapy experiment.
Assuntos
Genes Dominantes , Doenças Genéticas Inatas/terapia , Terapia Genética/tendências , RNA/genética , Animais , Doenças Genéticas Inatas/genética , Humanos , Mutação , RNA/administração & dosagem , Interferência de RNA , RNA Antissenso/administração & dosagem , RNA Antissenso/genética , RNA Catalítico/administração & dosagem , RNA Catalítico/genéticaRESUMO
The past several years have witnessed the evolution of gene medicine from an experimental technology into a viable strategy for developing therapeutics for a wide range of human disorders. Numerous prototype DNA-based biopharmaceuticals can now control disease progression by induction and/or inhibition of genes. These potent therapeutics include plasmids containing transgenes, oligonucleotides, aptamers, ribozymes, DNAzymes, and small interfering RNAs. Although only 2 DNA-based pharmaceuticals (an antisense oligonucleotide formulation, Vitravene, (USA, 1998), and an adenoviral gene therapy treatment, Gendicine (China, 2003), have received approval from regulatory agencies; numerous candidates are in advanced stages of human clinical trials. Selection of drugs on the basis of DNA sequence and structure has a reduced potential for toxicity, should result in fewer side effects, and therefore should eventually yield safer drugs than those currently available. These predictions are based on the high selectivity and specificity of such molecules for recognition of their molecular targets. However, poor cellular uptake and rapid in vivo degradation of DNA-based therapeutics necessitate the use of delivery systems to facilitate cellular internalization and preserve their activity. This review discusses the basis of structural design, mode of action, and applications of DNA-based therapeutics. The mechanisms of cellular uptake and intracellular trafficking of DNA-based therapeutics are examined, and the constraints these transport processes impose on the choice of delivery systems are summarized. Finally, the development of some of the most promising currently available DNA delivery platforms is discussed, and the merits and drawbacks of each approach are evaluated.
Assuntos
DNA/uso terapêutico , Terapia Genética/métodos , Elementos Antissenso (Genética)/administração & dosagem , Elementos Antissenso (Genética)/farmacocinética , Elementos Antissenso (Genética)/uso terapêutico , Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/farmacocinética , Aptâmeros de Nucleotídeos/uso terapêutico , Transporte Biológico , DNA/administração & dosagem , DNA/genética , DNA/farmacocinética , DNA Catalítico/administração & dosagem , DNA Catalítico/farmacocinética , DNA Catalítico/uso terapêutico , DNA Recombinante/administração & dosagem , DNA Recombinante/genética , DNA Recombinante/farmacocinética , DNA Recombinante/uso terapêutico , Formas de Dosagem , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Genes Transgênicos Suicidas , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacocinética , Vetores Genéticos/uso terapêutico , Humanos , Lipossomos/administração & dosagem , Lipossomos/classificação , Plasmídeos/administração & dosagem , Plasmídeos/genética , Plasmídeos/uso terapêutico , RNA Catalítico/administração & dosagem , RNA Catalítico/farmacocinética , RNA Catalítico/uso terapêutico , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/uso terapêutico , TransgenesRESUMO
Ribozymes are catalytic RNA that bind and cleave specific regions of target RNA. Therefore, protein synthesis by the target RNA may be specifically inhibited by ribozymes. In this study, we have investigated if ribozymes possess therapeutic activity on inflammatory processes in vivo, as judged from effects on an arthritis model. A hammerhead ribozyme against TNF-alpha was designed and its catalytic activity in vitro was verified. The ribozyme was employed in vivo without any delivery system, as the plasmid-based ribozyme was taken up adequately by various tissues in mice by intravenous injection. The ability of the ribozyme to regulate the development of collagen-induced arthritis (CIA), a model largely dependent on TNF-alpha, was investigated. Systemic administration of the ribozyme to mice immunized with collagen type II in CFA significantly reduced the development of CIA. No effect was observed with a catalytically inactive variant of the ribozyme. Furthermore, the ribozyme efficiently blocked cartilage and bone destruction in the joints and ameliorated established CIA. These data demonstrate for the first time that gene targeting by a ribozyme to inactivate TNF-alpha in vivo is highly efficient in suppressing autoimmune arthritis, thus providing proof of concept that it may be used as therapeutic tool for TNF-alpha-dependent chronic inflammatory disorders.
Assuntos
Artrite Reumatoide/terapia , Marcação de Genes , Terapia Genética/métodos , RNA Catalítico/administração & dosagem , RNA Mensageiro , Fator de Necrose Tumoral alfa/genética , Animais , Anticorpos Anti-Idiotípicos/sangue , Artrite Reumatoide/imunologia , Feminino , Imunoglobulina G/imunologia , Injeções Intra-Arteriais , Masculino , Camundongos , Camundongos Endogâmicos DBA , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: Anti-proliferative drugs released from endo-vascular stents have substantially contributed to reduce in-stent restenosis rates in coronary arteries bearing single primary lesions by down-regulating coronary smooth muscle cell (CSMC) growth. However, the considerably lower drug efficacy shown in treatment of more complex coronary lesions suggests that alternative anti-proliferative approaches can be beneficial. Thus, we explored the use of hammerhead ribozymes as tools to knock down cyclin E and E2F1, two potent activators of cell proliferation which cooperate to promote the G1 to S phase transition. METHODS: Two ribozymes, one directed against cyclin E and the other against E2F1 mRNAs, were delivered by liposomes to cultured human CSMCs. The influences on cell proliferation were measured evaluating BrdU incorporation into newly synthesised DNA. The effects on cell cycle phase distribution were determined by BrdU and 7-aminoactinomycin D incorporation into DNA. RESULTS: Both ribozymes exhibited a sequence-specific and dose-dependent reduction in BrdU incorporation, which, at a concentration of 280 nM, persisted up to 4 days after transfection of CSMCs. A combined administration of the two ribozymes (210+210 nM) resulted in a more pronounced decrease in BrdU incorporation compared to the administration of an equimolar amount (420 nM) of each of them. Finally, both ribozymes induced a significant (P<0.05) reduction in S phase cells with a concomitant increase of G1/G0 and G2-M phase cells, compared to controls. CONCLUSIONS: The ribozymes selected represent potent tools to prevent CSMC proliferation, especially when administered together, and thus are ideal candidates for in vivo application.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Ciclina E/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Miócitos de Músculo Liso/efeitos dos fármacos , RNA Catalítico/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Estenose Coronária/tratamento farmacológico , Regulação para Baixo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Humanos , Lipossomos , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , RNA Catalítico/administração & dosagem , RNA Catalítico/genética , TransfecçãoRESUMO
Ribozymes are catalytically active nucleic acids capable of site-specific cleavage of target mRNAs. They have widely been employed as tools in functional studies and for therapeutic purposes. Different classes of ribozymes distinguished by size and mechanism of action have been discovered in natural systems or obtained by in vitro selection. After an introduction to different types of ribozymes with a special focus on the hammerhead and hairpin ribozyme, major challenges in the process of developing ribozymes for medical purposes will be described in the present review. Subsequently, examples of ribozyme applications in animal models for various diseases including cancer, viral infections, rheumatoid arthritis and cardiovascular diseases will be given. The course of phase I and II clinical trials with ribozymes designed to treat patients with virus infections or cancer will be outlined. Finally, the current significance of ribozymes will be discussed in the light of the emergence of new powerful anti-mRNA strategies, particularly RNA interference (RNAi).
Assuntos
DNA Catalítico/uso terapêutico , RNA Catalítico/uso terapêutico , Animais , Ensaios Clínicos como Assunto , DNA Catalítico/administração & dosagem , DNA Catalítico/farmacocinética , Estabilidade de Medicamentos , Vírus Delta da Hepatite/genética , Humanos , Íntrons , Modelos Animais , RNA Catalítico/administração & dosagem , RNA Catalítico/farmacocinética , Ribonuclease P/metabolismoRESUMO
Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (D,L-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5'-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.
Assuntos
DNA Catalítico/administração & dosagem , Inativação Gênica , Ácido Láctico/química , Oligonucleotídeos Antissenso/administração & dosagem , Ácido Poliglicólico/química , Polímeros/química , RNA Catalítico/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Autorradiografia , DNA Catalítico/química , Sistemas de Liberação de Medicamentos , Fluoruracila/administração & dosagem , Fluoruracila/farmacocinética , Técnicas In Vitro , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microesferas , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RNA Catalítico/química , RNA Interferente Pequeno/química , Baço/metabolismo , Fatores de TempoRESUMO
Ribozymes are RNA molecules with the capacity to effect sequence-specific cleavage of other transcripts. Since their initial discovery, there has been considerable interest in the development of ribozymes and other RNA therapeutics for gene therapy, particularly in the realm of cancer. However, as with other gene therapy applications, the delivery of ribozyme-based therapeutics to the target tissues of interest has represented a significant obstacle to the maturation of this technology to the clinical arena. This review will discuss the progress made so far in the use of non-viral methods for the systemic delivery of ribozymes for cancer gene therapy.
Assuntos
Terapia Genética/métodos , Neoplasias/terapia , RNA Catalítico/uso terapêutico , Animais , Ensaios Clínicos Fase II como Assunto , Portadores de Fármacos , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Humanos , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Metástase Neoplásica/tratamento farmacológico , Neoplasias/genética , Células Neoplásicas Circulantes , Plasmídeos/genética , Plasmídeos/uso terapêutico , RNA Catalítico/administração & dosagem , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cervical cancer is the second most common neoplastic disease affecting women worldwide. Basic, clinical and epidemiological analyses indicate that expression of high-risk human papillomaviruses (HPVs) E6/E7 genes is the primary cause of cervical cancer and represent ideal targets for the application of therapeutic nucleic acids (TNAs). Antisense oligodeoxyribonucleotides (AS-ODNs) and ribozymes (RZs) are the most effective TNAs able to inhibit in vivo tumour growth by eliminating HPV-16 and HPV-18 E6/E7 transcripts. Expression of multiple RZs directed against alternative target sites by triplex expression systems may result in the abrogation of highly variable HPVs. More recently, RNA interference (RNAi) gene knockdown phenomenon, induced by small interfering RNA (siRNA), has demonstrated its potential value as an effective TNA for cervical cancer. siRNA and aptamers as TNAs will have a place in the armament for cervical cancer. TNAs against cervical cancer is in a dynamic state, and clinical trials will define the TNAs in preventive and therapeutic roles to control tumour growth, debulk tumour mass, prevent metastasis and facilitate immune interaction.
Assuntos
Terapia Genética , Ácidos Nucleicos/uso terapêutico , Neoplasias do Colo do Útero/terapia , Feminino , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Marcação de Genes , Humanos , Conformação de Ácido Nucleico , Ácidos Nucleicos/administração & dosagem , Ácidos Nucleicos/genética , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Proteínas Oncogênicas Virais/antagonistas & inibidores , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/fisiologia , Papillomaviridae/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/terapia , Interferência de RNA , RNA Catalítico/administração & dosagem , RNA Catalítico/genética , RNA Catalítico/uso terapêutico , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologiaRESUMO
The present study was undertaken to examine whether ribozymes cleaving specifically cathepsin L (CL) mRNA are able to decrease the synthesis of CL protease in rheumatoid arthritis synovial fibroblasts (RA-SF) and thereby reduce the invasiveness into cartilage both in vitro and in the SCID mouse coimplantation model of RA. Two different ribozymes that cleave CL mRNA specifically at positions 533 (RzCL533) and 790 (RzCL790) were generated. Using retroviral gene transfer, RA-SF were transduced with the ribozyme constructs or the empty vector. To examine the effect of the ribozymes on the mRNA level, quantitative analysis for CL mRNA was performed using real-time PCR. For evaluation on the protein level, ELISA using specific anti-CL antibodies was performed. In addition, transduced RA-SF were examined in vitro in a three-dimensional destruction assay evaluating their ability to degrade extracellular matrix produced by human chondrocytes. Matrix destruction was monitored by the release of soluble glycosaminoglycans (sGAG). Using the in vivo SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF and control cells were coimplanted with human cartilage for 60 days. After being killed, invasion of RA-SF into the cartilage was evaluated by using a semiquantitative score. Transduction of RA-SF with RzCL533 and RzCL790 ribozymes decreased significantly the expression of CL mRNA to 44% (range 25-62%) and 20% (range 1-43%), respectively, when compared to mock-transduced cells. The protein concentration of CL in the cell culture supernatants of transduced RA-SF was decreased from 16.0 ng/ml in the mock constructs to 4.1 and 8.2 ng/ml (mean), respectively. Using the in vitro cartilage destruction assay, the release of sGAG decreased to 46 and 60%, respectively, after 14 days when compared to mock-transduced cells. In the SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF revealed a significant lower cartilage invasion when compared to mock and untransduced cells. Using retroviral gene transfer, ribozymes cleaving CL mRNA inhibit specifically the synthesis of this matrix-degrading enzyme and reduce cartilage destruction in in vitro and in vivo models. Our study therefore suggests that ribozymes targeting CL could be a novel and efficient tool to inhibit joint destruction in RA.
Assuntos
Artrite Reumatoide/terapia , Catepsinas/genética , Terapia Genética/métodos , RNA Catalítico/administração & dosagem , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Cartilagem Articular/patologia , Catepsina L , Catepsinas/biossíntese , Células Cultivadas , Cisteína Endopeptidases , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos SCID , Membrana Sinovial/metabolismo , Transdução Genética/métodosRESUMO
We designed and synthesized a chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor (TGF)-beta 1 mRNA and found that this ribozyme effectively and specifically inhibited growth of vascular smooth muscle cells. We examined the effects of the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA on neointima formation and investigated the underlying mechanism to develop a possible gene therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty. Expression of mRNAs encoding TGF-beta 1, p27kip1, and connective tissue growth factor (CTGF) in carotid artery increased after balloon injury. Fluorescein-isothiocyanate (FITC)-labeled ribozyme was taken up into the midlayer smooth muscle of the injured carotid artery. Both 2 and 5 mg of ribozyme reduced neointima formation by 65% compared to that of controls. Ribozyme markedly decreased expression of TGF-beta 1 mRNA and protein in injured vessel. Mismatch ribozyme had no effect on expression of TGF-beta 1 mRNA protein in injured vessel. Ribozyme markedly decreased expression of fibronectin, p27kip1, and CTGF mRNAs in injured vessel, whereas a mismatch ribozyme had no effect on these mRNAs. These findings indicate that the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury with suppression of TGF-beta 1 and inhibition of extracellular matrix and CTGF. In conclusion, the hammerhead ribozyme against TGF-beta 1 may have promise as a therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty.