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1.
Mol Ther ; 27(7): 1242-1251, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31056400

RESUMO

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.


Assuntos
Citrulinemia/tratamento farmacológico , Citrulinemia/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Terapia Genética/métodos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , RNA Mensageiro/uso terapêutico , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Glucosefosfato Desidrogenase/genética , Células HeLa , Células Hep G2 , Humanos , Lipídeos/química , Mutação com Perda de Função , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Nanopartículas/química , Fases de Leitura Aberta/genética , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/genética , Transfecção , Resultado do Tratamento
2.
Blood Cells Mol Dis ; 76: 32-39, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30709626

RESUMO

The potential use of patient-specific induced pluripotent stem cells (hiPSCs) in the study and treatment of hematological diseases requires the setup of efficient and safe protocols for hiPSC generation. We aimed to adopt a reprogramming method for large-scale production of integration-free patient-specific hiPSC-lines in our stem cell processing laboratory, which supports a pediatric hematopoietic stem cell transplant unit located at a tertiary care children's hospital. We describe our 5-year experience in generation of hiPSC-lines from human bone marrow-derived mesenchymal stromal cells (BM-MSCs) using synthetic mRNAs encoding reprogramming factors. We generated hiPSC-lines from pediatric patients with ß-Thalassemia, Sickle Cell Anemia, Blackfan-Diamond Anemia, Severe Aplastic Anemia, DOCK8 Immunodeficiency and 1 healthy control. After optimization of the reprogramming procedure, average reprogramming efficiency of BM-MSCs was 0.29% (range 0.25-0.4). The complete reprogramming process lasted 14-16 days. Three to five hiPSC-colonies per sample were selected, expanded to 5 culture passages and then frozen. The whole procedure took an average time of 1.8 months (range 1.6-2.2). The hiPSC-lines expressed embryonic stem cell markers and exhibited pluripotency. This mRNA reprogramming method can be applicable in a hematopoietic stem cell culture lab setting and would be useful for the clinical translation of patient-specific hiPSCs.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Doenças Hematológicas/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , RNA Mensageiro/farmacologia , Criança , Transplante de Células-Tronco Hematopoéticas , Humanos , Métodos , Medicina de Precisão/métodos , RNA Mensageiro/síntese química , Fatores de Tempo
3.
J Immunol ; 202(2): 608-617, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30541883

RESUMO

Therapeutic strategies based on in vitro-transcribed mRNA (IVT) are attractive because they avoid the permanent signature of genomic integration that is associated with DNA-based therapy and result in the transient production of proteins of interest. To date, IVT has mainly been used in vaccination protocols to generate immune responses to foreign Ags. In this "proof-of-principle" study, we explore a strategy of combinatorial IVT to recruit and reprogram immune effector cells to acquire divergent biological functions in mice in vivo. First, we demonstrate that synthetic mRNA encoding CCL3 is able to recruit murine monocytes in a nonprogrammed state, exhibiting neither bactericidal nor tissue-repairing properties. However, upon addition of either Ifn-γ mRNA or Il-4 mRNA, we successfully polarized these cells to adopt either M1 or M2 macrophage activation phenotypes. This cellular reprogramming was demonstrated through increased expression of known surface markers and through the differential modulation of NADPH oxidase activity, or the superoxide burst. Our study demonstrates how IVT strategies can be combined to recruit and reprogram immune effector cells that have the capacity to fulfill complex biological tasks in vivo.


Assuntos
Reprogramação Celular , Macrófagos/imunologia , Monócitos/imunologia , RNA Mensageiro/imunologia , Animais , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL3/genética , Células HeLa , Humanos , Interferon gama/genética , Interleucina-4/genética , Linfócitos/imunologia , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Estudo de Prova de Conceito , RNA Mensageiro/síntese química , Transcrição Genética
4.
Stem Cell Res Ther ; 9(1): 277, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30359326

RESUMO

BACKGROUND: Transplantation of pancreatic ß cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been developed for this purpose, lentivirus-mediated forced expression of transcription factors (TF)-PDX1 and NKX6.1-has been at the forefront for its relatively fast and straightforward approach. However, considering that such cells will be used for therapeutic purposes in the future, it is desirable to develop a procedure that does not leave any footprint on the genome, as any changes of DNAs could potentially be a source of unintended, concerning effects such as tumorigenicity. In this study, we attempted to establish a novel protocol for rapid and footprint-free hESC differentiation into a pancreatic endocrine lineage by using synthetic mRNAs (synRNAs) encoding PDX1 and NKX6.1. We also tested whether siPOU5F1, which reduces the expression of pluripotency gene POU5F1 (also known as OCT4), can enhance differentiation as reported previously for mesoderm and endoderm lineages. METHODS: synRNA-PDX1 and synRNA-NKX6.1 were synthesized in vitro and were transfected five times to hESCs with a lipofection reagent in a modified differentiation culture condition. siPOU5F1 was included only in the first transfection. Subsequently, cells were seeded onto a low attachment plate and aggregated by an orbital shaker. At day 13, the degree of differentiation was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry for endocrine hormones such as insulin, glucagon, and somatostatin. RESULTS: Both PDX1 and NKX6.1 expression were detected in cells co-transfected with synRNA-PDX1 and synRNA-NKX6.1 at day 3. Expression levels of insulin in the transfected cells at day 13 were 450 times and 14 times higher by qRT-PCR compared to the levels at day 0 and in cells cultured without synRNA transfection, respectively. Immunohistochemically, pancreatic endocrine hormones were not detected in cells cultured without synRNA transfection but were highly expressed in cells transfected with synRNA-PDX1, synRNA-NKX6.1, and siPOU5F1 at as early as day 13. CONCLUSIONS: In this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , RNA Mensageiro/síntese química , Transativadores/genética , Diferenciação Celular , Engenharia Celular/métodos , Células Cultivadas , Regulação da Expressão Gênica , Glucagon/biossíntese , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células Secretoras de Insulina/citologia , Lipídeos/química , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Somatostatina/biossíntese , Transativadores/metabolismo , Transfecção
5.
ACS Chem Biol ; 13(12): 3243-3250, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29313662

RESUMO

The recent discovery of reversible chemical modifications on mRNA has opened a new era of post-transcriptional gene regulation in eukaryotes. Among the 15 types of modifications identified in mRNA of eukaryotes, N7-methylguanosine (m7G) is unique owing to its presence in the 5' cap structure. It remains unknown whether m7G is also present internally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal m7G in mRNA from that in the 5' cap. To address this analytical challenge, we developed a novel strategy of combining differential enzymatic digestion with liquid chromatography-tandem mass spectrometry analysis to quantify the levels of these two types of m7G modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5'-terminal m7G. By using this method, we found that internal m7G was present in mRNA of cultured human cells as well as plants and rat tissue. In addition, our results showed that plants contain higher levels of internal m7G in mRNA than mammals. We also observed that exposure of rice to cadmium (Cd) stimulated marked diminution in the levels of m7G at both the 5' cap and internal positions of mRNA, which was correlated with the Cd-induced elevated expression of m7G-decapping enzymes. Taken together, we reported here a strategy to distinguish internal and 5'-terminal m7G in mRNA, and by using this method, we demonstrated the prevalence of internal m7G modification in mRNA, which we believe will stimulate future functional studies of m7G on post-transcriptional gene regulation in eukaryotes.


Assuntos
Endorribonucleases/química , Guanina/análogos & derivados , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Animais , Cádmio/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Guanina/química , Humanos , Masculino , Espectrometria de Massas/métodos , Oryza/enzimologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/síntese química , RNA Mensageiro/genética , Ratos Sprague-Dawley
6.
Mol Pharm ; 15(2): 642-651, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29232147

RESUMO

Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.


Assuntos
Portadores de Fármacos/química , Composição de Medicamentos/métodos , RNA Mensageiro/administração & dosagem , Animais , Cátions/química , Linhagem Celular , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Bicamadas Lipídicas/química , Lipossomos , Luciferases/genética , Camundongos , Modelos Moleculares , Mioblastos , RNA Mensageiro/síntese química , Espalhamento a Baixo Ângulo , Transfecção/métodos , Difração de Raios X
7.
Mol Ther ; 25(9): 2028-2037, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28705346

RESUMO

Generation of functional dopamine (DA) neurons is an essential step for the development of effective cell therapy for Parkinson's disease (PD). The generation of DA neurons can be accomplished by overexpression of DA-inducible genes using virus- or DNA-based gene delivery methods. However, these gene delivery methods often cause chromosomal anomalies. In contrast, mRNA-based gene delivery avoids this problem and therefore is considered safe to use in the development of cell-based therapy. Thus, we used mRNA-based gene delivery method to generate safe DA neurons. In this study, we generated transformation-free DA neurons by transfection of mRNA encoding DA-inducible genes Nurr1 and FoxA2. The delivery of mRNA encoding dopaminergic fate inducing genes proved sufficient to induce naive rat forebrain precursor cells to differentiate into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neurons in vitro. Additionally, the generation efficiency of DA neurons was improved by the addition of small molecules, db-cAMP, and the adjustment of transfection timing. The successful generation of DA neurons using an mRNA-based method offers the possibility of developing clinical-grade cell sources for neuronal cell replacement treatment for PD.


Assuntos
Neurônios Dopaminérgicos/metabolismo , RNA Mensageiro/síntese química , RNA Mensageiro/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Expressão Gênica , Regulação da Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Ratos , Transfecção , Tirosina 3-Mono-Oxigenase/genética
8.
Angew Chem Int Ed Engl ; 55(44): 13808-13812, 2016 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-27690187

RESUMO

Therapeutic nucleic acids hold great promise for the treatment of disease but require vectors for safe and effective delivery. Synthetic nanoparticle vectors composed of poly(ß-amino esters) (PBAEs) and nucleic acids have previously demonstrated potential utility for local delivery applications. To expand this potential utility to include systemic delivery of mRNA, hybrid polymer-lipid nanoformulations for systemic delivery to the lungs were developed. Through coformulation of PBAEs with lipid-polyethylene glycol (PEG), mRNA formulations were developed with increased serum stability and increased in vitro potency. The formulations were capable of functional delivery of mRNA to the lungs after intravenous administration in mice. To our knowledge, this is the first report of the systemic administration of mRNA for delivery to the lungs using degradable polymer-lipid nanoparticles.


Assuntos
Lipídeos/química , Pulmão/química , Nanopartículas/química , Polímeros/química , RNA Mensageiro/química , Administração Intravenosa , Animais , Camundongos , Estrutura Molecular , Tamanho da Partícula , Polímeros/administração & dosagem , RNA Mensageiro/administração & dosagem , RNA Mensageiro/síntese química , Propriedades de Superfície
9.
Nat Methods ; 13(9): 792-8, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27502218

RESUMO

RNA sequencing (RNA-seq) can be used to assemble spliced isoforms, quantify expressed genes and provide a global profile of the transcriptome. However, the size and diversity of the transcriptome, the wide dynamic range in gene expression and inherent technical biases confound RNA-seq analysis. We have developed a set of spike-in RNA standards, termed 'sequins' (sequencing spike-ins), that represent full-length spliced mRNA isoforms. Sequins have an entirely artificial sequence with no homology to natural reference genomes, but they align to gene loci encoded on an artificial in silico chromosome. The combination of multiple sequins across a range of concentrations emulates alternative splicing and differential gene expression, and it provides scaling factors for normalization between samples. We demonstrate the use of sequins in RNA-seq experiments to measure sample-specific biases and determine the limits of reliable transcript assembly and quantification in accompanying human RNA samples. In addition, we have designed a complementary set of sequins that represent fusion genes arising from rearrangements of the in silico chromosome to aid in cancer diagnosis. RNA sequins provide a qualitative and quantitative reference with which to navigate the complexity of the human transcriptome.


Assuntos
Perfilação da Expressão Gênica/normas , Genes Sintéticos , Processamento de RNA , RNA Mensageiro/genética , Análise de Sequência de RNA/normas , Cromossomos Artificiais , Humanos , Controle de Qualidade , Processamento de RNA/genética , RNA Mensageiro/síntese química , RNA Mensageiro/química , Padrões de Referência , Análise de Sequência de RNA/métodos
10.
Sci Rep ; 6: 21991, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26902536

RESUMO

The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations.


Assuntos
Separação Celular/métodos , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , MicroRNAs/genética , RNA Mensageiro/genética , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Células MCF-7 , MicroRNAs/síntese química , MicroRNAs/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , RNA Mensageiro/síntese química , RNA Mensageiro/metabolismo , Transfecção
11.
Methods Mol Biol ; 1330: 17-28, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26621585

RESUMO

Reprogramming of somatic cells, such as skin fibroblasts, to pluripotency was first achieved by forced expression of four transcription factors using integrating retroviral or lentiviral vectors, which result in integration of exogenous DNA into cellular genome and present a formidable barrier to therapeutic application of induced pluripotent stem cells (iPSCs). To facilitate the translation of iPSC technology to clinical practice, mRNA reprogramming method that generates transgene-free iPSCs is a safe and efficient method, eliminating bio-containment concerns associated with viral vectors, as well as the need for weeks of screening of cells to confirm that viral material has been completely eliminated during cell passaging.


Assuntos
Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , RNA Mensageiro/genética , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Células Alimentadoras , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Mensageiro/síntese química , Transfecção , Transgenes
12.
Bioorg Med Chem ; 23(17): 5369-81, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26264844

RESUMO

The synthesis and biochemical properties of 17 new mRNA cap analogues are reported. Six of these nucleotides are m(7)GTP derivatives, whereas 11 are 'two headed' tetraphosphate dinucleotides based on a m(7)Gppppm(7)G structure. The compounds contain either a boranophosphate or phosphorothioate moiety in the nucleoside neighbouring position(s) and some of them possess an additional methylene group between ß and γ phosphorus atoms. The compounds were prepared by divalent metal chloride-mediated coupling of an appropriate m(7)GMP analogue with a given P(1),P(2)-di(1-imidazolyl) derivative. The analogues were evaluated as tools for studying cap-dependent processes in a number of biochemical assays, including determination of affinity to eukaryotic initiation factor eIF4E, susceptibility to enzymatic hydrolysis, and translational efficiency in vitro. The results indicate that modification in the phosphate chain can increase binding to cap-interacting proteins and provides higher resistance to degradation. Furthermore, modified derivatives of m(7)GTP were found to be potent inhibitors of cap-dependent translation in cell free systems.


Assuntos
Boranos/química , Fosfatos/química , Oligonucleotídeos Fosforotioatos/química , Análogos de Capuz de RNA/química , Boranos/síntese química , Boranos/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Hidrólise , Fosfatos/síntese química , Fosfatos/metabolismo , Oligonucleotídeos Fosforotioatos/síntese química , Oligonucleotídeos Fosforotioatos/metabolismo , Biossíntese de Proteínas , Análogos de Capuz de RNA/síntese química , Análogos de Capuz de RNA/metabolismo , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/metabolismo
13.
J Org Chem ; 80(8): 3982-97, 2015 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-25816092

RESUMO

To broaden the scope of existing methods based on (19)F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)nucleotide analogues containing an O to F substitution at the terminal position of the (oligo)phosphate moiety and evaluated them as tools for (19)F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-n-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in nucleobase type (A, G, C, U, m(7)G), phosphate chain length (from mono to tetra), and presence of additional phosphate modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5'-phosphorylated oligos we also synthesized oligonucleotide 5'-(2-fluorodiphosphates), which are potentially useful as (19)F NMR hybridization probes. The compounds were characterized by (19)F NMR and evaluated as (19)F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E).


Assuntos
Endorribonucleases/química , Fator de Iniciação 4E em Eucariotos/química , Fluoretos/síntese química , Radioisótopos de Flúor/química , Nucleotídeos/síntese química , Oligonucleotídeos/síntese química , Fosfatos/síntese química , Compostos de Amônio Quaternário/química , RNA Mensageiro/química , Marcadores de Spin/síntese química , Endorribonucleases/farmacologia , Fluoretos/química , Humanos , Espectroscopia de Ressonância Magnética , Atrofia Muscular Espinal/enzimologia , Atrofia Muscular Espinal/metabolismo , Nucleotídeos/química , Oligonucleotídeos/química , Fosfatos/química , Ligação Proteica , RNA Mensageiro/síntese química
14.
J Vis Exp ; (93): e51943, 2014 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-25489992

RESUMO

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.


Assuntos
RNA Mensageiro/administração & dosagem , RNA Mensageiro/síntese química , Citometria de Fluxo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Plasmídeos/administração & dosagem , Plasmídeos/síntese química , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção
15.
Cold Spring Harb Perspect Med ; 5(1): a014035, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25301935

RESUMO

Over the past two decades, a host of new molecular pathways have been uncovered that guide mammalian heart development and disease. The ability to genetically manipulate these pathways in vivo have largely been dependent on the generation of genetically engineered mouse model systems or the transfer of exogenous genes in a variety of DNA vectors (plasmid, adenoviral, adeno-associated viruses, antisense oligonucleotides, etc.). Recently, a new approach to manipulate the gene program of the adult mammalian heart has been reported that will quickly allow the high-efficiency expression of virtually any protein in the intact heart of mouse, rat, porcine, nonhuman primate, and human heart cells via the generation of chemically modified mRNA (modRNA). The technology platform has important implications for delineating the specific paracrine cues that drive human cardiogenesis, and the pathways that might trigger heart regeneration via the rapid generation of modRNA libraries of paracrine factors for direct in vivo administration. In addition, the strategy can be extended to a variety of other cardiovascular tissues and solid organs across multiple species, and recent improvements in the core technology have supported moving toward the first human studies of modRNA in the next 2 years. These recent advances are reviewed along with projections of the potential impact of the technology for a host of other biomedical problems in the cardiovascular system.


Assuntos
Cardiopatias/terapia , Coração/fisiologia , RNA Mensageiro/síntese química , Animais , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Camundongos , Músculo Esquelético/fisiologia , Miocárdio/citologia , Miócitos Cardíacos/fisiologia , Comunicação Parácrina/fisiologia , Proteínas/metabolismo , Ratos , Regeneração/fisiologia , Células-Tronco/fisiologia , Suínos , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
PLoS One ; 9(4): e94231, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24718618

RESUMO

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.


Assuntos
Técnicas de Cultura de Células/métodos , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , RNA Mensageiro/farmacologia , Adulto , Animais , Técnicas de Cultura de Células/normas , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Corpos Embrioides , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Perfilação da Expressão Gênica , Camadas Germinativas/citologia , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/transplante , Recém-Nascido , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Fator 3 de Transcrição de Octâmero/genética , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/síntese química , RNA Mensageiro/isolamento & purificação , Proteínas de Ligação a RNA/genética , Fatores de Transcrição SOXB1/genética , Pele/citologia , Teratoma/etiologia , Teratoma/patologia , Transfecção
17.
Bioorg Khim ; 39(2): 247-52, 2013.
Artigo em Russo | MEDLINE | ID: mdl-23964527

RESUMO

We have developed methods for producing recombinant proteins of Noggin family (Noggin1 and Noggin2 of the Xenopus laevis frog) that can interact with BMP factors of TGF-beta superfamily. The genetic constructs which allow one to effectively obtain Noggin1 and Noggin2 from synthetic mRNA microinjected into Xenopus laevis early embryos, as well as in the prokaryotic expression system, were generated. The obtained proteins contain three Myc-tag epitopes on their N-terminus. This allow one to compare the expression levels of Noggin1 and Noggin 2 constructs, to purify them on the affine immunosorbent and to show the activity of Noggin proteins by analyzing their ability to bind BMP4 factor TGF-beta surperfamily by co-immunoprecipitation.


Assuntos
Proteínas de Transporte/genética , Desenvolvimento Embrionário/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas de Xenopus/genética , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Ligação Proteica , RNA Mensageiro/síntese química , RNA Mensageiro/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis/genética , Xenopus laevis/metabolismo
18.
Methods Mol Biol ; 969: 55-72, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296927

RESUMO

The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modified cap structures at the 5'-end. mRNAs are synthesized in vitro by a phage RNA polymerase transcribing a plasmid containing the mRNA sequence in the presence of all four NTPs plus a cap dinucleotide. Modifications in the cap dinucleotide at the 2'- or 3'-positions of m(7)Guo, or modifications in the polyphosphate chain, can improve both translational efficiency and stability of the mRNA, thereby increasing the amount and duration of protein expression. In the context of RNA-based immunotherapy, the latter is especially important for antigen production and presentation by dendritic cells. Protocols are presented for synthesis of modified mRNAs, their introduction into cells and whole animals, and measurement of their translational efficiency and stability.


Assuntos
Biossíntese de Proteínas , Análogos de Capuz de RNA/química , Estabilidade de RNA , RNA Mensageiro , Transfecção/métodos , Animais , Humanos , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/genética
19.
Methods Mol Biol ; 969: 73-88, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296928

RESUMO

Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery where transient gene expression is desirable. However, cationic lipid-mediated delivery of DNA to post-mitotic cells is often of low efficiency, due to the difficulty of DNA translocation to the nucleus. Rapid lipid-mediated delivery of RNA is preferable to nonviral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary. Here we describe techniques for cationic lipid-mediated delivery of RNA encoding reporter genes in a variety of in vitro cell lines and in vivo. We describe optimized formulations and transfection procedures that we have previously assessed by flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in nondividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. These results are consistent with our in vivo delivery results, techniques for which are shown as well. Longer duration and the higher mean levels of expression per cell that are ultimately obtained following DNA delivery confirm a continuing role for DNA gene delivery in clinical applications that require long term transient gene expression. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression, and greater efficiency, particularly in nondividing cells.


Assuntos
Expressão Gênica , Lipídeos/química , RNA Mensageiro , Transfecção/métodos , DNA/química , DNA/genética , Biossíntese de Proteínas , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/genética , Fatores de Tempo
20.
Methods Mol Biol ; 969: 221-33, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296937

RESUMO

Changing cell fate without altering its genome is very desirable in many experimental systems and for cell therapy. Compared to DNA plasmid or viral-based approach, mRNA has the advantage of high transfection efficiency, no danger of changing the genome or creating mutational insertions. Here, we describe a straightforward protocol to synthesize mRNA of genes of interest, and use them to induce pluripotency and direct cell differentiation.


Assuntos
Diferenciação Celular/genética , Engenharia Celular/métodos , Células-Tronco Pluripotentes Induzidas , RNA Mensageiro , Transfecção/métodos , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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