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1.
Nat Med ; 25(8): 1266-1273, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285633

RESUMO

The ability to safely control transgene expression with simple synthetic gene switches is critical for effective gene- and cell-based therapies. In the present study, the signaling pathway controlled by human transient receptor potential (TRP) melastatin 8 (hTRPM8), a TRP channel family member1, is harnessed to control transgene expression. Human TRPM8 signaling is stimulated by menthol, an innocuous, natural, cooling compound, or by exposure to a cool environment (15-18 °C). By functionally linking hTRPM8-induced signaling to a synthetic promoter containing elements that bind nuclear factor of activated T cells, a synthetic gene circuit was designed that can be adjusted by exposure to either a cool environment or menthol. It was shown that this gene switch is functional in various cell types and human primary cells, as well as in mice implanted with engineered cells. In response to transdermal delivery of menthol, microencapsulated cell implants harboring this gene circuit, coupled to expression of either of two therapeutic proteins, insulin or a modified, activin type IIB, receptor ligand trap protein (mActRIIBECD-hFc), could alleviate hyperglycemia in alloxan-treated mice (a model of type 1 diabetes) or reverse muscle atrophy in dexamethasone-treated mice (a model of muscle wasting), respectively. This fully human-derived orthogonal transgene switch should be amenable to a wide range of clinical applications.


Assuntos
Receptores de Activinas Tipo II/sangue , Insulina/biossíntese , Canais de Cátion TRPM/fisiologia , Transgenes , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/uso terapêutico , Fosfatase Alcalina/genética , Animais , Temperatura Baixa , Diabetes Mellitus Tipo 1/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Insulina/genética , Insulina/uso terapêutico , Mentol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Distrofias Musculares/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos
2.
Nat Commun ; 9(1): 2318, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29921872

RESUMO

Programming cellular behavior using trigger-inducible gene switches is integral to synthetic biology. Although significant progress has been achieved in trigger-induced transgene expression, side-effect-free remote control of transgenes continues to challenge cell-based therapies. Here, utilizing a caffeine-binding single-domain antibody we establish a caffeine-inducible protein dimerization system, enabling synthetic transcription factors and cell-surface receptors that enable transgene expression in response to physiologically relevant concentrations of caffeine generated by routine intake of beverages such as tea and coffee. Coffee containing different caffeine concentrations dose-dependently and reversibly controlled transgene expression by designer cells with this caffeine-stimulated advanced regulators (C-STAR) system. Type-2 diabetic mice implanted with microencapsulated, C-STAR-equipped cells for caffeine-sensitive expression of glucagon-like peptide 1 showed substantially improved glucose homeostasis after coffee consumption compared to untreated mice. Biopharmaceutical production control by caffeine, which is non-toxic, inexpensive and only present in specific beverages, is expected to improve patient compliance by integrating therapy with lifestyle.


Assuntos
Cafeína/química , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica , Animais , Índice de Massa Corporal , Linhagem Celular , Sobrevivência Celular , Café , Feminino , Genes de Troca , Teste de Tolerância a Glucose , Células HEK293 , Humanos , Imunoglobulina G , Estilo de Vida , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Medicina de Precisão , Receptores para Leptina/genética , Fatores de Risco , Biologia Sintética , Fatores de Transcrição/metabolismo , Transgenes
3.
Nat Commun ; 9(1): 1305, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29610454

RESUMO

Exosomes are cell-derived nanovesicles (50-150 nm), which mediate intercellular communication, and are candidate therapeutic agents. However, inefficiency of exosomal message transfer, such as mRNA, and lack of methods to create designer exosomes have hampered their development into therapeutic interventions. Here, we report a set of EXOsomal transfer into cells (EXOtic) devices that enable efficient, customizable production of designer exosomes in engineered mammalian cells. These genetically encoded devices in exosome producer cells enhance exosome production, specific mRNA packaging, and delivery of the mRNA into the cytosol of target cells, enabling efficient cell-to-cell communication without the need to concentrate exosomes. Further, engineered producer cells implanted in living mice could consistently deliver cargo mRNA to the brain. Therapeutic catalase mRNA delivery by designer exosomes attenuated neurotoxicity and neuroinflammation in in vitro and in vivo models of Parkinson's disease, indicating the potential usefulness of the EXOtic devices for RNA delivery-based therapeutic applications.


Assuntos
Cérebro/patologia , Sistemas de Liberação de Medicamentos , Exossomos/metabolismo , Doença de Parkinson/terapia , Regiões 3' não Traduzidas , Animais , Encéfalo/metabolismo , Catalase/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Cérebro/metabolismo , Citosol/metabolismo , Eletroporação , Feminino , Terapia Genética , Células HEK293 , Células HeLa , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs , Nanopartículas , RNA Mensageiro/metabolismo , Biologia Sintética
4.
Nucleic Acids Res ; 42(14): e116, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25030908

RESUMO

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.


Assuntos
Proteínas de Bactérias/metabolismo , Aditivos Alimentares/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Animais , Proteínas de Bactérias/genética , Ácido Benzoico/farmacologia , Células Cultivadas , Feminino , Redes Reguladoras de Genes/efeitos dos fármacos , Inativação Gênica , Células HEK293 , Células HeLa , Proteína Vmw65 do Vírus do Herpes Simples/genética , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Humanos , Camundongos , Proteínas Repressoras/genética , Biologia Sintética/métodos , Ativação Transcricional , Transgenes , Ácido Vanílico/farmacologia
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