RESUMEN
Electrogenetics, the combination of electronics and genetics, is an emerging field of mammalian synthetic biology in which electrostimulation is used to remotely program user-designed genetic elements within designer cells to generate desired outputs. Here, we describe recent advances in electro-induced therapeutic gene expression and therapeutic protein secretion in engineered mammalian cells. We also review available tools and strategies to engineer electro-sensitive therapeutic designer cells that are able to sense electrical pulses and produce appropriate clinically relevant outputs in response. We highlight current limitations facing mammalian electrogenetics and suggest potential future directions for research.
Asunto(s)
Ingeniería Celular , Células , Estimulación Eléctrica , Genética , Mamíferos , Biología Sintética , Animales , Ingeniería Celular/métodos , Fenómenos Fisiológicos Celulares/genética , Células/metabolismo , Estimulación Eléctrica/métodos , Terapia por Estimulación Eléctrica , Electrónica , Regulación de la Expresión Génica , Mamíferos/genética , Biosíntesis de Proteínas , Biología Sintética/métodos , TelemetríaRESUMEN
Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Evolución Molecular Dirigida/métodos , Escherichia coli/genética , Fenilalanina/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia/genética , Aminoácidos/metabolismo , Aminoácidos/farmacología , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Emparejamiento Base , Materiales Biomiméticos/metabolismo , Materiales Biomiméticos/farmacología , Ingeniería Celular , Escherichia coli/metabolismo , Expresión Génica , Genes Reporteros , Vida Libre de Gérmenes , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Conformación de Ácido Nucleico , Fenilalanina/farmacología , Plásmidos/química , Plásmidos/metabolismo , ARN de Transferencia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Engineered living materials represent a new generation of human-made biotherapeutics that are highly attractive for a myriad of medical applications. In essence, such cell-rich platforms provide encodable bioactivities with extended lifetimes and environmental multi-adaptability currently unattainable in conventional biomaterial platforms. Emerging cell bioengineering tools are herein discussed from the perspective of materializing living cells as cooperative building blocks that drive the assembly of multiscale living materials. Owing to their living character, pristine cellular units can also be imparted with additional therapeutically-relevant biofunctionalities. On this focus, the most recent advances on the engineering of mammalian living materials and their biomedical applications are herein outlined, alongside with a critical perspective on major roadblocks hindering their realistic clinical translation. All in all, transposing the concept of leveraging living materials as autologous tissue-building entities and/or self-regulated biotherapeutics opens new realms for improving precision and personalized medicine strategies in the foreseeable future.
Asunto(s)
Ingeniería Celular/métodos , Animales , Terapia Biológica , Humanos , Mamíferos , Medicina RegenerativaRESUMEN
Intratumoral hypoxia significantly constrains the susceptibility of solid tumors to oxygen-dependent photodynamic therapy (PDT), and effort to reverse such hypoxia has achieved limited success to date. Herein, we developed a novel engineered bacterial system capable of targeting hypoxic tumor tissues and efficiently mediating the photodynamic treatment of these tumors. For this system, we genetically engineered Escherichia coli to express catalase, after which we explored an electrostatic adsorption approach to link black phosphorus quantum dots (BPQDs) to the surface of these bacteria, thereby generating an engineered E. coli/BPQDs (EB) system. Following intravenous injection, EB was able to target hypoxic tumor tissues. Subsequent 660 nm laser irradiation drove EB to generate reactive oxygen species (ROS) and destroy the membranes of these bacteria, leading to the release of catalase that subsequently degrades hydrogen peroxide to yield oxygen. Increased oxygen levels alleviate intratumoral hypoxia, thereby enhancing BPQD-mediated photodynamic therapy. This system was able to efficiently kill tumor cells in vivo, exhibiting good therapeutic efficacy. In summary, this study is the first to report the utilization of engineered bacteria to facilitate PDT, and our results highlight new avenues for BPQD-mediated cancer treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Hipoxia/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Fósforo/uso terapéutico , Fármacos Fotosensibilizantes/uso terapéutico , Puntos Cuánticos/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/efectos de la radiación , Catalasa/genética , Catalasa/metabolismo , Ingeniería Celular , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Hipoxia/etiología , Ratones Endogámicos BALB C , Neoplasias/complicaciones , Oxígeno/metabolismo , Fósforo/química , Fósforo/efectos de la radiación , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/efectos de la radiación , Puntos Cuánticos/química , Puntos Cuánticos/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismoAsunto(s)
Anticuerpos , Linfocitos B , Terapia Biológica , Ingeniería Celular , Ingeniería de Proteínas , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , COVID-19/terapia , Control de Enfermedades Transmisibles , Enfermedades Transmisibles/inmunología , Humanos , Inmunización Pasiva , Sueroterapia para COVID-19RESUMEN
Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.
Asunto(s)
Antineoplásicos/uso terapéutico , Genes Transgénicos Suicidas , Terapia Genética/métodos , Neoplasias/terapia , Profármacos/administración & dosificación , Animales , Antineoplásicos/farmacología , Ingeniería Celular/métodos , Línea Celular Tumoral , Medios de Cultivo Condicionados , Citosina Desaminasa/genética , Exosomas/genética , Flucitosina/administración & dosificación , Flucitosina/metabolismo , Fluorouracilo/metabolismo , Proteínas Fúngicas/genética , Vectores Genéticos/genética , Humanos , Ratones , Pentosiltransferasa/genética , Profármacos/metabolismo , Proteínas Recombinantes de Fusión/genética , Retroviridae/genética , Transducción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Adipose-derived stem cells are considered as candidate cells for regenerative plastic surgery. Measures to influence cellular properties and thereby direct their regenerative potential remain elusive. Hyperbaric oxygen therapy-the exposure to 100% oxygen at an increased atmospheric pressure-has been propagated as a noninvasive treatment for a multitude of indications and presents a potential option to condition cells for tissue-engineering purposes. The present study evaluates the effect of hyperbaric oxygen therapy on human adipose-derived stem cells. METHODS: Human adipose-derived stem cells from healthy donors were treated with hyperbaric oxygen therapy at 2 and 3 atm. Viability before and after each hyperbaric oxygen therapy, proliferation, expression of surface markers and protein contents of transforming growth factor (TGF)-ß, tumor necrosis factor-α, hepatocyte growth factor, and epithelial growth factor in the supernatants of treated adipose-derived stem cells were measured. Lastly, adipogenic, osteogenic, and chondrogenic differentiation with and without use of differentiation-inducing media (i.e., autodifferentiation) was examined. RESULTS: Hyperbaric oxygen therapy with 3 atm increased viability, proliferation, and CD34 expression and reduced the CD31/CD34/CD45 adipose-derived stem cell subset and endothelial progenitor cell population. TGF-ß levels were significantly decreased after two hyperbaric oxygen therapy sessions in the 2-atm group and decreased after three hyperbaric oxygen therapy sessions in the 3-atm group. Hepatocyte growth factor secretion remained unaltered in all groups. Although the osteogenic and chondrogenic differentiation were not influenced, adipogenic differentiation and autodifferentiation were significantly enhanced, with osteogenic autodifferentiation significantly alleviated by hyperbaric oxygen therapy with 3 atm. CONCLUSION: Hyperbaric oxygen therapy with 3 atm increases viability and proliferation of adipose-derived stem cells, alters marker expression and subpopulations, decreases TGF-ß secretion, and skews adipose-derived stem cells toward adipogenic differentiation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.
Asunto(s)
Adipogénesis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Ingeniería Celular/métodos , Células Madre Mesenquimatosas/efectos de los fármacos , Oxígeno/administración & dosificación , Tejido Adiposo/citología , Adulto , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Células Madre Mesenquimatosas/fisiología , Persona de Mediana Edad , Presión , Cultivo Primario de Células/métodosRESUMEN
Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34+ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca2+ endpoint) and -independent (ß-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca2+ and ß-arrestin endpoints and a specificity of 93% (ß-arrestin endpoint) and 83% (Ca2+ endpoint) compared to histamine release in CD34+ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.
Asunto(s)
Degranulación de la Célula/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Mastocitos/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Péptidos/efectos adversos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Antígenos CD34/metabolismo , Degranulación de la Célula/inmunología , Ingeniería Celular , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad/métodos , Evaluación Preclínica de Medicamentos/métodos , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/metabolismo , Histamina/análisis , Histamina/metabolismo , Humanos , Mastocitos/inmunología , Mastocitos/metabolismo , Cultivo Primario de Células , Sensibilidad y EspecificidadRESUMEN
Plants are a source of food for many animals, and they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ERß) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Receptor beta de Estrógeno/genética , Genes Reporteros/genética , Fitoestrógenos/farmacología , Animales , Ingeniería Celular , Humanos , Luciferasas/genética , Extractos Vegetales/farmacología , TransfecciónRESUMEN
Efficient cancer vaccines not only require the co-delivery of potent antigens and highly immunostimulatory adjuvants to initiate robust tumor-specific host immune response but also solve the spatiotemporal consistency of host immunity and tumor microenvironment (TME) immunomodulation. Here, we designed a biomaterials-based strategy for converting tumor-derived antigenic microparticles (T-MPs) into a cancer vaccine to meet this conundrum and demonstrated its therapeutic potential in multiple murine tumor models. The internal cavity of T-MPs was employed to store nano-Fe3O4 (Fe3O4/T-MPs), and then dense adjuvant CpG-loaded liposome arrays (CpG/Lipo) were tethered on the surface of Fe3O4/T-MP through mild surface engineering to get a vaccine (Fe3O4/T-MPs-CpG/Lipo), demonstrating that co-delivery of Fe3O4/T-MPs and CpG/Lipo to antigen presenting cells (APCs) could elicit strong tumor antigen-specific host immune response. Meanwhile, vaccines distributed in the TME could reverse infiltrated tumor-associated macrophages into a tumor-suppressive M1 phenotype by nano-Fe3O4, amazingly induce abundant infiltration of cytotoxic T lymphocytes, and transform a "cold" tumor into a "hot" tumor. Furthermore, amplified antitumor immunity was realized by the combination of an Fe3O4/T-MPs-CpG/Lipo vaccine and immune checkpoint PD-L1 blockade, specifically inhibiting â¼83% of the progression of B16F10-bearing mice and extending the median survival time to 3 months. Overall, this study synergistically modulates the tumor immunosuppressive network and host antitumor immunity in a spatiotemporal manner, which suggests a general cell-engineering strategy tailored to a personalized vaccine from autologous cancer cell materials of each individual patient.
Asunto(s)
Antígenos de Neoplasias , Vacunas contra el Cáncer , Inmunomodulación/inmunología , Inmunoterapia/métodos , Microambiente Tumoral/inmunología , Animales , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Ingeniería Celular , Sistemas de Liberación de Medicamentos , Femenino , Óxido Ferrosoférrico/química , Ratones , Ratones Endogámicos C57BLRESUMEN
Engineering approaches were adopted for liver microsystems to recapitulate cell arrangements and culture microenvironments in vivo for sensitive, high-throughput and biomimetic drug screening. This review introduces liver microsystems in vitro for drug hepatotoxicity, drug-drug interactions, metabolic function and enzyme induction, based on cell micropatterning, hydrogel biofabrication and microfluidic perfusion. The engineered microsystems provide varied microenvironments for cell culture that feature cell coculture with non-parenchymal cells, in a heterogeneous extracellular matrix and under controllable perfusion. The engineering methods described include cell micropatterning with soft lithography and dielectrophoresis, hydrogel biofabrication with photolithography, micromolding and 3D bioprinting, and microfluidic perfusion with endothelial-like structures and gradient generators. We discuss the major challenges and trends of liver microsystems to study drug response in vitro.
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Ingeniería Celular/métodos , Evaluación Preclínica de Medicamentos/métodos , Microtecnología/instrumentación , Preparaciones Farmacéuticas/metabolismo , Evaluación Preclínica de Medicamentos/instrumentación , Humanos , HígadoRESUMEN
Although photothermal therapy (PTT) is preclinically applied in solid tumor treatment, incomplete tumor removal of PTT and heat endurance of tumor cells induces significant tumor relapse after treatment, therefore lowering the therapeutic efficiency of PTT. Herein, a programmable therapeutic strategy that integrates photothermal therapeutic agents (PTAs), DNAzymes, and artificial engineered natural killer (A-NK) cells for immunotherapy of hepatocellular carcinoma (HCC) is designed. The novel PTAs, termed as Mn-CONASHs, with 2D structure are synthesized by the coordination of tetrahydroxyanthraquinone and Mn2+ ions. By further adsorbing polyetherimide/DNAzymes on the surface, the DNAzymes@Mn-CONASHs exhibit excellent light-to-heat conversion ability, tumor microenvironment enhanced T1 -MRI guiding ability, and antiheat endurance ability. Furthermore, the artificial engineered NK cells with HCC specific targeting TLS11a-aptamer decoration are constructed for specifically eliminating any possible residual tumor cells after PTT, to systematically enhance the therapeutic efficacy of PTT and avoid tumor relapse. Taken together, the potential of A-NK cells combined with antiheat endurance as a powerful strategy for immuno-enhancing photothermal therapy efficiency of solid tumors is highlighted, and the current strategy might provide promising prospects for cancer therapy.
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Células Artificiales , Carcinoma Hepatocelular/terapia , Ingeniería Celular , Hipertermia Inducida , Inmunoterapia , Células Asesinas Naturales/inmunología , Neoplasias Hepáticas/terapia , Fototerapia , Carcinoma Hepatocelular/diagnóstico por imagen , ADN Catalítico/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Neoplasias Hepáticas/diagnóstico por imagen , Imagen por Resonancia Magnética , Manganeso/química , Nanopartículas/ultraestructura , Espectrometría de Fluorescencia , Microambiente TumoralRESUMEN
Fibrosis is defined by excessive production of type I collagen in various organs. Excessive type I collagen production in fibrosis is stimulated by binding of RNA protein LARP6 to the structural element of collagen mRNAs, the 5' stem loop (5'SL). The LARP6-dependent regulation is specific for type I collagen and critical for fibrosis development. Inhibitors of LARP6 binding have potential to be specific antifibrotic drugs, as evidenced by the discovery of one such inhibitor. To create technology for phenotypic screening of additional compounds we developed an inverted yeast three hybrid system. The system is based on expression of human LARP6 and a short RNA containing the 5'SL of human collagen α1(I) mRNA in Saccharomyces cerevisiae cells. The cells were engineered in such a way that when LARP6 is bound to 5'SL RNA they fail to grow in a specific synthetic medium. Dissociation of LARP6 from 5'SL RNA permits the cell growth, allowing identification of the inhibitors of LARP6 binding. The assay simply involves measuring optical density of cells growing in multiwall plates and is pertinent for high throughput applications. We describe the specificity of the system and its characteristics for high throughput screening. As a proof of principle, the result of one screen using collection of FDA approved drugs is also presented. This screen demonstrates that using this technology discovery of novel LARP6 inhibitors is possible.
Asunto(s)
Descubrimiento de Drogas , Ribonucleoproteínas/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Técnicas del Sistema de Dos Híbridos , Autoantígenos/biosíntesis , Ingeniería Celular , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Fenotipo , Ribonucleoproteínas/biosíntesis , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Antígeno SS-BRESUMEN
IL-6 has an important role in the pathogenesis of autoimmunity and chronic inflammation. Several mAbs that target IL-6 or the IL-6 receptor (IL-6R) have been established and approved for the treatment of various diseases such as multicentric Castleman's disease and rheumatoid arthritis. Quality control of therapeutic antibodies requires accurate determination of bioactivity. However, current cell-based anti-proliferation assays are tedious, time consuming, and result in high variation. We therefore developed a reporter gene assay (RGA) based on an IL-6-dependent DS-1 cell line that stably expressed the reporter luciferase controlled by the serum-induced element (SIE) response element, which was a key element located downstream of the IL-6 signaling pathway. The RGA method demonstrated good performance characteristics after careful optimization, including high specificity, stability, accuracy, precision, and robustness. It also had superior precision and sensitivity. The assay is simple compared with the traditional anti-proliferation assay. This novel RGA based on the IL-6-IL-6R-STAT3 pathway can be useful, in conjunction with the anti-proliferation bioassay, to determine the bioactivity of anti-IL-6/anti-IL-6R therapeutic mAbs. Graphical abstract The mechanism sketch of the reporter gene assay for the bioactivity determination of anti-IL-6/anti-IL-6Rα mAbs.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Evaluación Preclínica de Medicamentos , Interleucina-6/antagonistas & inhibidores , Receptores de Interleucina-6/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados/inmunología , Ingeniería Celular , Línea Celular , Proliferación Celular , Evaluación Preclínica de Medicamentos/métodos , Genes Reporteros , Humanos , Interleucina-6/inmunología , Luciferasas/genética , Luciferasas/inmunología , Receptores de Interleucina-6/inmunología , Proteínas Recombinantes/inmunologíaRESUMEN
Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.
Asunto(s)
Alopecia/terapia , Diferenciación Celular , Ingeniería Celular/métodos , Cabello/citología , Cabello/crecimiento & desarrollo , Células Madre Pluripotentes Inducidas/citología , Animales , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Línea Celular , Doxiciclina/farmacología , Evaluación Preclínica de Medicamentos , Células Madre Pluripotentes Inducidas/metabolismo , Queratinas Específicas del Pelo/genética , Queratinas Específicas del Pelo/metabolismo , Queratinas Tipo I/genética , Queratinas Tipo I/metabolismo , Luciferasas/metabolismo , Sustancias Luminiscentes/metabolismo , Ratones , Regiones Promotoras Genéticas , Tetraciclina/farmacología , Tretinoina/farmacologíaRESUMEN
Plant natural products (PNPs) are widely used as pharmaceuticals, nutraceuticals, seasonings, pigments, etc., with a huge commercial value on the global market. However, most of these PNPs are still being extracted from plants. A resource-conserving and environment-friendly synthesis route for PNPs that utilizes microbial cell factories has attracted increasing attention since the 1940s. However, at the present only a handful of PNPs are being produced by microbial cell factories at an industrial scale, and there are still many challenges in their large-scale application. One of the challenges is that most biosynthetic pathways of PNPs are still unknown, which largely limits the number of candidate PNPs for heterologous microbial production. Another challenge is that the metabolic fluxes toward the target products in microbial hosts are often hindered by poor precursor supply, low catalytic activity of enzymes and obstructed product transport. Consequently, despite intensive studies on the metabolic engineering of microbial hosts, the fermentation costs of most heterologously produced PNPs are still too high for industrial-scale production. In this paper, we review several aspects of PNP production in microbial cell factories, including important design principles and recent progress in pathway mining and metabolic engineering. In addition, implemented cases of industrial-scale production of PNPs in microbial cell factories are also highlighted.
Asunto(s)
Bacterias/metabolismo , Productos Biológicos , Ingeniería Celular , Hongos/metabolismo , Microbiología Industrial , Preparaciones de Plantas/aislamiento & purificación , Bacterias/genética , Vías Biosintéticas , Suplementos Dietéticos , Fermentación , Hongos/genética , Ingeniería Metabólica , Preparaciones de Plantas/química , Preparaciones de Plantas/metabolismo , Preparaciones de Plantas/uso terapéutico , Biología SintéticaRESUMEN
BACKGROUND: Malaria is causing more than half of a million deaths and 214 million clinical cases annually. Despite tremendous efforts for the control of malaria, the global morbidity and mortality have not been significantly changed in the last 50 years. Artemisinin, extracted from the medicinal plant Artemisia sp. is an effective anti-malarial drug. In 2015, elucidation of the effectiveness of artemisinin as a potent anti-malarial drug was acknowledged with a Nobel prize. Owing to the tight market and low yield of artemisinin, an economical way to increase its production is to increase its content in Artemisia sp. through different biotechnological approaches including genetic transformation. METHODS: Artemisia annua and Artemisia dubia were transformed with rol ABC genes through Agrobacterium tumefacienes and Agrobacterium rhizogenes methods. The artemisinin content was analysed and compared between transformed and untransformed plants with the help of LC-MS/MS. Expression of key genes [Cytochrome P450 (CYP71AV1), aldehyde dehydrogenase 1 (ALDH1), amorpha-4, 11 diene synthase (ADS)] in the biosynthetic pathway of artemisinin and gene for trichome development and sesquiterpenoid biosynthetic (TFAR1) were measured using Quantitative real time PCR (qRT-PCR). Trichome density was analysed using confocal microscope. RESULTS: Artemisinin content was significantly increased in transformed material of both Artemisia species when compared to un-transformed plants. The artemisinin content within leaves of transformed lines was increased by a factor of nine, indicating that the plant is capable of synthesizing much higher amounts than has been achieved so far through traditional breeding. Expression of all artemisinin biosynthesis genes was significantly increased, although variation between the genes was observed. CYP71AV1 and ALDH1 expression levels were higher than that of ADS. Levels of the TFAR1 expression were also increased in all transgenic lines. Trichome density was also significantly increased in the leaves of transformed plants, but no trichomes were found in control roots or transformed roots. The detection of significantly raised levels of expression of the genes involved in artemisinin biosynthesis in transformed roots correlated with the production of significant amounts of artemisinin in these tissues. This suggests that synthesis is occurring in tissues other than the trichomes, which contradicts previous theories. CONCLUSION: Transformation of Artemisia sp. with rol ABC genes can lead to the increased production of artemisinin, which will help to meet the increasing demand of artemisinin because of its diverse pharmacological and anti-malarial importance.
Asunto(s)
Antimaláricos/metabolismo , Artemisia/metabolismo , Artemisininas/metabolismo , Proteínas Bacterianas/genética , Ingeniería Celular/métodos , Ingeniería Metabólica/métodos , Plantas Modificadas Genéticamente/metabolismo , Agrobacterium/genética , Artemisia/química , Artemisia/genética , Cromatografía Liquida , Perfilación de la Expresión Génica , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en Tándem , Transformación GenéticaRESUMEN
Diabetes now is the most common chronic disease in the world inducing heavy burden for the people's health. Based on this, diabetes research such as islet function has become a hot topic in medical institutes of the world. Today, in medical institutes, the conventional experiment platform in vitro is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have been developed to fabricate three-dimensional (3D) islet models in vitro which can better mimic the islet of pancreases in vivo. These in vitro islet models have shown better cell function than monolayer cells, indicating their great potential as better experimental platforms to elucidate islet behaviors under both physiological and pathological conditions, such as the molecular mechanisms of diabetes and clinical islet transplantation. In this review, we present the state-of-the-art advances in the microengineering methods for fabricating microscale islet models in vitro. We hope this will help researchers to better understand the progress in the engineering 3D islet models and their biomedical applications such as drug screening and islet transplantation.
Asunto(s)
Ingeniería Celular , Islotes Pancreáticos , Animales , Evaluación Preclínica de Medicamentos , Matriz Extracelular , HumanosRESUMEN
Higher multicellular organisms have evolved sophisticated intracellular and intercellular biological networks that enable cell growth and survival to fulfill an organism's needs. Although such networks allow the assembly of complex tissues and even provide healing and protective capabilities, malfunctioning cells can have severe consequences for an organism's survival. In humans, such events can result in severe disorders and diseases, including metabolic and immunological disorders, as well as cancer. Dominating the therapeutic frontier for these potentially lethal disorders, cell and gene therapies aim to relieve or eliminate patient suffering by restoring the function of damaged, diseased, and aging cells and tissues via the introduction of healthy cells or alternative genes. However, despite recent success, these efforts have yet to achieve sufficient therapeutic effects, and further work is needed to ensure the safe and precise control of transgene expression and cellular processes. In this review, we describe the biological tools and devices that are at the forefront of synthetic biology and discuss their potential to advance the specificity, efficiency, and safety of the current generation of cell and gene therapies, including how they can be used to confer curative effects that far surpass those of conventional therapeutics. We also highlight the current therapeutic delivery tools and the current limitations that hamper their use in human applications.
Asunto(s)
Terapia Biológica/métodos , Ingeniería Celular/métodos , Trasplante de Células/métodos , Terapia Genética/métodos , Biología Sintética/métodos , Investigación Biomédica/tendencias , HumanosRESUMEN
Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis.