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1.
Nat Commun ; 12(1): 4415, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285228

RESUMO

Complex dynamical fluctuations, from intracellular noise, brain dynamics or computer traffic display bursting dynamics consistent with a critical state between order and disorder. Living close to the critical point has adaptive advantages and it has been conjectured that evolution could select these critical states. Is this the case of living cells? A system can poise itself close to the critical point by means of the so-called self-organized criticality (SOC). In this paper we present an engineered gene network displaying SOC behaviour. This is achieved by exploiting the saturation of the proteolytic degradation machinery in E. coli cells by means of a negative feedback loop that reduces congestion. Our critical motif is built from a two-gene circuit, where SOC can be successfully implemented. The potential implications for both cellular dynamics and behaviour are discussed.


Assuntos
Engenharia Celular/métodos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Retroalimentação Fisiológica , Modelos Genéticos , Proteólise , Análise de Célula Única
2.
Philos Trans R Soc Lond B Biol Sci ; 376(1831): 20200228, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34176326

RESUMO

The goal of achieving enhanced diagnosis and continuous monitoring of human health has led to a vibrant, dynamic and well-funded field of research in medical sensing and biosensor technologies. The field has many sub-disciplines which focus on different aspects of sensor science; engaging engineers, chemists, biochemists and clinicians, often in interdisciplinary teams. The trends which dominate include the efforts to develop effective point of care tests and implantable/wearable technologies for early diagnosis and continuous monitoring. This review will outline the current state of the art in a number of relevant fields, including device engineering, chemistry, nanoscience and biomolecular detection, and suggest how these advances might be employed to develop effective systems for measuring physiology, detecting infection and monitoring biomarker status in wild animals. Special consideration is also given to the emerging threat of antimicrobial resistance and in the light of the current SARS-CoV-2 outbreak, zoonotic infections. Both of these areas involve significant crossover between animal and human health and are therefore well placed to seed technological developments with applicability to both human and animal health and, more generally, the reviewed technologies have significant potential to find use in the measurement of physiology in wild animals. This article is part of the theme issue 'Measuring physiology in free-living animals (Part II)'.


Assuntos
Técnicas Biossensoriais/instrumentação , COVID-19/diagnóstico , Biologia Sintética/métodos , Dispositivos Eletrônicos Vestíveis , Infecção por Zika virus/veterinária , Zoonoses/diagnóstico , Animais , Animais Selvagens/microbiologia , Animais Selvagens/parasitologia , Animais Selvagens/virologia , Biomarcadores/análise , Engenharia Celular/métodos , Humanos , Monitorização Fisiológica/instrumentação , Monitorização Fisiológica/métodos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Testes Imediatos , Infecção por Zika virus/diagnóstico
3.
Nat Commun ; 12(1): 3388, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099676

RESUMO

Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.


Assuntos
Proteínas de Bactérias/efeitos da radiação , Diabetes Mellitus Tipo 2/terapia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Optogenética/métodos , Transativadores/efeitos da radiação , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Celular , Diabetes Mellitus Tipo 2/genética , Feminino , Engenharia Genética , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células HEK293 , Humanos , Luz , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Obesos , Optogenética/instrumentação , Fotopletismografia/instrumentação , Domínios Proteicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Thermus thermophilus/genética , Transativadores/genética , Transativadores/metabolismo , Transgenes , Dispositivos Eletrônicos Vestíveis
4.
Nat Commun ; 12(1): 2637, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976146

RESUMO

Checkpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E711-19, 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential 'off-the-shelf' in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.


Assuntos
Células Apresentadoras de Antígenos/transplante , Engenharia Celular/métodos , Eritrócitos/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Ligante 4-1BB/genética , Ligante 4-1BB/imunologia , Ligante 4-1BB/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Modelos Animais de Doenças , Eritrócitos/metabolismo , Feminino , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Ativação Linfocitária , Neoplasias/imunologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/metabolismo , Cultura Primária de Células , Linfócitos T/imunologia , Linfócitos T/transplante , Transplante Homólogo/métodos
5.
Sheng Wu Gong Cheng Xue Bao ; 37(4): 1443-1449, 2021 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-33973457

RESUMO

We introduce the portfolio assessment into the classroom teaching reform in the curriculum of Cell Engineering, a specialty course in bioengineering & biotechnology. We established a complete classroom evaluation system that was divided the classroom assessment system of portfolio into four stages including the preparation stage, training stage, implementation stage and exhibition stage. We also discuss the feasibility and necessity of implementing the portfolio evaluation method in the course of cell engineering, the construction of evaluation system, and the key points and matters needing attention in the implementation process. The classroom reform is very productive, not only the classroom atmosphere has been activated, students' learning initiative and autonomy has been enhanced, but also the students' ability to analyze and solve professional problems related to cell engineering technology has been improved. The implementation of classroom teaching reform of this course can provide reference for other similar professional courses in colleges and universities.


Assuntos
Currículo , Universidades , Engenharia Celular , Humanos , Aprendizagem , Estudantes
6.
Aging (Albany NY) ; 13(10): 14399-14415, 2021 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-34031263

RESUMO

BACKGROUND: Cavernosa injury is a common cause of organic erectile dysfunction (ED), which requires safe and effective treatments. In the present study, the therapeutic efficiency of muscle-derived stem cells (MDSCs) modified with microRNA-126 (miR-126) was determined in rats with cavernosa injury. METHODS: MDSCs were transfected with miR-126 and then were transplanted into rats with cavernosa injury. Erectile function, vascular function (western blot and immunofluorescence), extraction, and detection of exosomes were then undertaken. RESULTS: On the 28th day after transplantation, the highest value of intra-cavernous pressure (ICP)/mean arterial pressure (MAP) in rats of miRNA-126 group (0.84 ± 0.14) was observed (Control: 0.38 ± 0.07; MDSC: 0.54 ± 0.11, Vector: 0.60 ± 0.02; respectively). Treatment of miRNA-126-modified-MDSCs remarkably strengthened vascular structure, supported by hematoxylin-eosin staining. The expression of CD31, von Willebrand Factor and vascular endothelial factors were higher than those in other groups, indicating improved vascular function. In vitro mechanism studies showed that exosomes containing miR-126 isolated from MDSCs promoted angiogenesis and attenuated apoptosis of human umbilical venous endothelial cells. Finally, insulin receptor substrate 1 and Krüppel-like factor 10 were determined as the direct target genes of miR-126. CONCLUSIONS: MiR-126 engineered MDSCs notably repaired cavernosa injury in rats via vascular reconstruction by directly targeting IRS1 and KLF10, in which the exosomes secreted by MDSCs played a critical role.


Assuntos
Engenharia Celular , Disfunção Erétil/etiologia , Disfunção Erétil/terapia , MicroRNAs/metabolismo , Músculos/patologia , Pênis/lesões , Transplante de Células-Tronco , Células-Tronco/metabolismo , Animais , Apoptose , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Disfunção Erétil/genética , Exossomos/metabolismo , Exossomos/ultraestrutura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Neovascularização Fisiológica , Pênis/irrigação sanguínea , Ratos Sprague-Dawley , Fatores de Transcrição/metabolismo
7.
Biotechnol Adv ; 49: 107760, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33932532

RESUMO

Genetically engineered T cells have sparked interest in advanced cancer treatment, reaching a milestone in 2017 with two FDA-approvals for CD19-directed chimeric antigen receptor (CAR) T cell therapeutics. It is becoming clear that the next generation of CAR T cell therapies will demand more complex engineering strategies and combinations thereof, including the use of revolutionary gene editing approaches. To date, manufacturing of CAR T cells mostly relies on γ-retroviral or lentiviral vectors, but their use is associated with several drawbacks, including safety issues, high manufacturing cost and vector capacity constraints. Non-viral approaches, including membrane permeabilization and carrier-based techniques, have therefore gained a lot of interest to replace viral transductions in the manufacturing of T cell therapeutics. This review provides an in-depth discussion on the avid search for alternatives to viral vectors, discusses key considerations for T cell engineering technologies, and provides an overview of the emerging spectrum of non-viral transfection technologies for T cells. Strengths and weaknesses of each technology will be discussed in relation to T cell engineering. Altogether, this work emphasizes the potential of non-viral transfection approaches to advance the next-generation of genetically engineered T cells.


Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T , Engenharia Celular , Receptores de Antígenos de Linfócitos T/genética , Tecnologia , Transfecção
8.
Nat Commun ; 12(1): 3275, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045451

RESUMO

Despite advancements in human pluripotent stem cells (hPSCs) differentiation protocols to generate appropriate neuronal progenitors suitable for transplantation in Parkinson's disease, resultant grafts contain low proportions of dopamine neurons. Added to this is the tumorigenic risk associated with the potential presence of incompletely patterned, proliferative cells within grafts. Here, we utilised a hPSC line carrying a FailSafeTM suicide gene (thymidine kinase linked to cyclinD1) to selectively ablate proliferative cells in order to improve safety and purity of neural transplantation in a Parkinsonian model. The engineered FailSafeTM hPSCs demonstrated robust ventral midbrain specification in vitro, capable of forming neural grafts upon transplantation. Activation of the suicide gene within weeks after transplantation, by ganciclovir administration, resulted in significantly smaller grafts without affecting the total yield of dopamine neurons, their capacity to innervate the host brain or reverse motor deficits at six months in a rat Parkinsonian model. Within ganciclovir-treated grafts, other neuronal, glial and non-neural populations (including proliferative cells), were significantly reduced-cell types that may pose adverse or unknown influences on graft and host function. These findings demonstrate the capacity of a suicide gene-based system to improve both the standardisation and safety of hPSC-derived grafts in a rat model of Parkinsonism.


Assuntos
Engenharia Celular/métodos , Genes Transgênicos Suicidas , Doença de Parkinson Secundária/terapia , Transplante de Células-Tronco/métodos , Animais , Apoptose/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células/genética , Modelos Animais de Doenças , Neurônios Dopaminérgicos/fisiologia , Feminino , Genes bcl-1/genética , Xenoenxertos/citologia , Xenoenxertos/patologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Masculino , Mesencéfalo/citologia , Mesencéfalo/patologia , Oxidopamina/administração & dosagem , Oxidopamina/toxicidade , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/patologia , Ratos , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/normas , Timidina Quinase/genética
9.
Nat Commun ; 12(1): 3236, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050151

RESUMO

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.


Assuntos
Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptor A2A de Adenosina/genética , Linfócitos T/transplante , Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Sistemas CRISPR-Cas/genética , Engenharia Celular/métodos , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Edição de Genes , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/imunologia , RNA Interferente Pequeno/metabolismo , RNA-Seq , Receptor A2A de Adenosina/metabolismo , Receptor ErbB-2/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/genética
10.
Sci Rep ; 11(1): 5376, 2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33686154

RESUMO

Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Engenharia Celular , Descoberta de Drogas , Modelos Biológicos , SARS-CoV-2/fisiologia , Animais , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Linhagem Celular , Humanos , SARS-CoV-2/efeitos dos fármacos , Proteínas Virais/biossíntese , Replicação Viral/efeitos dos fármacos
12.
Nat Commun ; 12(1): 792, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542232

RESUMO

The immune system is a sophisticated network of different cell types performing complex biocomputation at single-cell and consortium levels. The ability to reprogram such an interconnected multicellular system holds enormous promise in treating various diseases, as exemplified by the use of chimeric antigen receptor (CAR) T cells as cancer therapy. However, most CAR designs lack computation features and cannot reprogram multiple immune cell types in a coordinated manner. Here, leveraging our split, universal, and programmable (SUPRA) CAR system, we develop an inhibitory feature, achieving a three-input logic, and demonstrate that this programmable system is functional in diverse adaptive and innate immune cells. We also create an inducible multi-cellular NIMPLY circuit, kill switch, and a synthetic intercellular communication channel. Our work highlights that a simple split CAR design can generate diverse and complex phenotypes and provide a foundation for engineering an immune cell consortium with user-defined functionalities.


Assuntos
Engenharia Celular/métodos , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/genética , Proteínas Recombinantes de Fusão/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Cultura Primária de Células , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Biologia Sintética/métodos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Nucleic Acids Res ; 49(5): 2642-2654, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33591308

RESUMO

The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement. Here, we describe paired gRNA deletions along the PIGA locus on the human X chromosome ranging from 17 kb to 2 Mb. We found no compelling linear correlation between deletion size and the deletion efficiency, and there is no substantial impact of topologically associating domains on deletion frequency. Using this precise deletion technique, we have engineered a series of designer deletion cell lines, including one with deletions of two X-chromosomal counterselectable (negative selection) markers, PIGA and HPRT1, and additional cell lines bearing each individual deletion. PIGA encodes a component of the glycosylphosphatidylinositol (GPI) anchor biosynthetic apparatus. The PIGA gene counterselectable marker has unique features, including existing single cell level assays for both function and loss of function of PIGA and the existence of a potent counterselectable agent, proaerolysin, which we use routinely for selection against cells expressing PIGA. These designer cell lines may serve as a general platform with multiple selection markers and may be particularly useful for large scale genome engineering projects such as Genome Project-Write (GP-write).


Assuntos
Sistemas CRISPR-Cas , Engenharia Celular , Proteínas de Membrana/genética , Deleção de Sequência , Toxinas Bacterianas/toxicidade , Linhagem Celular , Cromossomos Humanos X , Marcadores Genéticos , Heterozigoto , Humanos , Mutação , N-Acetilglucosaminiltransferases/genética , Proteínas Citotóxicas Formadoras de Poros/toxicidade , RNA/genética
14.
ACS Appl Mater Interfaces ; 13(7): 7879-7889, 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33587612

RESUMO

Polymer-stabilized complex coacervate microdroplets have emerged as a robust platform for synthetic cell research. Their unique core-shell properties enable the sequestration of high concentrations of biologically relevant macromolecules and their subsequent release through the semipermeable membrane. These unique properties render the synthetic cell platform highly suitable for a range of biomedical applications, as long as its biocompatibility upon interaction with biological cells is ensured. The purpose of this study is to investigate how the structure and formulation of these coacervate-based synthetic cells impact the viability of several different cell lines. Through careful examination of the individual synthetic cell components, it became evident that the presence of free polycation and membrane-forming polymer had to be prevented to ensure cell viability. After closely examining the structure-toxicity relationship, a set of conditions could be found whereby no detrimental effects were observed, when the artificial cells were cocultured with RAW264.7 cells. This opens up a range of possibilities to use this modular system for biomedical applications and creates design rules for the next generation of coacervate-based, biomedically relevant particles.


Assuntos
Amilose/química , Células Artificiais/química , Materiais Biocompatíveis/química , Engenharia Celular , Polímeros/química , Amilose/síntese química , Amilose/farmacologia , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células HeLa , Humanos , Teste de Materiais , Camundongos , Estrutura Molecular , Tamanho da Partícula , Polímeros/síntese química , Polímeros/farmacologia , Células RAW 264.7 , Propriedades de Superfície
15.
Science ; 371(6534): 1166-1171, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33632893

RESUMO

Overexpressed tumor-associated antigens [for example, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)] are attractive targets for therapeutic T cells, but toxic "off-tumor" cross-reaction with normal tissues that express low levels of target antigen can occur with chimeric antigen receptor (CAR)-T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive-feedback circuit that allows human cytotoxic T cells to discriminate targets on the basis of a sigmoidal antigen-density threshold. In this circuit, a low-affinity synthetic Notch receptor for HER2 controls the expression of a high-affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells-it increases both CAR expression and activation-leading to a sigmoidal response. T cells with this circuit show sharp discrimination between target cells expressing normal amounts of HER2 and cancer cells expressing 100 times as much HER2, both in vitro and in vivo.


Assuntos
Engenharia Celular , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva , Células K562 , Camundongos , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Receptores Artificiais/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Esferoides Celulares , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cancer Sci ; 112(5): 1723-1734, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33609296

RESUMO

T cells could be engineered to overcome the aberrant metabolic milieu of solid tumors and tip the balance in favor of a long-lasting clinical response. Here, we explored the therapeutic potential of stably overexpressing cystathionine-gamma-lyase (CTH, CSE, or cystathionase), a pivotal enzyme of the transsulfuration pathway, in antitumor CD8+ T cells with the initial aim to boost intrinsic cysteine metabolism. Using a mouse model of adoptive cell transfer (ACT), we found that CTH-expressing T cells showed a superior control of tumor growth compared to control T cells. However, contrary to our hypothesis, this effect was not associated with increased T cell expansion in vivo or proliferation rescue in the absence of cysteine/cystine in vitro. Rather than impacting methionine or cysteine, ACT with CTH overexpression unexpectedly reduced glycine, serine, and proline concentration within the tumor interstitial fluid. Interestingly, in vitro tumor cell growth was mostly impacted by the combination of serine/proline or serine/glycine deprivation. These results suggest that metabolic gene engineering of T cells could be further investigated to locally modulate amino acid availability within the tumor environment while avoiding systemic toxicity.


Assuntos
Transferência Adotiva/métodos , Linfócitos T CD8-Positivos/metabolismo , Cistationina gama-Liase/metabolismo , Cisteína/biossíntese , Animais , Engenharia Celular , Linhagem Celular Tumoral , Proliferação de Células , Líquido Extracelular/metabolismo , Feminino , Glicina/metabolismo , Metionina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Neoplasias Ovarianas/terapia , Prolina/metabolismo , Serina/metabolismo , Microambiente Tumoral/imunologia
17.
ACS Appl Mater Interfaces ; 13(8): 10564-10573, 2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33605723

RESUMO

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.


Assuntos
Antineoplásicos/uso terapêutico , Hipóxia/tratamento farmacológico , Neoplasias/tratamento farmacológico , Fósforo/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Pontos Quânticos/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/efeitos da radiação , Catalase/genética , Catalase/metabolismo , Engenharia Celular , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Hipóxia/etiologia , Camundongos Endogâmicos BALB C , Neoplasias/complicações , Oxigênio/metabolismo , Fósforo/química , Fósforo/efeitos da radiação , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/efeitos da radiação , Pontos Quânticos/química , Pontos Quânticos/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo
18.
Methods Mol Biol ; 2229: 313-330, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33405229

RESUMO

RNA-seq enables the analysis of gene expression profiles across different conditions and organisms. Gene expression burden slows down growth, which results in poor predictability of gene constructs and product yields. Here, we describe how we applied RNA-seq to study the transcriptional profiles of Escherichia coli when burden is elicited during heterologous gene expression. We then present how we selected early responsive promoters from our RNA-seq results to design sensors for gene expression burden. Finally, we describe how we used one of these sensors to develop a burden-driven feedback regulator to improve cellular fitness in engineered E. coli.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Perfilação da Expressão Gênica/métodos , Engenharia Celular , Regulação Bacteriana da Expressão Gênica , Análise de Sequência de RNA , Biologia Sintética
19.
Nucleic Acids Res ; 49(3): 1769-1783, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33423048

RESUMO

The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes synthetic neochromosomes as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular neochromosomes of 50 and 100 kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. Neochromosomes made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding neochromosomes were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.


Assuntos
Engenharia Celular , Cromossomos , Saccharomyces cerevisiae/genética , Glicólise/genética
20.
Analyst ; 146(2): 454-462, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33491017

RESUMO

Neuronal cell microengineering involving micropatterning and polydimethylsiloxane (PDMS) microfluidics enables promising advances in microscale neuron control. However, a facile methodology for the precise and effective manipulation of neurons on a cell-repellent PDMS substrate remains challenging. Herein, a simple and straightforward strategy for neuronal cell patterning and neuronal network construction on PDMS based on microfluidics-assisted modification of functionalized Pluronic is described. The cell patterning process simply involves a one-step microfluidic modification and routine in vitro culture. It is demonstrated that multiple types of neuronal cell arrangements with various spatial profiles can be conveniently produced using this patterning tool. The precise control of neuronal cells with high patterning fidelity up to single cell resolution, as well as high adhesion and differentiation, is achieved too. Furthermore, neuronal network construction using the respective cell population and single cell patterning prove to be applicable. This achievement provides a convenient and feasible methodology for engineering neuronal cells on PDMS substrates, which will be useful for applications in many neuron-related microscale analytical research fields, including cell engineering, neurobiology, neuropharmacology, and neuronal sensing.


Assuntos
Engenharia Celular/instrumentação , Dimetilpolisiloxanos/química , Dimetilpolisiloxanos/farmacologia , Dispositivos Lab-On-A-Chip , Rede Nervosa/citologia , Neurônios/citologia , Poloxâmero/química , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Imagem Óptica
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