Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
1.
Cell ; 167(1): 248-259.e12, 2016 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-27662092

RESUMEN

Synthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines and present combinatorial methods for the production of antibody conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world.


Asunto(s)
Formación de Anticuerpos , Péptidos Catiónicos Antimicrobianos/biosíntesis , Vacunas/biosíntesis , Animales , Péptidos Catiónicos Antimicrobianos/genética , Sistema Libre de Células , Técnicas Químicas Combinatorias , Humanos , Biosíntesis de Proteínas , Biología Sintética , Transcripción Genética , Vacunas/genética
2.
Proc Natl Acad Sci U S A ; 116(47): 23505-23511, 2019 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-31685628

RESUMEN

Comorbidity is common as age increases, and currently prescribed treatments often ignore the interconnectedness of the involved age-related diseases. The presence of any one such disease usually increases the risk of having others, and new approaches will be more effective at increasing an individual's health span by taking this systems-level view into account. In this study, we developed gene therapies based on 3 longevity associated genes (fibroblast growth factor 21 [FGF21], αKlotho, soluble form of mouse transforming growth factor-ß receptor 2 [sTGFßR2]) delivered using adeno-associated viruses and explored their ability to mitigate 4 age-related diseases: obesity, type II diabetes, heart failure, and renal failure. Individually and combinatorially, we applied these therapies to disease-specific mouse models and found that this set of diverse pathologies could be effectively treated and in some cases, even reversed with a single dose. We observed a 58% increase in heart function in ascending aortic constriction ensuing heart failure, a 38% reduction in α-smooth muscle actin (αSMA) expression, and a 75% reduction in renal medullary atrophy in mice subjected to unilateral ureteral obstruction and a complete reversal of obesity and diabetes phenotypes in mice fed a constant high-fat diet. Crucially, we discovered that a single formulation combining 2 separate therapies into 1 was able to treat all 4 diseases. These results emphasize the promise of gene therapy for treating diverse age-related ailments and demonstrate the potential of combination gene therapy that may improve health span and longevity by addressing multiple diseases at once.


Asunto(s)
Envejecimiento , Diabetes Mellitus Experimental/terapia , Factores de Crecimiento de Fibroblastos/fisiología , Terapia Genética , Glucuronidasa/genética , Insuficiencia Cardíaca/terapia , Fallo Renal Crónico/terapia , Obesidad/terapia , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1/genética , Animales , Dependovirus/genética , Diabetes Mellitus Experimental/etiología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Fibrosis , Vectores Genéticos/uso terapéutico , Glucuronidasa/sangre , Glucuronidasa/fisiología , Resistencia a la Insulina , Fallo Renal Crónico/etiología , Fallo Renal Crónico/patología , Médula Renal/patología , Proteínas Klotho , Longevidad/genética , Masculino , Ratones Endogámicos C57BL , Obesidad/etiología , Fenotipo , Receptor Tipo II de Factor de Crecimiento Transformador beta/fisiología , Factor de Crecimiento Transformador beta1/sangre , Factor de Crecimiento Transformador beta1/fisiología , Obstrucción Ureteral/complicaciones
3.
Proc Natl Acad Sci U S A ; 113(19): 5245-50, 2016 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-27114509

RESUMEN

The design of cell-targeted protein therapeutics can be informed by natural protein-protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics.


Asunto(s)
Anemia/sangre , Anemia/tratamiento farmacológico , Eritropoyetina/administración & dosificación , Terapia Molecular Dirigida/métodos , Ingeniería de Proteínas/métodos , Receptores de Eritropoyetina/metabolismo , Animales , Diseño de Fármacos , Eritropoyesis/efectos de los fármacos , Eritropoyesis/fisiología , Eritropoyetina/genética , Eritropoyetina/farmacocinética , Humanos , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión , Resultado del Tratamiento
4.
bioRxiv ; 2024 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-38260393

RESUMEN

Current SARS-CoV-2 vaccines have demonstrated robust induction of neutralizing antibodies and CD4+ T cell activation, however CD8+ responses are variable, and the duration of immunity and protection against variants are limited. Here we repurposed our DNA origami vaccine platform, DoriVac, for targeting infectious viruses, namely SARS-CoV-2, HIV, and Ebola. The DNA origami nanoparticle, conjugated with infectious-disease-specific HR2 peptides, which act as highly conserved antigens, and CpG adjuvant at precise nanoscale spacing, induced neutralizing antibodies, Th1 CD4+ T cells, and CD8+ T cells in naïve mice, with significant improvement over a bolus control. Pre-clinical studies using lymph-node-on-a-chip systems validated that DoriVac, when conjugated with antigenic peptides or proteins, induced promising cellular immune responses in human cells. These results suggest that DoriVac holds potential as a versatile, modular vaccine platform, capable of inducing both humoral and cellular immunities. The programmability of this platform underscores its potential utility in addressing future pandemics.

5.
Nat Nanotechnol ; 19(7): 1055-1065, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38491184

RESUMEN

Multivalent presentation of ligands often enhances receptor activation and downstream signalling. DNA origami offers a precise nanoscale spacing of ligands, a potentially useful feature for therapeutic nanoparticles. Here we use a square-block DNA origami platform to explore the importance of the spacing of CpG oligonucleotides. CpG engages Toll-like receptors and therefore acts to activate dendritic cells. Through in vitro cell culture studies and in vivo tumour treatment models, we demonstrate that square blocks induce Th1 immune polarization when CpG is spaced at 3.5 nm. We observe that this DNA origami vaccine enhances DC activation, antigen cross-presentation, CD8 T-cell activation, Th1-polarized CD4 activation and natural-killer-cell activation. The vaccine also effectively synergizes with anti-PD-L1 for improved cancer immunotherapy in melanoma and lymphoma models and induces long-term T-cell memory. Our results suggest that DNA origami may serve as a platform for controlling adjuvant spacing and co-delivering antigens in vaccines.


Asunto(s)
Vacunas contra el Cáncer , Oligodesoxirribonucleótidos , Animales , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Ratones , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacología , ADN/química , ADN/inmunología , Células Dendríticas/inmunología , Humanos , Ratones Endogámicos C57BL , Islas de CpG , Vacunas de ADN/química , Vacunas de ADN/inmunología , Vacunas de ADN/farmacología , Linfocitos T CD8-positivos/inmunología , Vacunación/métodos , Línea Celular Tumoral , Femenino
6.
Nat Protoc ; 16(4): 2088-2108, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33692551

RESUMEN

Classic approaches to mapping the developmental history of cells in vivo have relied on techniques that require complex interventions and often capture only a single trajectory or moment in time. We have previously described a developmental barcoding system to address these issues using synthetically induced mutations to record information about each cell's lineage in its genome. This system uses MARC1 mouse lines, which have multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Here, we detail two MARC1 lines that are available from a public repository. We describe strategies for using MARC1 mice and experimental design considerations. We provide a protocol for barcode retrieval and sequencing as well as the analysis of the sequencing data. This protocol generates barcodes based on synthetically induced mutations in mice to enable lineage analysis.


Asunto(s)
Sistemas CRISPR-Cas/genética , Código de Barras del ADN Taxonómico/métodos , Filogenia , Animales , Ratones , Mutación/genética , ARN Guía de Kinetoplastida/genética
7.
Protein Eng Des Sel ; 342021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-34725710

RESUMEN

Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10-14 M, allowing an estimate of the number of receptor-ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia.


Asunto(s)
Eritropoyetina , Animales , Eritropoyesis , Eritropoyetina/genética , Eritropoyetina/metabolismo , Hipoxia , Ratones , Unión Proteica , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo
8.
Nat Biotechnol ; 39(4): 510-519, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33257861

RESUMEN

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.


Asunto(s)
Técnicas de Reprogramación Celular/métodos , Oligodendroglía/citología , Células Madre Pluripotentes/citología , Factores de Transcripción/genética , Empalme Alternativo , Diferenciación Celular , Ingeniería Celular , Células Cultivadas , Técnicas de Cocultivo , Humanos , Oligodendroglía/metabolismo , Células Madre Pluripotentes/metabolismo , Biología de Sistemas
9.
Sci Transl Med ; 13(580)2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33568518

RESUMEN

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.


Asunto(s)
Dependovirus , Vectores Genéticos , Animales , Dependovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética , Inmunidad Innata , Ratones , Porcinos
10.
ACS Synth Biol ; 9(2): 191-197, 2020 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-31834794

RESUMEN

Protein "AND-gate" systems, in which a ligand acts only on cells with two different receptors, direct signaling activity to a particular cell type and avoid action on other cells. In a bifunctional AND-gate protein, the molecular geometry of the protein domains is crucial. Here we constructed a tissue-targeted erythropoietin (EPO) that stimulates red blood cell (RBC) production without triggering thrombosis. The EPO was directed to RBC precursors and mature RBCs by fusion to an anti-glycophorin A antibody V region. Many such constructs activated EPO receptors in vitro and stimulated RBC and not platelet production in mice but nonetheless enhanced thrombosis in mice and caused adhesion between RBCs and EPO-receptor-bearing cells. On the basis of a protein-structural model of the RBC surface, we rationally designed an anti-glycophorin-EPO fusion that does not induce cell adhesion in vitro or enhance thrombosis in vivo. Thus, mesoscale geometry can inform the design of synthetic-biological systems.


Asunto(s)
Comunicación Celular/fisiología , Eritrocitos/metabolismo , Eritropoyetina/metabolismo , Glicoforinas/inmunología , Animales , Línea Celular , Darbepoetina alfa/uso terapéutico , Epítopos/genética , Epítopos/metabolismo , Eritrocitos/citología , Eritropoyetina/genética , Glicoforinas/metabolismo , Hemorragia/tratamiento farmacológico , Humanos , Ligandos , Ratones , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/uso terapéutico , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo
11.
Sci Transl Med ; 11(479)2019 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-30760580

RESUMEN

Platelets are crucial for normal hemostasis; however, their hyperactivation also contributes to many potentially lethal pathologies including myocardial infarction, stroke, and cancer. We hypothesized that modified platelets lacking their aggregation and activation capacity could act as reversible inhibitors of platelet activation cascades. Here, we describe the development of detergent-extracted human modified platelets (platelet decoys) that retained platelet binding functions but were incapable of functional activation and aggregation. Platelet decoys inhibited aggregation and adhesion of platelets on thrombogenic surfaces in vitro, which could be immediately reversed by the addition of normal platelets; in vivo in a rabbit model, pretreatment with platelet decoys inhibited arterial injury-induced thromboembolism. Decoys also interfered with platelet-mediated human breast cancer cell aggregation, and their presence decreased cancer cell arrest and extravasation in a microfluidic human microvasculature on a chip. In a mouse model of metastasis, simultaneous injection of the platelet decoys with tumor cells inhibited metastatic tumor growth. Thus, our results suggest that platelet decoys might represent an effective strategy for obtaining antithrombotic and antimetastatic effects.


Asunto(s)
Plaquetas/patología , Trombosis/patología , Animales , Plaquetas/ultraestructura , Línea Celular Tumoral , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Adhesividad Plaquetaria , Agregación Plaquetaria , Conejos , Receptores de Superficie Celular/metabolismo
12.
Nat Commun ; 7: 10176, 2016 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-26733371

RESUMEN

Accurate assessment of blood haemostasis is essential for the management of patients who use extracorporeal devices, receive anticoagulation therapy or experience coagulopathies. However, current monitoring devices do not measure effects of haemodynamic forces that contribute significantly to platelet function and thrombus formation. Here we describe a microfluidic device that mimics a network of stenosed arteriolar vessels, permitting evaluation of blood clotting within small sample volumes under pathophysiological flow. By applying a clotting time analysis based on a phenomenological mathematical model of thrombus formation, coagulation and platelet function can be accurately measured in vitro in patient blood samples. When the device is integrated into an extracorporeal circuit in pig endotoxemia or heparin therapy models, it produces real-time readouts of alterations in coagulation ex vivo that are more reliable than standard clotting assays. Thus, this disposable device may be useful for personalized diagnostics and for real-time surveillance of antithrombotic therapy in clinic.


Asunto(s)
Pruebas de Coagulación Sanguínea/instrumentación , Plaquetas/efectos de los fármacos , Hemostasis/efectos de los fármacos , Dispositivos Laboratorio en un Chip , Abciximab , Animales , Anticuerpos Monoclonales/farmacología , Pruebas de Coagulación Sanguínea/métodos , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Técnicas Analíticas Microfluídicas , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Pruebas de Función Plaquetaria/instrumentación , Pruebas de Función Plaquetaria/métodos , Sistemas de Atención de Punto , Resistencia al Corte , Porcinos
13.
Comp Med ; 65(2): 96-113, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25926395

RESUMEN

Hematologic parameters are important markers of disease in human and veterinary medicine. Biomedical research has benefited from mouse models that recapitulate such disease, thus expanding knowledge of pathogenetic mechanisms and investigative therapies that translate across species. Mice in health have many notable hematologic differences from humans and other veterinary species, including smaller erythrocytes, higher percentage of circulating reticulocytes or polychromasia, lower peripheral blood neutrophil and higher peripheral blood and bone marrow lymphocyte percentages, variable leukocyte morphologies, physiologic splenic hematopoiesis and iron storage, and more numerous and shorter-lived erythrocytes and platelets. For accurate and complete hematologic analyses of disease and response to investigative therapeutic interventions, these differences and the unique features of murine hematopathology must be understood. Here we review murine hematology and hematopathology for practical application to translational investigation.


Asunto(s)
Enfermedades Hematológicas/sangre , Hematología , Ratones/sangre , Patología Veterinaria , Animales , Médula Ósea/patología , Modelos Animales de Enfermedad , Enfermedades Hematológicas/etiología , Enfermedades Hematológicas/terapia , Hematología/métodos , Hematopoyesis , Humanos , Patología Veterinaria/métodos , Investigación Biomédica Traslacional/métodos
14.
Comp Med ; 64(2): 99-105, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24674583

RESUMEN

Urinary biomarkers may offer a more sensitive and less invasive means to monitor kidney disease than traditional blood chemistry biomarkers such as creatinine. CD1(pcy/pcy) (pcy) mice have a slowly progressive disease phenotype that resembles human autosomal dominant polycystic kidney disease with renal cyst formation and inflammation. Previous reports suggest that dietary protein restriction may slow disease progression in mice and humans with polycystic kidney disease. Accordingly, we fed pcy mice either a standard chow (22.5% protein) or a protein-restricted (11.5% soy-based protein) diet from weaning until 34 wk of age. Every 6 wk we measured markers of kidney disease, including serum creatinine, BUN, and serum albumin as well as urinary monocyte chemoattractant protein 1 (MCP1), microalbumin, and specific gravity. Progression of kidney disease was equivalent for both diet groups despite dietary protein restriction. Urinary biomarkers proved useful for early detection of disease, in that urinary microalbumin was elevated as early as 22 wk of age and urinary MCP1 was increased by 28 wk of age, whereas increases in serum creatinine and BUN were detected later (at 34 wk of age) in both diet groups. Thus, urinary microalbumin and MCP1 analyses provided earlier, noninvasive indicators for detection of kidney disease and disease progression in pcy mice than did serum creatinine and BUN.


Asunto(s)
Azotemia/orina , Biomarcadores/orina , Enfermedades Renales Poliquísticas/complicaciones , Enfermedades Renales Poliquísticas/diagnóstico , Albuminuria , Análisis de Varianza , Animales , Azotemia/etiología , Nitrógeno de la Urea Sanguínea , Quimiocina CCL2/orina , Creatinina/sangre , Dieta con Restricción de Proteínas , Ratones , Enfermedades Renales Poliquísticas/dietoterapia , Albúmina Sérica
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA