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1.
Mol Ther ; 23(9): 1507-18, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26061646

RESUMEN

The adoptive transfer of chimeric antigen receptor (CAR) T cell represents a highly promising strategy to fight against multiple cancers. The clinical outcome of such therapies is intimately linked to the ability of effector cells to engraft, proliferate, and specifically kill tumor cells within patients. When allogeneic CAR T-cell infusion is considered, host versus graft and graft versus host reactions must be avoided to prevent rejection of adoptively transferred cells, host tissue damages and to elicit significant antitumoral outcome. This work proposes to address these three requirements through the development of multidrug-resistant T cell receptor αß-deficient CAR T cells. We demonstrate that these engineered T cells displayed efficient antitumor activity and proliferated in the presence of purine and pyrimidine nucleoside analogues, currently used in clinic as preconditioning lymphodepleting regimens. The absence of TCRαß at their cell surface along with their purine nucleotide analogues-resistance properties could prevent their alloreactivity and enable them to resist to lymphodepleting regimens that may be required to avoid their ablation via HvG reaction. By providing a basic framework to develop a universal T cell compatible with allogeneic adoptive transfer, this work is laying the foundation stone of the large-scale utilization of CAR T-cell immunotherapies.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Resistencia a Múltiples Medicamentos/genética , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Antígenos CD19/genética , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Terapia Combinada , Citotoxicidad Inmunológica , Desoxicitidina Quinasa/deficiencia , Desoxicitidina Quinasa/genética , Expresión Génica , Silenciador del Gen , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Concentración 50 Inhibidora , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/efectos de los fármacos , Trasplante Homólogo
2.
Nucleic Acids Res ; 42(8): 5390-402, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24569350

RESUMEN

A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Aminoácidos/química , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cricetulus , ADN/química , ADN/metabolismo , División del ADN , Mutación , Análisis por Matrices de Proteínas , Ingeniería de Proteínas , Levaduras/genética
3.
Nat Commun ; 15(1): 4965, 2024 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-38862518

RESUMEN

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing ß-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.


Asunto(s)
Anemia de Células Falciformes , Edición Génica , Terapia Genética , Células Madre Hematopoyéticas , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Anemia de Células Falciformes/terapia , Anemia de Células Falciformes/genética , Edición Génica/métodos , Animales , Células Madre Hematopoyéticas/metabolismo , Humanos , Femenino , Ratones , Terapia Genética/métodos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Trasplante de Células Madre Hematopoyéticas , Globinas beta/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Reparación del ADN , Mutación , Talasemia beta/terapia , Talasemia beta/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen
4.
J Biol Chem ; 287(46): 38427-32, 2012 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-23019344

RESUMEN

Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it.


Asunto(s)
Citosina/química , Metilación de ADN , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Animales , Secuencia de Bases , Células CHO , Cricetinae , ADN/genética , Epigénesis Genética , Silenciador del Gen , Ingeniería Genética/métodos , Terapia Genética/métodos , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutagénesis , Unión Proteica , Ingeniería de Proteínas/métodos , Estructura Terciaria de Proteína , Proteínas Recombinantes/química
6.
Nat Commun ; 10(1): 5100, 2019 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-31723132

RESUMEN

Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner. Here, we explore this concept by repurposing TCR, CD25 and PD1, three major players of the T cell activation pathway. We insert the CAR into the TCRα gene (TRACCAR), and IL-12P70 into either IL2Rα or PDCD1 genes. This process results in transient, antigen concentration-dependent IL-12P70 secretion, increases TRACCAR T cell cytotoxicity and extends survival of tumor-bearing mice. This gene network repurposing strategy can be extended to other cellular pathways, thus paving the way for generating smart CAR T cells able to integrate biological inputs and to translate them into therapeutic outputs in a highly regulated manner.


Asunto(s)
Sistema Inmunológico/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Edición Génica , Humanos , Interleucina-12/genética , Activación de Linfocitos/inmunología , Ratones , Neoplasias/inmunología , Neoplasias/patología , Receptores de Antígenos de Linfocitos T/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo
7.
Sci Rep ; 7: 39833, 2017 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-28106050

RESUMEN

A key to the success of chimeric antigen receptor (CAR) T-cell based therapies greatly rely on the capacity to identify and target antigens with expression restrained to tumor cells. Here we present a strategy to generate CAR T-cells that are only effective locally (tumor tissue), potentially also increasing the choice of targetable antigens. By fusing an oxygen sensitive subdomain of HIF1α to a CAR scaffold, we generated CAR T-cells that are responsive to a hypoxic environment, a hallmark of certain tumors. Along with the development of oxygen-sensitive CAR T-cells, this work also provides a basic framework to use a multi-chain CAR as a platform to create the next generation of smarter self-decision making CAR T-cells.


Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Oxígeno/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T Citotóxicos/fisiología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Ingeniería Genética , Humanos , Activación de Linfocitos , Neoplasias/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T Citotóxicos/trasplante , Microambiente Tumoral
8.
Sci Rep ; 6: 18950, 2016 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-26750734

RESUMEN

The ability to control T cells engineered to permanently express chimeric antigen receptors (CARs) is a key feature to improve safety. Here, we describe the development of a new CAR architecture with an integrated switch-on system that permits to control the CAR T-cell function. This system offers the advantage of a transient CAR T-cell for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug.


Asunto(s)
Citotoxicidad Inmunológica/efectos de los fármacos , Ingeniería Genética/métodos , Proteínas Mutantes Quiméricas/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Antígenos CD8/genética , Antígenos CD8/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/inmunología , Humanos , Proteínas Mutantes Quiméricas/inmunología , Dominios Proteicos , Receptores de Antígenos de Linfocitos T/inmunología , Anticuerpos de Cadena Única/genética , Sirolimus/análogos & derivados , Sirolimus/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Tacrolimus/farmacología , Transfección
9.
Sci Rep ; 5: 8150, 2015 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-25632877

RESUMEN

A key feature when designing DNA targeting tools and especially nucleases is specificity. The ability to control and tune this important parameter represents an invaluable advance to the development of such molecular scissors. Here, we identified and characterized new non-conventional RVDs (ncRVDs) that possess novel intrinsic targeting specificity features. We further report a strategy to control TALEN targeting based on the exclusion capacities of ncRVDs (discrimination between different nucleotides). By implementing such ncRVDs, we demonstrated in living cells the possibility to efficiently promote TALEN-mediated processing of a target in the HBB locus and alleviate undesired off-site cleavage. We anticipate that this method can greatly benefit to designer nucleases, especially for therapeutic applications and synthetic biology.


Asunto(s)
Desoxirribonucleasas/metabolismo , Repeticiones de Dinucleótido , Marcación de Gen , Secuencia de Bases , Línea Celular , Hemoglobinas/química , Hemoglobinas/genética , Humanos , Hidrólisis , Datos de Secuencia Molecular , Especificidad por Sustrato , Levaduras/genética , Levaduras/metabolismo
10.
Nat Commun ; 5: 3831, 2014 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-24871200

RESUMEN

Diatoms, a major group of photosynthetic microalgae, have a high biotechnological potential that has not been fully exploited because of the paucity of available genetic tools. Here we demonstrate targeted and stable modifications of the genome of the marine diatom Phaeodactylum tricornutum, using both meganucleases and TALE nucleases. When nuclease-encoding constructs are co-transformed with a selectable marker, high frequencies of genome modifications are readily attained with 56 and 27% of the colonies exhibiting targeted mutagenesis or targeted gene insertion, respectively. The generation of an enhanced lipid-producing strain (45-fold increase in triacylglycerol accumulation) through the disruption of the UDP-glucose pyrophosphorylase gene exemplifies the power of genome engineering to harness diatoms for biofuel production.


Asunto(s)
Biotecnología , Diatomeas/genética , Ingeniería Genética/métodos , Genoma , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Roturas del ADN de Doble Cadena , Endonucleasas/metabolismo , Citometría de Flujo , Genotipo , Lípidos/biosíntesis , Datos de Secuencia Molecular , Mutagénesis/genética , Tasa de Mutación , Espectrometría de Masas en Tándem , Transactivadores/metabolismo , Triglicéridos/análisis
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