RESUMEN
CRISPR technology has opened a new era of genome interrogation and genome engineering. Discovered in bacteria, where it protects against bacteriophage by cleaving foreign nucleic acid sequences, the CRISPR system has been repurposed as an adaptable tool for genome editing and multiple other applications. CRISPR's ease of use, precision, and versatility have led to its widespread adoption, accelerating biomedical research and discovery in human cells and model organisms. Here we review CRISPR-based tools and discuss how they are being applied to decode the genetic circuits that control immune function in health and disease. Genetic variation in immune cells can affect autoimmune disease risk, infectious disease pathogenesis, and cancer immunotherapies. CRISPR provides unprecedented opportunities for functional mechanistic studies of coding and noncoding genome sequence function in immunity. Finally, we discuss the potential of CRISPR technology to engineer synthetic cellular immunotherapies for a wide range of human diseases.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Infecciones/inmunología , Neoplasias/inmunología , Animales , Enfermedades Autoinmunes/genética , Sistemas CRISPR-Cas , Edición Génica , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Inmunidad , Infecciones/genética , Neoplasias/genéticaRESUMEN
CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.
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Ingeniería Metabólica , Linfocitos T , Humanos , Perfilación de la Expresión Génica , Ingeniería Metabólica/métodos , ARN , TranscriptomaRESUMEN
The therapeutic potential for human type 2 innate lymphoid cells (ILC2s) has been underexplored. Although not observed in mouse ILC2s, we found that human ILC2s secrete granzyme B (GZMB) and directly lyse tumor cells by inducing pyroptosis and/or apoptosis, which is governed by a DNAM-1-CD112/CD155 interaction that inactivates the negative regulator FOXO1. Over time, the high surface density expression of CD155 in acute myeloid leukemia cells impairs the expression of DNAM-1 and GZMB, thus allowing for immune evasion. We describe a reliable platform capable of up to 2,000-fold expansion of human ILC2s within 4 weeks, whose molecular and cellular ILC2 profiles were validated by single-cell RNA sequencing. In both leukemia and solid tumor models, exogenously administered expanded human ILC2s show significant antitumor effects in vivo. Collectively, we demonstrate previously unreported properties of human ILC2s and identify this innate immune cell subset as a member of the cytolytic immune effector cell family.
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Granzimas , Inmunidad Innata , Linfocitos , Neoplasias , Animales , Humanos , Ratones , Apoptosis , Citocinas , Neoplasias/inmunología , Neoplasias/terapiaRESUMEN
Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.
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Supervivencia Celular , Neuronas Dopaminérgicas , FN-kappa B , Factor de Necrosis Tumoral alfa , Proteína p53 Supresora de Tumor , Neuronas Dopaminérgicas/metabolismo , Animales , Humanos , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ratones , Supervivencia Celular/efectos de los fármacos , Transducción de Señal , Enfermedad de Parkinson/metabolismo , Células Madre Pluripotentes/metabolismo , Apoptosis , Modelos Animales de Enfermedad , Sistemas CRISPR-CasRESUMEN
Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.
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Elementos Transponibles de ADN , Humanos , Elementos Transponibles de ADN/genética , Ingeniería Genética/métodos , Genoma Humano , Animales , Evolución MolecularRESUMEN
Anti-NMDA receptor (NMDAR) autoantibodies cause NMDAR encephalitis, the most common autoimmune encephalitis, leading to psychosis, seizures, and autonomic dysfunction. Current treatments comprise broad immunosuppression or non-selective antibody removal. We developed NMDAR-specific chimeric autoantibody receptor (NMDAR-CAAR) T cells to selectively eliminate anti-NMDAR B cells and disease-causing autoantibodies. NMDAR-CAARs consist of an extracellular multi-subunit NMDAR autoantigen fused to intracellular 4-1BB/CD3ζ domains. NMDAR-CAAR T cells recognize a large panel of human patient-derived autoantibodies, release effector molecules, proliferate, and selectively kill antigen-specific target cell lines even in the presence of high autoantibody concentrations. In a passive transfer mouse model, NMDAR-CAAR T cells led to depletion of an anti-NMDAR B cell line and sustained reduction of autoantibody levels without notable off-target toxicity. Treatment of patients may reduce side effects, prevent relapses, and improve long-term prognosis. Our preclinical work paves the way for CAAR T cell phase I/II trials in NMDAR encephalitis and further autoantibody-mediated diseases.
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Autoanticuerpos , Encefalitis , Linfocitos T , Animales , Humanos , Ratones , Autoanticuerpos/metabolismo , Encefalitis/metabolismo , Encefalitis/terapia , Receptores de N-Metil-D-Aspartato , Enfermedades Autoinmunes , Modelos Animales de EnfermedadRESUMEN
Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors that we call synthetic intramembrane proteolysis receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the therapeutic potential of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.
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Tratamiento Basado en Trasplante de Células y Tejidos , Receptores Artificiales , Humanos , Receptores de Antígenos de Linfocitos T/genética , Receptores Artificiales/genética , Biología Sintética , Linfocitos TRESUMEN
A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
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Sistemas CRISPR-Cas , Reprogramación Celular , Epigénesis Genética , Epigenoma , Edición Génica , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Diferenciación Celular , Islas de CpG , Metilación de ADN , Silenciador del Gen , Código de Histonas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.
Asunto(s)
Técnicas de Sustitución del Gen/métodos , Ingeniería Genética/métodos , Inmunoterapia/métodos , Animales , Células Sanguíneas , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , ARN Guía de Kinetoplastida/genética , Análisis de la Célula Individual/métodos , Linfocitos T , Transcriptoma/genéticaRESUMEN
Current approaches to reducing the latent HIV reservoir entail first reactivating virus-containing cells to become visible to the immune system. A critical second step is killing these cells to reduce reservoir size. Endogenous cytotoxic T-lymphocytes (CTLs) may not be adequate because of cellular exhaustion and the evolution of CTL-resistant viruses. We have designed a universal CAR-T cell platform based on CTLs engineered to bind a variety of broadly neutralizing anti-HIV antibodies. We show that this platform, convertibleCAR-T cells, effectively kills HIV-infected, but not uninfected, CD4 T cells from blood, tonsil, or spleen and only when armed with anti-HIV antibodies. convertibleCAR-T cells also kill within 48 h more than half of the inducible reservoir found in blood of HIV-infected individuals on antiretroviral therapy. The modularity of convertibleCAR-T cell system, which allows multiplexing with several anti-HIV antibodies yielding greater breadth and control, makes it a promising tool for attacking the latent HIV reservoir.
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Anticuerpos Antiidiotipos/farmacología , Infecciones por VIH/terapia , Inmunoterapia Adoptiva , Replicación Viral/genética , Animales , Anticuerpos Antiidiotipos/inmunología , Células HEK293 , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Ratones , Tonsila Palatina/inmunología , Tonsila Palatina/metabolismo , Cultivo Primario de Células , Bazo/inmunología , Bazo/metabolismo , Linfocitos T Citotóxicos/inmunología , Latencia del Virus/inmunología , Replicación Viral/inmunologíaRESUMEN
Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.
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Carcinoma Ductal Pancreático , Claudinas , Activación de Linfocitos , Neoplasias Pancreáticas , Linfocitos T Citotóxicos , Animales , Humanos , Ratones , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Claudinas/metabolismo , Claudinas/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Sinapsis Inmunológicas/metabolismo , Sinapsis Inmunológicas/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Microdominios de Membrana/metabolismo , Microdominios de Membrana/inmunología , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.
Asunto(s)
Antígenos CD28 , Redes Reguladoras de Genes , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Antígenos CD28/metabolismo , Transducción de Señal , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Ligando CD27/genética , Ligando CD27/metabolismo , Linfocitos T CD8-positivosRESUMEN
Many community- and hospital-acquired bacterial infections are caused by antibiotic-resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) predisposes humans to invasive infections that are difficult to eradicate. We designed a closed-loop gene network programming mammalian cells to autonomously detect and eliminate bacterial infections. The genetic circuit contains human Toll-like receptors as the bacterial sensor and a synthetic promoter driving reversible and adjustable expression of lysostaphin, a bacteriolytic enzyme highly lethal to S. aureus. Immunomimetic designer cells harboring this genetic circuit exhibited fast and robust sense-and-destroy kinetics against live staphylococci. When tested in a foreign-body infection model in mice, microencapsulated cell implants prevented planktonic MRSA infection and reduced MRSA biofilm formation by 91%. Notably, this system achieved a 100% cure rate of acute MRSA infections, whereas conventional vancomycin treatment failed. These results suggest that immunomimetic designer cells could offer a therapeutic approach for early detection, prevention, and cure of pathogenic infections in the post-antibiotic era.
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Biomimética/métodos , Staphylococcus aureus Resistente a Meticilina/fisiología , Infecciones Estafilocócicas/prevención & control , Fosfatasa Alcalina/sangre , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Pruebas Antimicrobianas de Difusión por Disco , Femenino , Células HEK293 , Humanos , Receptores de Lipopolisacáridos/genética , Lisostafina/metabolismo , Lisostafina/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Plásmidos/genética , Plásmidos/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Receptor Toll-Like 1/genética , Receptor Toll-Like 2/genética , Receptor Toll-Like 6/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Tumor immune escape limits durable responses to T cell therapy. Here, we examined how regulation and function of gene products that provide the target epitopes for CD8+ T cell anti-tumor immunity influence therapeutic efficacy and resistance. We used a CRISPR-Cas9-based method (CRISPitope) in syngeneic melanoma models to fuse the same model CD8+ T cell epitope to the C-termini of different endogenous gene products. Targeting melanosomal proteins or oncogenic CDK4R24C (Cyclin-dependent kinase 4) by adoptive cell transfer (ACT) of the same epitope-specific CD8+ T cells revealed diverse genetic and non-genetic immune escape mechanisms. ACT directed against melanosomal proteins, but not CDK4R24C, promoted melanoma dedifferentiation, and increased myeloid cell infiltration. CDK4R24C antigen persistence was associated with an interferon-high and T-cell-rich tumor microenvironment, allowing for immune checkpoint inhibition as salvage therapy. Thus, the choice of target antigen determines the phenotype and immune contexture of recurrent melanomas, with implications to the design of cancer immunotherapies.
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Traslado Adoptivo/métodos , Linfocitos T CD8-positivos/trasplante , Epítopos de Linfocito T/inmunología , Melanoma/inmunología , Melanoma/terapia , Escape del Tumor/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Epítopos de Linfocito T/genética , Técnicas de Inactivación de Genes , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/citología , Células Mieloides/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Human Vγ9Vδ2 T cells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, triggering extracellular interactions with the Vγ9Vδ2 T cell receptor (TCR). Here, we examined the molecular basis of this "inside-out" triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ T cell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ T cell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ T cell and a target cell during "outside" signaling, as measured by single-cell force microscopy. Our findings provide insight into the "inside-out" triggering of Vγ9Vδ2 T cell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design.
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Antígenos CD/química , Butirofilinas/química , Activación de Linfocitos , Organofosfatos/metabolismo , Subgrupos de Linfocitos T/inmunología , Antígenos CD/metabolismo , Sitios de Unión , Butirofilinas/metabolismo , Cristalografía por Rayos X , Dimerización , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Inmunoterapia , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Procesamiento Proteico-Postraduccional , Receptores de Antígenos de Linfocitos T gamma-delta , Análisis de la Célula Individual , Relación Estructura-Actividad , Subgrupos de Linfocitos T/metabolismoRESUMEN
Two opposing descriptions of so-called mesenchymal stem cells (MSCs) exist at this time. One sees MSCs as the postnatal, self-renewing, and multipotent stem cells for the skeleton. This cell coincides with a specific type of bone marrow perivascular cell. In skeletal physiology, this skeletal stem cell is pivotal to the growth and lifelong turnover of bone and to its native regeneration capacity. In hematopoietic physiology, its role as a key player in maintaining hematopoietic stem cells in their niche and in regulating the hematopoietic microenvironment is emerging. In the alternative description, MSCs are ubiquitous in connective tissues and are defined by in vitro characteristics and by their use in therapy, which rests on their ability to modulate the function of host tissues rather than on stem cell properties. Here, I discuss how the two views developed, conceptually and experimentally, and attempt to clarify the confusion arising from their collision.
Asunto(s)
Células Madre Mesenquimatosas/citología , Animales , Células de la Médula Ósea/clasificación , Células de la Médula Ósea/citología , Huesos/citología , Antígeno CD146/análisis , Separación Celular/métodos , Tratamiento Basado en Trasplante de Células y Tejidos , Células Cultivadas , Células Clonales/citología , Tejido Conectivo/inmunología , Humanos , Inmunomodulación , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/clasificación , Ratones , Modelos Biológicos , Pericitos/citología , Células Madre Pluripotentes/citología , Quimera por Radiación , Nicho de Células Madre , Células del Estroma/clasificación , Células del Estroma/citología , Trasplante HeterotópicoRESUMEN
Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.
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Antígeno B7-H1/genética , Interferón gamma/metabolismo , ARN Largo no Codificante/genética , Anciano , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoterapia , Inmunoterapia Adoptiva/métodos , Interferón gamma/genética , Interferones/genética , Interferones/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Persona de Mediana Edad , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T CitotóxicosRESUMEN
The translational stem cell research field has progressed immensely in the past decade. Development and refinement of differentiation protocols now allows the generation of a range of cell types, such as pancreatic ß-cells and dopaminergic neurons, from human pluripotent stem cells (hPSCs) in an efficient and good manufacturing practice-compliant fashion. This has led to the initiation of several clinical trials using hPSC-derived cells to replace lost or dysfunctional cells, demonstrating evidence of both safety and efficacy. Here, we highlight successes from some of the hPSC-based trials reporting early signs of efficacy and discuss common challenges in clinical translation of cell therapies.
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Células Madre Pluripotentes , Humanos , Línea Celular , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Investigación con Células MadreRESUMEN
Population aging, a pervasive global demographic trend, is anticipated to challenge health and social systems worldwide. This phenomenon is due to medical advancements enabling longer lifespans, with 20% of the US population soon to be over 65 years old. Consequently, there will be a surge in age-related diseases. Senescence, characterized by the loss of biological maintenance and homeostasis at molecular and cellular levels, either correlates with or directly causes age-related phenotypic changes. Decline of the immune system is a critical factor in the senescence process, with cancer being a primary cause of death in elderly populations. Chimeric antigen receptor (CAR) T cell therapy, an innovative approach, has demonstrated success mainly in pediatric and young adult hematological malignancies but remains largely ineffective for diseases affecting older populations, such as late-in-life B cell malignancies and most solid tumor indications. This limitation arises because CAR T cell efficacy heavily relies on the fitness of the patient-derived starting T cell material. Numerous studies suggest that T cell senescence may be a key driver of CAR T cell deficiency. This review examines correlates and underlying factors associated with favorable CAR T cell outcomes and explores potential experimental and clinically actionable strategies for T cell rejuvenation.
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Neoplasias , Receptores de Antígenos de Linfocitos T , Adolescente , Humanos , Niño , Anciano , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T , Inmunoterapia Adoptiva , EnvejecimientoRESUMEN
Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation.