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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.
Assuntos
Anemia Falciforme , Edição de Genes , Terapia Genética , Células-Tronco Hematopoéticas , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Anemia Falciforme/terapia , Anemia Falciforme/genética , Edição de Genes/métodos , Animais , Células-Tronco Hematopoéticas/metabolismo , Humanos , Feminino , Camundongos , Terapia Genética/métodos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Transplante de Células-Tronco Hematopoéticas , Globinas beta/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Reparo do DNA , Mutação , Talassemia beta/terapia , Talassemia beta/genética , Modelos Animais de Doenças , Técnicas de Transferência de GenesRESUMO
One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles.
Assuntos
Citosina , Edição de Genes , Timina , Edição de Genes/métodos , Humanos , Citosina/metabolismo , Citosina/química , Timina/metabolismo , Timina/química , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Efetores Semelhantes a Ativadores de Transcrição/genética , DNA/metabolismo , DNA/genética , Células HEK293RESUMO
Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoterless intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of CD11b and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA ex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.
Assuntos
Edição de Genes , Células-Tronco Hematopoéticas , Íntrons , Células Mieloides , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Transgenes , Animais , Edição de Genes/métodos , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Mieloides/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Diferenciação Celular/genética , Terapia Genética/métodos , Iduronidase/genética , Iduronidase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Expressão Gênica , Linhagem da Célula/genética , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Mucopolissacaridose I/terapia , Mucopolissacaridose I/genéticaRESUMO
Gain-of-function mutations in the PIK3CD gene result in activated phosphoinositide 3-kinase δ syndrome type 1 (APDS1). This syndrome is a life-threatening combined immunodeficiency and today there are neither optimal nor long-term therapeutic solutions for APDS1 patients. Thus, new alternative treatments are highly needed. The aim of the present study is to explore one therapeutic avenue that consists of the correction of the PIK3CD gene through gene editing. Our proof-of-concept shows that TALEN-mediated gene correction of the mutated PIK3CD gene in APDS1 T cells results in normalized phospho-AKT levels in basal and activated conditions. Normalization of PI3K signaling was correlated to restored cytotoxic functions of edited CD8+ T cells. At the transcriptomic level, single-cell RNA sequencing revealed corrected signatures of CD8+ effector memory and CD8+ proliferating T cells. This proof-of-concept study paves the way for the future development of a gene therapy candidate to cure activated phosphoinositide 3-kinase δ syndrome type 1.
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Adoptive cell therapy based on chimeric antigen receptor (CAR)-engineered T-cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has so far been restricted to only a few malignancies, with solid tumors proving to be especially recalcitrant to efficient therapy. Poor intra-tumor infiltration by T cells and T cell dysfunction due to a desmoplastic, immunosuppressive microenvironment are key barriers for CAR T-cell success against solid tumors. Cancer-associated fibroblasts (CAFs) are critical components of the tumor stroma, evolving specifically within the tumor microenvironment (TME) in response to tumor cell cues. The CAF secretome is a significant contributor to the extracellular matrix and a plethora of cytokines and growth factors that induce immune suppression. Together they form a physical and chemical barrier which induces a T cell-excluding 'cold' TME. CAF depletion in stroma rich solid tumors can thus provide an opportunity to convert immune evasive tumors susceptible to tumor-antigen CAR T-cell cytotoxicity. Using our TALEN-based gene editing platform we engineered non-alloreactive, immune evasive CAR T-cells (termed UCAR T-cells) targeting the unique CAF marker Fibroblast Activation Protein, alpha (FAP). In an orthotopic mouse model of triple-negative breast cancer (TNBC) composed of patient derived-CAFs and tumor cells, we demonstrate the efficacy of our engineered FAP UCAR T-cells in CAF depletion, reduction of desmoplasia and successful tumor infiltration. Furthermore, while previously resistant, pre-treatment with FAP UCAR T-cells now sensitized these tumors to Mesothelin (Meso) UCAR T-cell infiltration and anti-tumor cytotoxicity. Combination therapy of FAP UCAR, Meso UCAR T cells and the checkpoint inhibitor anti-PD-1 significantly reduced tumor burden and prolonged mice survival. Our study thus proposes a novel treatment paradigm for successful CAR T-cell immunotherapy against stroma-rich solid tumors.
Assuntos
Receptores de Antígenos Quiméricos , Animais , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Imunoterapia , Linfócitos T , Antígenos de NeoplasiasRESUMO
Resistance to potyviruses in plants has been largely provided by the selection of natural variant alleles of eukaryotic translation initiation factors (eIF) 4E in many crops. However, the sources of such variability for breeding can be limited for certain crop species, while new virus isolates continue to emerge. Different methods of mutagenesis have been applied to inactivate the eIF4E genes to generate virus resistance, but with limited success due to the physiological importance of translation factors and their redundancy. Here, we employed genome editing approaches at the base level to induce non-synonymous mutations in the eIF4E1 gene and create genetic diversity in cherry tomato (Solanum lycopersicum var. cerasiforme). We sequentially edited the genomic sequences coding for two regions of eIF4E1 protein, located around the cap-binding pocket and known to be important for susceptibility to potyviruses. We show that the editing of only one of the two regions, by gene knock-in and base editing, respectively, is not sufficient to provide resistance. However, combining amino acid mutations in both regions resulted in resistance to multiple potyviruses without affecting the functionality in translation initiation. Meanwhile, we report that extensive base editing in exonic region can alter RNA splicing pattern, resulting in gene knockout. Altogether our work demonstrates that precision editing allows to design plant factors based on the knowledge on evolutionarily selected alleles and enlarge the gene pool to potentially provide advantageous phenotypes such as pathogen resistance.
Assuntos
Potyvirus , Solanum lycopersicum , Edição de Genes , Solanum lycopersicum/genética , Fator de Iniciação 4E em Eucariotos/genética , Potyvirus/genética , Proteínas de Plantas/genética , Melhoramento Vegetal , Mutação , Doenças das Plantas/genéticaRESUMO
TALE base editors are a recent addition to the genome editing toolbox. These molecular tools are fusions of a transcription activator-like effector domain (TALE), split-DddA deaminase halves, and an uracil glycosylase inhibitor (UGI) that have the distinct ability to directly edit double strand DNA, converting a cytosine (C) to a thymine (T). To dissect the editing rules of TALE-BE, we combined the screening of dozens of TALE-BE targeting nuclear genomic loci with a medium/high throughput strategy based on precise knock-in of TALE-BE target site collections into the cell genome. This latter approach allowed us to gain in depth insight of the editing rules in cellulo, while excluding confounding factors such as epigenetic and microenvironmental differences among different genomic loci. Using the knowledge gained, we designed TALE-BE targeting CD52 and achieved very high frequency of gene knock-out (up to 80% of phenotypic CD52 knock out). We further demonstrated that TALE-BE generate only insignificant levels of Indels and byproducts. Finally, we combined two molecular tools, a TALE-BE and a TALEN, for multiplex genome engineering, generating high levels of double gene knock-out (â¼75%) without creation of translocations between the two targeted sites.
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Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive universal CAR T-cells scaffold using precise TALEN-mediated gene editing and DNA matrices vectorized by recombinant adeno-associated virus 6. We simultaneously disrupt and repurpose the endogenous TRAC and B2M loci to generate TCRαß- and HLA-ABC-deficient T-cells expressing the CAR construct and the NK-inhibitor named HLA-E. This highly efficient gene editing process enables the engineered T-cells to evade NK cell and alloresponsive T-cell attacks and extend their persistence and antitumor activity in the presence of cytotoxic levels of NK cell in vivo and in vitro, respectively. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for clinical applications.
Assuntos
Edição de Genes , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Humanos , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/genética , Linfócitos TRESUMO
The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock-in approaches using homology-directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene-edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double-stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator-like effector nuclease (TALEN)-mediated gene editing in primary T cells to improve nonviral gene knock-in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single-stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time-controlled two-step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to Ë 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T-cell receptor alpha constant region (TRAC) locus to generate 'off-the shelf' CAR-T cells.
Assuntos
Edição de Genes , Linfócitos T , Eletroporação/métodos , Edição de Genes/métodos , Mutagênese Insercional , Linfócitos T/metabolismo , TransfecçãoRESUMO
Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains. Here, an engineered transcription activator-like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN-encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5-edited cells, and genome-wide off-target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5-tropic HIV, protection in a dose-dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts.
Assuntos
Infecções por HIV , HIV-1 , Receptores CCR5 , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Resistência à Doença , Infecções por HIV/genética , Infecções por HIV/prevenção & controle , HIV-1/genética , Humanos , Receptores CCR5/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/farmacologia , Efetores Semelhantes a Ativadores de TranscriçãoRESUMO
We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.
Assuntos
Desoxirribonucleases/genética , Dependovirus/genética , Infecções Oculares/terapia , Edição de Genes/métodos , Herpes Simples/terapia , Herpesvirus Humano 1/genética , Latência Viral/genética , Animais , Sistemas CRISPR-Cas/genética , Células Cultivadas , Chlorocebus aethiops , Infecções Oculares/genética , Infecções Oculares/virologia , Feminino , Células HEK293 , Herpes Simples/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/virologia , RNA-Seq , Análise de Célula Única , Gânglio Cervical Superior/metabolismo , Gânglio Cervical Superior/virologia , Células VeroRESUMO
Here, we developed a straightforward methodology to generate TCRαß negative (allogeneic) cells for CAR-T cell therapies. With an early and transient expression of an anti-CD3 CAR in the engineered donor T cells, we programmed these cells to self-eliminate the TCR+ cell population and obtained an ultrapure TCRαß- population (99-99.9%) at the end of the CAR-T production. This novel and easy-to-implement procedure preserves the production yield and cell fitness and has the potential to streamline the manufacturing of "off-the-shelf" CAR T-cell therapies.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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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.
Assuntos
Sistema Imunitário/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Edição de Genes , Humanos , Interleucina-12/genética , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.
Assuntos
Edição de Genes/métodos , Células-Tronco Hematopoéticas/citologia , Piruvato Quinase/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Células Cultivadas , Células HEK293 , Células-Tronco Hematopoéticas/química , Humanos , CamundongosRESUMO
BACKGROUND: Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. Chimeric antigen receptor (CAR) T-cells are designed to detect and kill tumor cells that present a specific, predefined antigen. The rapid expansion of targeted antigen beyond CD19, has highlighted new challenges, such as autoactivation and T-cell fratricide, that could impact the capacity to manufacture engineered CAR T-cells. Therefore, the development of strategies to control CAR expression at the surface of T-cells and their functions is under intense investigations. RESULTS: Here, we report the development and evaluation of an off-switch directly embedded within a CAR construct (SWIFF-CAR). The incorporation of a self-cleaving degradation moiety controlled by a protease/protease inhibitor pair allowed the ex vivo tight and reversible control of the CAR surface presentation and the subsequent CAR-induced signaling and cytolytic functions of the engineered T-cells using the cell permeable Asunaprevir (ASN) small molecule. CONCLUSIONS: The strategy described in this study could, in principle, be broadly adapted to CAR T-cells development to circumvent some of the possible hurdle of CAR T-cell manufacturing. This system essentially creates a CAR T-cell with an integrated functional rheostat.
Assuntos
Antígenos CD19/imunologia , Expressão Gênica/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Isoquinolinas/farmacologia , Inibidores de Proteases/farmacologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Sulfonamidas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Clinical success of autologous CD19-directed chimeric antigen receptor T cells (CAR Ts) in acute lymphoblastic leukemia and non-Hodgkin lymphoma suggests that CAR Ts may be a promising therapy for hematological malignancies, including multiple myeloma. However, autologous CAR T therapies have limitations that may impact clinical use, including lengthy vein-to-vein time and manufacturing constraints. Allogeneic CAR T (AlloCAR T) therapies may overcome these innate limitations of autologous CAR T therapies. Unlike autologous cell therapies, AlloCAR T therapies employ healthy donor T cells that are isolated in a manufacturing facility, engineered to express CARs with specificity for a tumor-associated antigen, and modified using gene-editing technology to limit T cell receptor (TCR)-mediated immune responses. Here, transcription activator-like effector nuclease (TALEN) gene editing of B cell maturation antigen (BCMA) CAR Ts was used to confer lymphodepletion resistance and reduced graft-versus-host disease (GvHD) potential. The safety profile of allogeneic BCMA CAR Ts was further enhanced by incorporating a CD20 mimotope-based intra-CAR off switch enabling effective CAR T elimination in the presence of rituximab. Allogeneic BCMA CAR Ts induced sustained antitumor responses in mice supplemented with human cytokines, and, most importantly, maintained their phenotype and potency after scale-up manufacturing. This novel off-the-shelf allogeneic BCMA CAR T product is a promising candidate for clinical evaluation.
Assuntos
Antígeno de Maturação de Linfócitos B/imunologia , Transplante de Células/métodos , Imunoterapia Adotiva/métodos , Mieloma Múltiplo/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Animais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno de Maturação de Linfócitos B/genética , Doadores de Sangue , Linhagem Celular Tumoral , Transplante de Células/efeitos adversos , Citotoxicidade Imunológica/genética , Edição de Genes , Vetores Genéticos , Doença Enxerto-Hospedeiro/terapia , Humanos , Imunoterapia Adotiva/efeitos adversos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/patologia , Intervalo Livre de Progressão , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Rituximab/uso terapêutico , Linfócitos T/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Transdução Genética , Transplante Homólogo/métodosRESUMO
Chimeric antigen receptor T-cell (CAR T-cell) therapy has been shown to be clinically effective for managing a variety of hematological cancers. However, CAR T-cell therapy is associated with multiple adverse effects, including neurotoxicity and cytokine release syndrome (CRS). CRS arises from massive cytokine secretion and can be life-threatening, but it is typically managed with an anti-IL-6Ra mAb or glucocorticoid administration. However, these treatments add to a patient's medication burden and address only the CRS symptoms. Therefore, alternative strategies that can prevent CRS and neurotoxicity associated with CAR T-cell treatment are urgently needed. Here, we explored a therapeutic route aimed at preventing CRS rather than limiting its consequences. Using a cytokine-profiling assay, we show that granulocyte-macrophage colony-stimulating factor (GMCSF) is a key CRS-promoting protein. Through a combination of in vitro experiments and gene-editing technology, we further demonstrate that antibody-mediated neutralization or TALEN-mediated genetic inactivation of GMCSF in CAR T-cells drastically decreases available GMCSF and abolishes macrophage-dependent secretion of CRS biomarkers, including monocyte chemoattractant protein 1 (MCP-1), interleukin (IL) 6, and IL-8. Of note, we also found that the genetic inactivation of GMCSF does not impair the antitumor function or proliferative capacity of CAR T-cells in vitro We conclude that it is possible to prevent CRS by using "all-in-one" GMCSF-knockout CAR T-cells. This approach may eliminate the need for anti-CRS treatment and may improve the overall safety of CAR T-cell therapies for cancer patients.
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Citocinas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/terapia , Imunoterapia Adotiva , Monócitos , Proteínas de Neoplasias/imunologia , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacologia , Citocinas/genética , Edição de Genes , Técnicas de Silenciamento de Genes , Glucocorticoides/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Monócitos/imunologia , Monócitos/patologia , Proteínas de Neoplasias/genética , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologiaRESUMO
CAR T-cell therapies hold great promise for treating a range of malignancies but are however challenged by the complexity of their production and by the adverse events related to their activity. Here we report the development of the CubiCAR, a tri-functional CAR architecture that enables CAR T-cell detection, purification and on-demand depletion by the FDA-approved antibody Rituximab. This novel architecture has the potential to streamline the manufacturing of CAR T-cells, allow their tracking and improve their overall safety.
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Imunoterapia Adotiva , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/cirurgia , Receptores de Antígenos Quiméricos/imunologia , Rituximab/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/patologiaRESUMO
Using a TALEN-mediated gene-editing approach, we have previously described a process for the large-scale manufacturing of "off-the-shelf" CAR T cells from third-party donor T cells by disrupting the gene encoding TCRα constant chain (TRAC). Taking advantage of a previously described strategy to control TALEN targeting based on the exclusion capacities of non-conventional RVDs, we have developed highly efficient and specific nucleases targeting a key T cell immune checkpoint, PD-1, to improve engineered CAR T cells' functionalities. Here, we demonstrate that this approach allows combined TRAC and PDCD1 TALEN processing at the desired locus while eliminating low-frequency off-site processing. Thus, by replacing few RVDs, we provide here an easy and rapid redesign of optimal TALEN combinations. We anticipate that this method can greatly benefit multiplex editing, which is of key importance especially for therapeutic applications where high editing efficiencies need to be associated with maximal specificity and safety.