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BACKGROUND: Adoptive cell therapy using genetically modified T cells to express chimeric antigen receptors (CAR-T) has shown encouraging results, particularly in certain blood cancers. Nevertheless, over 40% of B cell malignancy patients experience a relapse after CAR-T therapy, likely due to inadequate persistence of the modified T cells in the body. IL15, known for its pro-survival and proliferative properties, has been suggested for incorporation into the fourth generation of CAR-T cells to enhance their persistence. However, the potential systemic toxicity associated with this cytokine warrants further evaluation. METHODS: We analyzed the persistence, antitumor efficacy and potential toxicity of anti-mouse CD19 CAR-T cells which express a membrane-bound IL15-IL15Rα chimeric protein (CD19/mbIL15q CAR-T), in BALB/c mice challenged with A20 tumor cells as well as in NSG mice. RESULTS: Conventional CD19 CAR-T cells showed low persistence and poor efficacy in BALB/c mice treated with mild lymphodepletion regimens (total body irradiation (TBI) of 1 Gy). CD19/mbIL15q CAR-T exhibits prolonged persistence and enhanced in vivo efficacy, effectively eliminating established A20 B cell lymphoma. However, this CD19/mbIL15q CAR-T displays important long-term toxicities, with marked splenomegaly, weight loss, transaminase elevations, and significant inflammatory findings in some tissues. Mice survival is highly compromised after CD19/mbIL15q CAR-T cell transfer, particularly if a high TBI regimen is applied before CAR-T cell transfer. CONCLUSION: Tethered IL15-IL15Rα augments the antitumor activity of CD19 CAR-T cells but displays long-term toxicity in immunocompetent mice. Inducible systems to regulate IL15-IL15Rα expression could be considered to control this toxicity.
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Antígenos CD19 , Imunoterapia Adotiva , Interleucina-15 , Animais , Camundongos , Antígenos CD19/imunologia , Imunoterapia Adotiva/métodos , Humanos , Modelos Animais de Doenças , Linhagem Celular Tumoral , Feminino , Subunidade alfa de Receptor de Interleucina-15 , Receptores de Antígenos Quiméricos/imunologia , Linfoma/terapia , Linfoma/imunologia , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Linfócitos T/transplanteRESUMO
Hematological toxicity is a common side effect of CAR-T therapies, particularly severe in relapsed/refractory multiple myeloma (MM) patients. In this study, we analyzed a cohort of 48 patients treated with BCMA CAR-T cells to characterize the kinetics of cytopenia, identify predictive factors and determine potential mechanism underlying these toxicities. The overall incidence of cytopenia was 95.7%, and grade>3 thrombocytopenia and neutropenia, one month after infusion, was observed in 57% and 53% of the patients, being still present after one year in 4 and 3 patients respectively. Presence of cytopenia at baseline and high peak inflammatory markers highly correlated with cytopenia persisting up to three months. To determine potential mechanisms underpinning cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on HSPCs differentiation using an ex-vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPCs differentiation, promoting more immature phenotypes, with reduced expression of granulocytic, monocytic and erythroid markers, which could be prevented with a combination of IFNγ, TNFα/ß, TGFß, IL-6 and IL-17 inhibitors. Single-cell RNA-seq demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and decreased activity of key regulons involved in neutrophil and monocytic maturation (ID2, MAFB). Our results suggest that CAR-T cell activation negatively influences hematopoietic differentiation through paracrine effects inducing HSPCs maturation arrest. Moreover, our study contributes to the understanding of severe cytopenia observed after CAR-T therapy in MM and provides potential treatments to prevent or decrease its severity.
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Naive human pluripotent stem cells (hPSCs) are defined as the in vitro counterpart of the human preimplantation embryo's epiblast and are used as a model system to study developmental processes. In this study, we report the discovery and characterization of distinct cell populations coexisting with epiblast-like cells in 5iLAF naive human induced PSC (hiPSC) cultures. It is noteworthy that these populations closely resemble different cell types of the human embryo at early developmental stages. While epiblast-like cells represent the main cell population, interestingly we detect a cell population with gene and transposable element expression profile closely resembling the totipotent eight-cell (8C)-stage human embryo, and three cell populations analogous to trophectoderm cells at different stages of their maturation process: transition, early, and mature stages. Moreover, we reveal the presence of cells resembling primitive endoderm. Thus, 5iLAF naive hiPSC cultures provide an excellent opportunity to model the earliest events of human embryogenesis, from the 8C stage to the peri-implantation period.
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Embrião de Mamíferos , Células-Tronco Pluripotentes , Humanos , Desenvolvimento Embrionário/genética , Endoderma , Camadas Germinativas , Diferenciação Celular/genética , BlastocistoRESUMO
The high metabolic activity and insufficient perfusion of tumors leads to the acidification of the tumor microenvironment (TME) that may inhibit the antitumor T cell activity. We found that pharmacological inhibition of the acid loader chloride/bicarbonate anion exchanger 2 (Ae2), with 4,4'-diisothiocyanatostilbene-2,2'-disulfonicacid (DIDS) enhancedCD4+ andCD8+ T cell function upon TCR activation in vitro, especially under low pH conditions. In vivo, DIDS administration delayed B16OVA tumor growth in immunocompetent mice as monotherapy or when combined with adoptive T cell transfer of OVA-specificT cells. Notably, genetic Ae2 silencing in OVA-specificT cells improvedCD4+/CD8+ T cell function in vitro as well as their antitumor activity in vivo. Similarly, genetic modification of OVA-specificT cells to overexpress Hvcn1, a selectiveH+ outward current mediator that prevents cell acidification, significantly improved T cell function in vitro, even at low pH conditions. The adoptive transfer of OVA-specificT cells overexpressing Hvcn1 exerted a better antitumor activity in B16OVA tumor-bearingmice. Hvcn1 overexpression also improved the antitumor activity of CAR T cells specific for Glypican 3 (GPC3) in mice bearing PM299L-GPC3tumors. Our results suggest that preventing intracellular acidification by regulating the expression of acidifier ion channels such as Ae2 or alkalinizer channels like Hvcn1 in tumor-specificlymphocytes enhances their antitumor response by making them more resistant to the acidic TME.
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Linfócitos T CD8-Positivos , Imunoterapia Adotiva , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Concentração de Íons de Hidrogênio , Imunoterapia Adotiva/métodos , CamundongosRESUMO
Single-cell RNA-Sequencing has the potential to provide deep biological insights by revealing complex regulatory interactions across diverse cell phenotypes at single-cell resolution. However, current single-cell gene regulatory network inference methods produce a single regulatory network per input dataset, limiting their capability to uncover complex regulatory relationships across related cell phenotypes. We present SimiC, a single-cell gene regulatory inference framework that overcomes this limitation by jointly inferring distinct, but related, gene regulatory dynamics per phenotype. We show that SimiC uncovers key regulatory dynamics missed by previously proposed methods across a range of systems, both model and non-model alike. In particular, SimiC was able to uncover CAR T cell dynamics after tumor recognition and key regulatory patterns on a regenerating liver, and was able to implicate glial cells in the generation of distinct behavioral states in honeybees. SimiC hence establishes a new approach to quantitating regulatory architectures between distinct cellular phenotypes, with far-reaching implications for systems biology.
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Redes Reguladoras de Genes , Neoplasias , Animais , Abelhas , Regulação da Expressão Gênica , Fenótipo , Biologia de SistemasRESUMO
Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.
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BACKGROUND: One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues. METHODS: EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC. RESULTS: EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3ζ endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice. CONCLUSIONS: These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer.
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Receptores de Antígenos Quiméricos , Teratocarcinoma , Animais , Células Endoteliais , Fibronectinas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T , Teratocarcinoma/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immunotherapy with gene engineered CAR and TCR transgenic T-cells is a transformative treatment in cancer medicine. There is a rich pipeline with target antigens and sophisticated technologies that will enable establishing this novel treatment not only in rare hematological malignancies, but also in common solid tumors. The T2EVOLVE consortium is a public private partnership directed at accelerating the preclinical development of and increasing access to engineered T-cell immunotherapies for cancer patients. A key ambition in T2EVOLVE is to assess the currently available preclinical models for evaluating safety and efficacy of engineered T cell therapy and developing new models and test parameters with higher predictive value for clinical safety and efficacy in order to improve and accelerate the selection of lead T-cell products for clinical translation. Here, we review existing and emerging preclinical models that permit assessing CAR and TCR signaling and antigen binding, the access and function of engineered T-cells to primary and metastatic tumor ligands, as well as the impact of endogenous factors such as the host immune system and microbiome. Collectively, this review article presents a perspective on an accelerated translational development path that is based on innovative standardized preclinical test systems for CAR and TCR transgenic T-cell products.
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Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia , Imunoterapia Adotiva , Neoplasias/terapia , Linfócitos TRESUMO
Primary hyperoxalurias (PHs) are a group of inherited alterations of the hepatic glyoxylate metabolism. PHs classification based on gene mutations parallel a variety of enzymatic defects, and all involve the harmful accumulation of calcium oxalate crystals that produce systemic damage. These geographically widespread rare diseases have a deep impact in the life quality of the patients. Until recently, treatments were limited to palliative measures and kidney/liver transplants in the most severe forms. Efforts made to develop pharmacological treatments succeeded with the biotechnological agent lumasiran, a siRNA product against glycolate oxidase, which has become the first effective therapy to treat PH1. However, small molecule drugs have classically been preferred since they benefit from experience and have better pharmacological properties. The development of small molecule inhibitors designed against key enzymes of glyoxylate metabolism is on the focus of research. Enzyme inhibitors are successful and widely used in several diseases and their pharmacokinetic advantages are well known. In PHs, effective enzymatic targets have been determined and characterized for drug design and interesting inhibitory activities have been achieved both in vitro and in vivo. This review describes the most recent advances towards the development of small molecule enzyme inhibitors in the treatment of PHs, introducing the multi-target approach as a more effective and safe therapeutic option.
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Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5GFP reporter cardiac fibroblasts. We first established a new NKX2.5GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.
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Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.
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Terapia Genética/métodos , Vírus da Hepatite B da Marmota/imunologia , Hepatite B/veterinária , Interleucina-12/biossíntese , Interleucina-12/imunologia , Marmota/virologia , Adenoviridae/genética , Animais , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Vetores Genéticos , Hepatite B/imunologia , Interferon gama/biossíntese , Fígado/patologia , Fígado/virologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Linfócitos T/imunologia , ViremiaRESUMO
Adeno-associated viruses (AAV) have become one of the most promising tools for gene transfer in clinics. Among all the serotypes, AAV9 has been described as the most efficient for cardiac transduction. In order to achieve optimal therapeutic delivery in heart disease, we have explored AAV9 transduction efficiency in an infarcted heart using different routes of administration and promoters, including a cardiac-specific one. AAV9 vectors carrying luciferase or green fluorescence protein under the control of the ubiquitous elongation-factor-1-alpha or the cardiac-specific troponin-T (TnT) promoters were administered by intramyocardial or intravenous injection, either in healthy or myocardial-infarcted mice. The transduction efficacy and specificity, the time-course expression, and the safety of each vector were tested. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV-TnT, which was significantly higher after intramyocardial injection regardless of ischemia-induction. On the contrary, high hepatic transgene expression levels were detected with the elongation-factor-1-alpha-promoter, independently of the administration route and heart damage. Moreover, tissue-specific green fluorescence protein expression was found in cardiomyocytes with the TnT vector, whereas minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that myocardial infarction greatly increased the transcriptional activity of AAV genomes. Our findings show that the use of cardiac promoters allows for specific and stable cardiac gene expression, which is optimal and robust when intramyocardially injected. Furthermore, our data indicate that the pathological status of the tissue can alter the transcriptional activity of AAV genomes, an aspect that should be carefully evaluated for clinical applications.
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Dependovirus/genética , Isquemia Miocárdica/patologia , Animais , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Genoma Viral , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Coração/fisiologia , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/citologia , Fator 1 de Elongação de Peptídeos/metabolismo , Regiões Promotoras Genéticas , Distribuição Tecidual , Transdução Genética , Transgenes , Troponina T/metabolismoRESUMO
Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.
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Pigment epithelium derived factor (PEDF) is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF) in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng) PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.
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The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.
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Reprogramação Celular , Genoma Humano , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas Repressoras/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Células Cultivadas , Antígenos de Histocompatibilidade , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.
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Antineoplásicos/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cristalografia por Raios X , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/mortalidade , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interferons/imunologia , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos , Simulação de Acoplamento Molecular , Análise de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The ability to cryopreserve and store for long term the structure and function of therapeutic cells and tissues plays a pivotal role in clinical medicine. In fact, it is an essential pre-requisite for the commercial and clinical application of stem cells since preserves cells at low temperature and creates a reserve for future uses. This requisite may also affect the encapsulated stem cells. Several parameters should be considered on encapsulated cell cryopreservation such as the time and temperature during the cryopreservation process, or the cryoprotectant solutions used. In this study, we have compared the influence of penetrating and nonpenetrating cryoprotectants on the viability and functionality of encapsulated mesenchymal stem cells genetically modified to secrete erythropoeitin. Several cryoprotectant solutions combining DMSO, glycerol and trehalose at different concentrations were studied. Although almost no differences among the studied cryoprotectant solutions were observed on the differentiation potential of encapsulated mesenchymal stem cells, the penetrating cryoprotectant DMSO at a concentration of 10% displayed the best viability and erythropoietin secretion profile compared to the other cryoprotectant solutions. These results were confirmed after subcutaneous implantation of thawed encapsulated mesenchymal stem cells secreting erythropoeitin on Balb/c mice. The hematocrit levels of these animals increased to similar levels of those detected on animals transplanted with noncryopreserved encapsulated cells. Therefore, DMSO 10% represents the most suitable cryoprotectant solution among the solutions here studied, for encapsulated mesenchymal stem cells cryopreservation and its translation into the clinic. Similar studies should be performed for the encapsulation of other cell types before they can be translated into the clinic.
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Criopreservação , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Eritropoetina/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Transfecção , Animais , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Eritropoetina/genética , Glicerol/farmacologia , Hematócrito , Hematopoese , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Tempo , Trealose/farmacologiaRESUMO
Stem cell-derived cardiomyocytes (CMs) are often electrophysiologically immature and heterogeneous, which represents a major barrier to their in vitro and in vivo application. Therefore, the purpose of this study was to examine whether Neuregulin-1ß (NRG-1ß) treatment could enhance in vitro generation of mature "working-type" CMs from induced pluripotent stem (iPS) cells and assess the regenerative effects of these CMs on cardiac tissue after acute myocardial infarction (AMI). With that purpose, adult mouse fibroblast-derived iPS from α-MHC-GFP mice were derived and differentiated into CMs through NRG-1ß and/or dimethyl sulfoxide (DMSO) treatment. Cardiac specification and maturation of the iPS was analyzed by gene expression array, quantitative real-time polymerase chain reaction, immunofluorescence, electron microscopy, and patch-clamp techniques. In vivo, the iPS-derived CMs or culture medium control were injected into the peri-infarct region of hearts after coronary artery ligation, and functional and histology changes were assessed from 1 to 8 weeks post-transplantation. On differentiation, the iPS displayed early and robust in vitro cardiogenesis, expressing cardiac-specific genes and proteins. More importantly, electrophysiological studies demonstrated that a more mature ventricular-like cardiac phenotype was achieved when cells were treated with NRG-1ß and DMSO compared with DMSO alone. Furthermore, in vivo studies demonstrated that iPS-derived CMs were able to engraft and electromechanically couple to heart tissue, ultimately preserving cardiac function and inducing adequate heart tissue remodeling. In conclusion, we have demonstrated that combined treatment with NRG-1ß and DMSO leads to efficient differentiation of iPS into ventricular-like cardiac cells with a higher degree of maturation, which are capable of preserving cardiac function and tissue viability when transplanted into a mouse model of AMI.