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1.
Cancer Immunol Immunother ; 68(11): 1891-1899, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31628525

RESUMO

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and overall survival remains poor. Chemotherapy is the standard of care for intensive induction therapy. Patients who achieve a complete remission require post-remission therapies to prevent relapse. There is no standard of care for patients with minimal residual disease (MRD), and stem cell transplantation is a salvage therapy. Considering the AML genetic heterogeneity and the leukemia immune-suppressive properties, novel cellular immune therapies to effectively harness immunological responses to prevent relapse are needed. We developed a novel modality of immune therapy consisting of monocytes reprogrammed with lentiviral vectors expressing GM-CSF, IFN-α and antigens. Preclinical studies in humanized mice showed that the reprogrammed monocytes self-differentiated into highly viable induced dendritic cells (iDCs) in vivo which migrated effectively to lymph nodes, producing remarkable effects in the de novo regeneration of T and B cell responses. For the first-in-man clinical trial, the patient's monocytes will be transduced with an integrase-defective tricistronic lentiviral vector expressing GM-CSF, IFN-α and a truncated WT1 antigen. For transplanted patients, pre-clinical development of iDCs co-expressing cytomegalovirus antigens is ongoing. To simplify the product chain for a de-centralized supply model, we are currently exploring a closed automated system for a short two-day manufacturing of iDCs. A phase I clinical trial study is in preparation for immune therapy of AML patients with MRD. The proposed cell therapy can fill an important gap in the current and foreseeable future immunotherapies of AML.


Assuntos
Antígenos de Neoplasias/metabolismo , Vetores Genéticos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunoterapia , Interferon-alfa/metabolismo , Leucemia Mieloide Aguda/terapia , Monócitos/imunologia , Células Dendríticas/imunologia , Humanos , Lentivirus/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Medicina de Precisão , Transplante de Células-Tronco
2.
Nat Med ; 25(9): 1396-1401, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501599

RESUMO

Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date1,2. Mutations in FANCA account for more than 60% of FA cases worldwide3,4. Clinically, FA is associated with congenital abnormalities and cancer predisposition. However, bone marrow failure is the primary pathological feature of FA that becomes evident in 70-80% of patients with FA during the first decade of life5,6. In this clinical study (ClinicalTrials.gov, NCT03157804 ; European Clinical Trials Database, 2011-006100-12), we demonstrate that lentiviral-mediated hematopoietic gene therapy reproducibly confers engraftment and proliferation advantages of gene-corrected hematopoietic stem cells (HSCs) in non-conditioned patients with FA subtype A. Insertion-site analyses revealed the multipotent nature of corrected HSCs and showed that the repopulation advantage of these cells was not due to genotoxic integrations of the therapeutic provirus. Phenotypic correction of blood and bone marrow cells was shown by the acquired resistance of hematopoietic progenitors and T lymphocytes to DNA cross-linking agents. Additionally, an arrest of bone marrow failure progression was observed in patients with the highest levels of gene marking. The progressive engraftment of corrected HSCs in non-conditioned patients with FA supports that gene therapy should constitute an innovative low-toxicity therapeutic option for this life-threatening disorder.


Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Adolescente , Adulto , Células da Medula Óssea/citologia , Criança , Pré-Escolar , Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatologia , Feminino , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Lentivirus/genética , Masculino , Mutação/genética , Espanha/epidemiologia , Reparo Gênico Alvo-Dirigido , Transdução Genética , Adulto Jovem
3.
Br J Anaesth ; 123(4): 439-449, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31383364

RESUMO

BACKGROUND: Nerve growth factor (NGF) has been implicated in hyperalgesia by sensitising nociceptors. A role for NGF in modulating myocardial injury through ischaemic nociceptive signalling is plausible. We examined whether inhibition of spinal NGF attenuates myocardial ischaemia-reperfusion injury and explored the underlying mechanisms. METHODS: In adult rats, lentivirus-mediated short-hairpin RNA targeted at reducing NGF gene expression (NGF-shRNA) or a transient receptor potential vanilloid 1 (TRPV1) antagonist (capsazepine) was injected intrathecally before myocardial ischaemia-reperfusion. Infarct size (expressed as the ratio of area at risk) and risk of arrhythmias were quantified. Whole-cell clamp patch electrophysiology was used to record capsaicin currents in primary dorsal root ganglion neurones. The co-expression of substance P (SP) and calcitonin gene-related peptide (CGRP), plus activation of TRPV1, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also quantified. RESULTS: NGF levels increased by 2.95 (0.34)-fold in dorsal root ganglion and 2.12 (0.27)-fold in spinal cord after myocardial ischaemia-reperfusion injury. Intrathecal injection of NGF-shRNA reduced infarct area at risk from 0.58 (0.02) to 0.37 (0.02) (P<0.01) and reduced arrhythmia score from 3.67 (0.33) to 1.67 (0.33) (P<0.01). Intrathecal capsazepine was similarly cardioprotective. NGF-shRNA suppressed expression of SP/CGRP and activation of Akt/ERK and TRPV1 in spinal cord. NGF increased capsaicin current amplitude from 144 (42) to 840 (132) pA (P<0.05), which was blocked by the TRPV1 antagonist 5'-iodoresiniferatoxin. Exogenous NGF enhanced capsaicin-induced Akt/ERK and TRPV1 activation in PC12 neuroendocrine tumour cells in culture. CONCLUSIONS: Spinal NGF contributes to myocardial ischaemia-reperfusion injury by mediating nociceptive signal transmission.


Assuntos
Terapia Genética/métodos , Lentivirus/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fator de Crescimento Neural/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Animais , Arritmias Cardíacas/prevenção & controle , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Cardiotônicos/administração & dosagem , Cardiotônicos/uso terapêutico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Injeções Espinhais , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/prevenção & controle , Fator de Crescimento Neural/biossíntese , Células PC12 , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo
4.
Prep Biochem Biotechnol ; 49(8): 822-829, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156045

RESUMO

Therapeutic monoclonal antibodies (mAbs) have become the dominant products in biopharmaceutical industry. Mammalian cell expression systems including Chinese hamster ovary (CHO) cells are the most commonly used hosts for the production of complex recombinant proteins. However, development of stable, high producing CHO cell lines suffers from the low expression level and instability of the transgene. The increasing efforts in the development of novel therapeutic antibodies and the advent of biosimilars have revealed the necessity for the development of improved platforms for rapid production of products for initial characterization and testing. In line with this premise, vector design and engineering has been applied to improve the expression level and stability of the transgene. This study reports the application of an improved lentiviral vector system containing the human interferon-ß scaffold attachment region (IFN-SAR) for the development of antibody producing stable CHO cells. mAb expressing clones producing 1100 µg/L of IgG1 monoclonal antibody were isolated without extensive screening of a large number of clones. Our results here indicate the positive effects of IFN-SAR on stable mAb expression using lentiviral based expression vectors. We also observed that although IFN-SAR can improve light chain (LC) and heavy chain (HC) gene copy numbers in stable cell pools, mAb expression in single cell clones was not affected by the transgene copy number.


Assuntos
Anticorpos Monoclonais/genética , Clonagem Molecular/métodos , Vetores Genéticos/genética , Lentivirus/genética , Animais , Células CHO , Linhagem Celular , Cricetulus , Dosagem de Genes , Humanos , Proteínas Recombinantes/genética , Transdução Genética
5.
Neuron ; 103(3): 459-472.e4, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31204083

RESUMO

Vocalizations are fundamental to mammalian communication, but the underlying neural circuits await detailed characterization. Here, we used an intersectional genetic method to label and manipulate neurons in the midbrain periaqueductal gray (PAG) that are transiently active in male mice when they produce ultrasonic courtship vocalizations (USVs). Genetic silencing of PAG-USV neurons rendered males unable to produce USVs and impaired their ability to attract females. Conversely, activating PAG-USV neurons selectively triggered USV production, even in the absence of any female cues. Optogenetic stimulation combined with axonal tracing indicates that PAG-USV neurons gate downstream vocal-patterning circuits. Indeed, activating PAG neurons that innervate the nucleus retroambiguus, but not those innervating the parabrachial nucleus, elicited USVs in both male and female mice. These experiments establish that a dedicated population of PAG neurons gives rise to a descending circuit necessary and sufficient for USV production while also demonstrating the communicative salience of male USVs. VIDEO ABSTRACT.


Assuntos
Corte , Rede Nervosa/fisiologia , Substância Cinzenta Periaquedutal/fisiologia , Vocalização Animal/fisiologia , Animais , Sinais (Psicologia) , Vias Eferentes/fisiologia , Feminino , Genes Reporter , Vetores Genéticos/genética , Lentivirus/genética , Masculino , Camundongos , Neurônios/fisiologia , Neurotransmissores/metabolismo , Optogenética , Centro Respiratório/fisiologia
6.
Gene ; 710: 265-272, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200085

RESUMO

Patients with leukocyte adhesion deficiency type 1 (LAD1) suffer from life-threatening bacterial infections due to mutations in the common ß2 integrin subunit (CD18/ITGB2 gene). We tested different fragments of the ubiquitous chromatin opening element (UCOE) from the human HNRPA2B1-CBX3 locus for their efficiency in driving the human CD18 gene expression and compared it with that of an elongation factor 1 alpha promoter (EF1αL, 1169 bp; EF1αS 248 bp) and a murine stem cell virus (MSCV) promoter within the context of the same lentiviral vector backbone. These vectors were tested in vitro for the human CD18 gene expression on the surface of CD34+ hematopoietic stem cells (HSCs) isolated from both moderate and severe LAD1 patients. Among the promoters tested in the patients' CD34+ HSCs, only U631 bp, U652 bp, U1262 bp, 5' 2.2 kb A2UCOE and EF1αS resulted in higher percentage of CD18+CD34+ cells comparable to that of the MSCV promoter. The U655 bp, U723 bp, U1296 bp, U2598 bp and EF1αL promoters resulted in comparatively lower numbers of CD18+CD34+ cells. This study would be useful in investigating the human CD18 gene expression in an ex vivo experiment to demonstrate the phenotypic correction of LAD1 in a pre-clinical model.


Assuntos
Antígenos CD18/genética , Proteínas Cromossômicas não Histona/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Lentivirus/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Fator 1 de Elongação de Peptídeos/genética , Terapia Genética , Vetores Genéticos/genética , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Síndrome da Aderência Leucocítica Deficitária/terapia , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Transdução Genética
7.
Lipids Health Dis ; 18(1): 118, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31122252

RESUMO

BACKGROUND: Fatty acid synthase (FASN) is overexpressed in most human carcinomas, including non-small cell lung cancer (NSCLC), and contributes to poor prognosis. An increasing number of studies have highlighted the potential function of FASN as both a biomarker and therapeutic target for cancers. However, the underlying molecular mechanisms of FASN in glucose metabolism and the malignant biological behavior of NSCLC remain the subjects of intensive investigation. METHODS: FASN expression was depleted by FASN-siRNA in A549 and NCI-H1299 cell lines to detect the function of glucose metabolism and the malignant biological behavior of NSCLC cells. Western-blot and qPCR were applied to determine the expressions of FASN, t-AKT, p-AKT, t-ERK, p-ERK, PKM2, HK2 and AZGP1. ATP and lactate were detected to determine the activation of glucose metabolism. CCK8 and transwell assays were used to detect the proliferation, invasion, and migration capacity of the two types of NSCLC cells. The xenograft mouse model was used to evaluate tumor weights after suppression of FASN. RESULTS: LV-FASN-siRNA and its control lentiviral vector were successfully transfected into the two types of NSCLC cells (A549 and NCI-H1299). LV-FASN siRNA significantly suppressed FASN expression in both NSCLC cell types, and expressions of p-AKT, p-ERK, PKM2, and AZGP1 were also significantly decreased. Notably, the levels of ATP and lactate were significantly decreased after transfection with LV-FASN siRNA. The proliferation of both NSCLC cell types was decreased after suppression of FASN. The invasion and migration capacity of A549, but not NCI-H1299, were inhibited following down-regulation of FASN. In vivo, inhibition of FASN caused a marked animal tumor weight loss. CONCLUSIONS: FASN was involved in glucose metabolism via down-regulation of the AKT/ERK pathway and eventually altered the malignant phenotype in lung cancer cells.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Glucose/metabolismo , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular , Ácido Graxo Sintase Tipo I/metabolismo , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos/metabolismo , Humanos , Lentivirus/genética , Neoplasias Pulmonares/genética , Camundongos Nus , Invasividade Neoplásica , Interferência de RNA , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Sensors (Basel) ; 19(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096615

RESUMO

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.


Assuntos
Técnicas Biossensoriais , Citocromo P-450 CYP1A1/isolamento & purificação , Citocromo P-450 CYP2B6/isolamento & purificação , Citocromo P-450 CYP3A/isolamento & purificação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP3A/genética , Proteínas de Fluorescência Verde/genética , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Lentivirus/genética , Taxa de Depuração Metabólica , Transgenes/genética
9.
Methods Mol Biol ; 1979: 395-406, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028650

RESUMO

The combination of single-cell RNA-seq and CRISPR allows for efficient interrogation of possibly any number of genes, only limited by the sequencing capability. Here we describe the current protocols for CRISPR screening in single cells, from cloning and virus production to generating sequencing data.


Assuntos
Sistemas CRISPR-Cas , Análise de Célula Única/métodos , Animais , Linhagem Celular , Clonagem Molecular/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Lentivirus/genética , RNA Guia/genética , Análise de Sequência de RNA/métodos , Transdução Genética/métodos , Transfecção/métodos
10.
Neurochem Res ; 44(7): 1690-1702, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31004260

RESUMO

Trigeminal neuralgia (TN) is a type of chronic neuropathic pain that is caused by peripheral nerve lesions that result from various conditions, including the compression of vessels, tumors and viral infections. MicroRNAs (miRs) are increasingly recognized as potential regulators of neuropathic pain. Previous evidence has demonstrated that miR-195 is involved in neuropathic pain, but the mechanism remains unclear. To investigate the pathophysiological role of miR-195 and Shh signaling in TN, persistent facial pain was induced by infraorbital nerve chronic constriction injury (CCI-IoN), and facial pain responses were evaluated by Von Frey hairs. qPCR and Western blotting were used to determine the relative expression of miR-195 and Patched1, the major receptor of the Sonic Hedgehog (Shh) signaling pathway, in the caudal brain stem at distinct time points after CCI-IoN. Here, we found that the expression of miR-195 was increased in a rat model of CCI-IoN. In contrast, the expression of Patched1 decreased significantly. Luciferase assays confirmed the binding of miR-195 to Patched1. In addition, the overexpression of miR-195 by an intracerebroventricular (i.c.v) administration of LV-miR-195 aggravated facial pain development, and this was reversed by upregulating the expression of Patched1. These results suggest that miR-195 is involved in the development of TN by targeting Patched1 in the Shh signaling pathway, thus regulating extracellular glutamate.


Assuntos
Proteínas Hedgehog/metabolismo , MicroRNAs/fisiologia , Receptor Patched-1/metabolismo , Transdução de Sinais/fisiologia , Neuralgia do Trigêmeo/fisiopatologia , Animais , Regulação para Baixo , Ácido Glutâmico/líquido cefalorraquidiano , Ácido Glutâmico/metabolismo , Infusões Intraventriculares , Lentivirus/genética , Masculino , MicroRNAs/administração & dosagem , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Receptor Patched-1/genética , Ratos Sprague-Dawley
11.
Int J Mol Sci ; 20(9)2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027376

RESUMO

We previously reported that upregulation of mortalin (HSPA9/GRP75), the mitochondrial HSP70 chaperone, facilitates tumor cell proliferation and survival in human medullary thyroid carcinoma (MTC), proposing mortalin as a novel therapeutic target for MTC. In this report, we show that mortalin is also upregulated in other thyroid tumor types, including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC), and that mortalin depletion can effectively induce growth arrest and cell death in human PTC (TPC-1), FTC (FTC133), and ATC (8505C and C643) cells in culture. Intriguingly, mortalin depletion induced varied effects on cell cycle arrest (G0/G1 phase arrest in TPC-1 and C643, G2/M phase arrest in 8505C, and mild G2/M phase arrest with increased sub-G0/G1 population in FTC133) and on the levels of TP53, E2F-1, p21CIP1, p27KIP1, and poly (ADP-ribose) polymerase cleavage in these cells, suggesting that thyroid tumor cells respond to mortalin depletion in a cell type-specific manner. In these cells, we also determined the efficacy of triphenyl-phosphonium-carboxy-proxyl (Mito-CP) because this mitochondria-targeted metabolism interfering agent exhibited similar tumor suppressive effects as mortalin depletion in MTC cells. Indeed, Mito-CP also induced robust caspase-dependent apoptosis in PTC and ATC cell lines in vitro, exhibiting IC50 lower than PLX4032 in 8505C cells and IC50 lower than vandetanib and cabozantinib in TPC-1 cells. Intriguingly, Mito-CP-induced cell death was partially rescued by mortalin overexpression, suggesting that Mito-CP may inactivate a mechanism that requires mortalin function. These findings support the significance of mortalin and mitochondrial activity in a broad spectrum of thyroid cancer.


Assuntos
Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Lentivirus/genética , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética
12.
J Exp Clin Cancer Res ; 38(1): 169, 2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-30999937

RESUMO

BACKGROUND: FGFR3 plays an important role in the development of bladder cancer (BCa). Hsa_circ_0068871 is a circRNA generated from several exons of FGFR3. However, the potential functional role of hsa_circ_0068871 in BCa remains largely unknown. Here we aim to evaluate the role of hsa_circ_0068871 in BCa. METHODS: We selected miR-181a-5p as the potential target miRNA of hsa_circ_0068871. The expression levels of hsa_circ_0068871 and miR-181a-5p were examined in BCa tissues and paired adjacent normal tissues by quantitative real-time PCR. To characterize the function of hsa_circ_0068871, BCa cell lines were stably infected with lentivirus targeting hsa_circ_0068871, followed by examinations of cell proliferation, migration and apoptosis. In addition, xenografts experiment in nude mice were performed to evaluate the effect of hsa_circ_0068871 in BCa. Biotinylated RNA probe pull-down assay, fluorescence in situ hybridization and luciferase reporter assay were conducted to confirm the relationship between hsa_circ_0068871, miR-181a-5p and FGFR3. RESULTS: Hsa_circ_0068871 is over-expressed in BCa tissues and cell lines, whereas miR-181a-5p expression is repressed. Depletion of has_circ_0068871 or upregulation of miR-181a-5p inhibited the proliferation and migration of BCa cells in vitro and in vivo. Mechanistically, hsa_circ_0068871 upregulated FGFR3 expression and activated STAT3 by targeting miR-181a-5p to promote BCa progression. CONCLUSIONS: Hsa_circ_0068871 regulates the miR-181a-5p/FGFR3 axis and activates STAT3 to promote BCa progression, and it may serve as a potential biomarker.


Assuntos
MicroRNAs/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Fator de Transcrição STAT3/genética , Neoplasias da Bexiga Urinária/genética , Animais , Apoptose/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Lentivirus/genética , Camundongos , RNA/genética , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Biomed Res Int ; 2019: 9540702, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31032368

RESUMO

Lentiviral vectors have been used for gene therapy in the clinical phase in recent years. These vectors provide a tool for gene insertion, deletion, or modification in organisms. The K562 human cell line has been used extensively in hematopoietic research. Despite its broad application, it is hard-to-transfection and transduction. So, this study presents a simple method to increase the transduction efficiency of K562 cells with a low multiplicity of infection (MOI) of the virus particle. For this purpose, 24-well plate was coated by 300 µl fetal bovine serum (FBS) before seeding. Then 2×104 K562 cells were seeded in each FBS coated plate. After 24h, K562 cells were attached and doubled. Different amount of lentivirus-based GFP vector according to MOI (5, 10, 15, and 20) along with 8 µg polybrene was added to the attached K562 cells and after 6h cells and viral particle complex were spinfected. Then cells were returned to the plate and incubated in 37°C overnight. After 48h transduction efficiency was established by measuring the GFP-expressing cells by flow cytometry. Flow cytometry analysis showed that, after plate treatment by FBS, 64.5% transduction rate in K562 cells was achieved at MOI=20. Therefore, this method can be an effective and simple way to increase the lentiviral transduction rate for suspended cells such as K562.


Assuntos
Técnicas de Cultura de Células/métodos , Terapia Genética , Células K562 , Lentivirus/genética , Meios de Cultura , Citometria de Fluxo , Vetores Genéticos/uso terapêutico , Humanos , Transdução Genética , Transfecção
14.
Lancet Haematol ; 6(5): e239-e253, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30981783

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy. METHODS: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32). FINDINGS: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 109 per L in one patient, 50-100 × 109 per L in five patients, and more than 100 × 109 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy. INTERPRETATION: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available. FUNDING: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Pré-Escolar , Feminino , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Itália , Masculino , Mutação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Condicionamento Pré-Transplante/métodos , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética
15.
Methods Mol Biol ; 1961: 93-109, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912042

RESUMO

Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.


Assuntos
Sistemas CRISPR-Cas/genética , RNA Guia/genética , Edição de Genes/métodos , Vetores Genéticos/genética , Lentivirus/genética
16.
Methods Mol Biol ; 1961: 307-328, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912054

RESUMO

Genome editing and knockout by virus-based delivery of CRISPR/Cas9 may provide a new option to cure inherited and acquired ocular diseases. Here we describe development and application of lentivirus-based delivery vectors enabling knockout of the Vegfa gene. We show that Streptococcus pyogenes (Sp) Cas9 and single-guide RNAs (sgRNAs) delivered by such vectors selectively can ablate the vascular endothelial growth factor A (Vegfa) gene in mouse retina following a single administration. These findings may contribute to the development of a new therapeutic path in the treatment of ocular diseases including exudative age-related macular degeneration (AMD).


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Edição de Genes , Terapia Genética , Lentivirus/genética , Camundongos , Camundongos Knockout , Retina/metabolismo , Retina/patologia
17.
Curr Protoc Neurosci ; 87(1): e66, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30883041

RESUMO

Advances in design and use of light-sensitive and light-emitting sensors have facilitated observation, measurement, and control of neuronal activities. Viruses are effective vectors for delivery of these valuable research tools to mammalian brains. Recombinant viruses are optimized to mediate regulatable, long-term, and cell-specific gene expression. Here, we describe production methods for three of the most commonly used types of recombinant viruses in neurobiology research: adeno-associated virus (AAV), retrovirus/lentivirus, and glycoprotein-deleted rabies virus. These viral constructs are frequently used for calcium imaging or to deliver neural tracers and optogenetic tools. Popular constructs are readily obtained commercially; however, customized virus production through commercial sources is time consuming and costly. This article aims to provide readers with detailed technical information for rapid production and validation of high-quality viral particles in a laboratory setting while highlighting advantages and limitations of each viral type. © 2019 by John Wiley & Sons, Inc.


Assuntos
Cálcio/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Neuroanatomia , Optogenética , Animais , Expressão Gênica/genética , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Optogenética/métodos
18.
Int J Mol Med ; 43(5): 2005-2014, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864693

RESUMO

The thoracic ossification of the posterior longitudinal ligament (T­OPLL) can cause thoracic spinal stenosis, which results in intractable myelopathy and radiculopathy. Our previous whole­genome sequencing study first reported rs199772854 in the interleukin 17 receptor C (IL17RC) gene as a potentially pathogenic loci for T­OPLL. The aim of the present study was to examine the effects of the IL17RC gene rs199772854A site mutation on osteogenesis by establishing a model of osteogenic differentiation. IL17RC gene mutation site and wild­type site mouse embryonic osteoblast (3T3­E1) models were constructed in order to induce the differentiation of the cells into osteoblasts. Whether the mutation site causes the abnormal expression of the IL17RC gene and osteogenic markers was analyzed by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis. The IL17RC gene rs199772854A site mutation was demonstrated to play a biological role through the overexpression of its own gene, and also to significantly increase the expression levels of osteogenic markers. Furthermore, the mutation upregulated the expression of the key proteins, tumor necrosis factor receptor (TNFR)­associated factor 6 (TRAF6) and nuclear factor (NF)­κB, in the interleukin (IL)­17 signaling axis. On the whole, the findings of this study suggest that the IL17RC gene rs199772854A loci mutation propels mouse embryonic osteoblasts towards osteogenic differentiation and may play an important role in the pathogenesis of T­OPLL. The IL17RC gene may promote osteogenesis through the IL­17 signaling pathway and may thus be involved in the process of ectopic osteogenesis in T­OPLL.


Assuntos
Ligamentos Longitudinais/patologia , Ossificação Heterotópica/genética , Receptores de Interleucina-17/genética , Tórax/patologia , Células 3T3 , Animais , Sequência de Bases , Diferenciação Celular , Lentivirus/genética , Camundongos , Mutação/genética , Osteogênese , Polimorfismo de Nucleotídeo Único/genética
19.
Int Immunopharmacol ; 70: 216-224, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30851701

RESUMO

OBJECTIVES: House dust mites, including Der p1, are common allergens. The current study was designed to explore the allergen-specific immune tolerance effects of Der p1-modified dendritic cells (DCs) through IL-4, IL-10 and IL-13 on an allergic rhinitis (AR) mouse model. METHODS: A lentivirus was modified to express Derp1. Then, immature DCs from mice were infected with this modified lentivirus to generate a lenti-Derp1-GFP DCs. 24 mice were random divided into four groups (n = 6 each), AR mouse were sensitized by Derp1 allergens and treated with lenti-GFP DCs (GFP-DC/AR group), or lenti-Derp1-GFP DCs (Der p1-DC/AR group) and dexamethasone (Dex/AR group), mice in the control group were treated with PBS instead of Der p1 then also intraperitoneally injected with 5 × 106 lenti-GFP DCs/mouse. AR symptoms expressed by each mouse were recorded. The proportions of CD4+CD25+Foxp3+ regulatory T cells among CD4+ T cells in the peripheral blood, and mRNA and protein expression levels of IL-4, IL-10, and IL-13 were measured. RESULTS: DCs infected with lenti-Derp1-GFP stimulated the maturation of DCs. Compared with the GFP-DC/AR group, mice in the Der p1-DC/AR group showed an ameliorated allergic response, a significant decrease in the levels of serum IgE, IgG1, and histamine, and a decrease in the expression of IL-4 and IL-13 mRNA and protein in the nasal mucosa. The expression of IL-10 increased in the Der p1-DC/AR group to a level similar to that observed in the Dex/AR group. CONCLUSIONS: These results indicate that Der p1-modified DCs have therapeutic potential for AR via downregulation of IL-4 and IL-13, and upregulation of IL-10.


Assuntos
Alérgenos/metabolismo , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/metabolismo , Cisteína Endopeptidases/metabolismo , Células Dendríticas/fisiologia , Mucosa Nasal/fisiologia , Rinite Alérgica/imunologia , Alérgenos/genética , Animais , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Células Cultivadas , Cisteína Endopeptidases/genética , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Tolerância Imunológica/genética , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Lentivirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Pyroglyphidae/imunologia
20.
Nat Commun ; 10(1): 1225, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874549

RESUMO

Although cortical interneurons are apparently well-placed to suppress seizures, several recent reports have highlighted a paradoxical role of perisomatic-targeting parvalbumin-positive (PV+) interneurons in ictogenesis. Here, we use an acute in vivo model of focal cortical seizures in awake behaving mice, together with closed-loop optogenetic manipulation of PV+ interneurons, to investigate their function during seizures. We show that photo-depolarization of PV+ interneurons rapidly switches from an anti-ictal to a pro-ictal effect within a few seconds of seizure initiation. The pro-ictal effect of delayed photostimulation of PV+ interneurons was not shared with dendrite-targeting somatostatin-positive (SOM+) interneurons. We also show that this switch can be prevented by overexpression of the neuronal potassium-chloride co-transporter KCC2 in principal cortical neurons. These results suggest that strategies aimed at improving the ability of principal neurons to maintain a trans-membrane chloride gradient in the face of excessive network activity can prevent interneurons from contributing to seizure perpetuation.


Assuntos
Interneurônios/fisiologia , Neocórtex/fisiologia , Inibição Neural/fisiologia , Convulsões/fisiopatologia , Simportadores/metabolismo , Animais , Cloretos/metabolismo , Modelos Animais de Doenças , Eletrocorticografia , Eletrodos , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Masculino , Camundongos , Neocórtex/citologia , Vias Neurais/fisiologia , Optogenética/instrumentação , Optogenética/métodos , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Estimulação Luminosa , Convulsões/diagnóstico , Somatostatina/metabolismo , Simportadores/genética
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