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1.
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
2.
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
3.
Nat Commun ; 10(1): 2315, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127098

RESUMO

Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Medo/fisiologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Comportamento Animal/fisiologia , Condicionamento (Psicologia)/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Modelos Animais , Neurônios/metabolismo , Técnicas de Patch-Clamp , Córtex Pré-Frontal/citologia , Técnicas Estereotáxicas
4.
Nat Commun ; 10(1): 2262, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118412

RESUMO

Most biomedical research aimed at understanding gene function uses the Cre-Lox system, which consists of the Cre recombinase-dependent deletion of genes containing LoxP sites. This system enables conditional genetic modifications because the expression and activity of the recombinase Cre/CreERT2 can be regulated in space by tissue-specific promoters and in time by the ligand tamoxifen. Since the precise Cre-Lox recombination event is invisible, methods were developed to report Cre activity and are widely used. However, numerous studies have shown that expression of a given Cre activity reporter cannot be assumed to indicate deletion of other LoxP-flanked genes of interest. Here, we report the generation of an inducible dual reporter-Cre mouse allele, iSuRe-Cre. By significantly increasing Cre activity in reporter-expressing cells, iSuRe-Cre provides certainty that these cells have completely recombined floxed alleles. This genetic tool increases the ease, efficiency, and reliability of conditional mutagenesis and gene function analysis.


Assuntos
Edição de Genes/métodos , Vetores Genéticos/genética , Integrases/genética , Plasmídeos/genética , Animais , Técnicas de Cultura de Células , Clonagem Molecular/métodos , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Recombinação Genética/efeitos dos fármacos , Tamoxifeno/farmacologia
5.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091742

RESUMO

Ceratocystis paradoxa, the causal agent of stem-bleeding disease of the coconut palm, causes great losses to the global coconut industry. As the mechanism of pathogenicity of C. paradoxa has not been determined, an exogenous gene marker was introduced into the fungus. In this study, pCT74-sGFP, which contains the green fluorescent protein (GFP) gene, and the hygromycin B resistance gene as a selective marker, was used as an expression vector. Several protoplast release buffers were compared to optimize protoplast preparation. The plasmid pCT74-sGFP was successfully transformed into the genome of C. paradoxa, which was verified using polymerase chain reaction and green fluorescence detection. The transformants did not exhibit any obvious differences from the wild-type isolates in terms of growth and morphological characteristics. Pathogenicity tests showed that the transformation process did not alter the virulence of the X-3314 C. paradoxa strain. This is the first report on the polyethylene glycol-mediated transformation of C. paradoxa carrying a 'reporter' gene GFP that was stably and efficiently expressed in the transformants. These findings provide a basis for future functional genomics studies of C. paradoxa and offer a novel opportunity to track the infection process of C. paradoxa.


Assuntos
Ascomicetos/genética , Técnicas de Transferência de Genes , Ascomicetos/patogenicidade , Cocos/microbiologia , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo
6.
Prep Biochem Biotechnol ; 49(8): 790-799, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31140364

RESUMO

The sweet-tasting protein brazzein is a candidate sugar substitute owing to its sweet, sugar-like taste and good stability. To commercialize brazzein as a sweetener, optimization of fermentation and purification procedure is necessary. Here, we report the expression conditions of brazzein in the yeast Kluyveromices lactis and purification method for maximum yield. Transformed K. lactis was cultured in YPGlu (pH 7.0) at 25 °C and induced by adding glucose:galactose at a weight ratio of 1:2 (%/%) during the stationary phase, which increased brazzein expression 2.5 fold compared to the previous conditions. Cultures were subjected to heat treatment at 80 °C for 1 h, and brazzein containing supernatant was purified using carboxymethyl-sepharose cation exchange chromatography using 50 mM NaCl in 50 mM sodium acetate buffer (pH 4.0) as a wash buffer and 400 mM NaCl (pH 7.0) for elution. The yield of purified brazzein under these conditions was 2.0-fold higher than that from previous purification methods. We also determined that the NanoOrange assay was a suitable method for quantifying tryptophan-deficient brazzein. Thus, it is possible to obtain pure recombinant brazzein with high yield in K. lactis using our optimized expression, purification, and quantification protocols, which has potential applications in the food industry.


Assuntos
Clonagem Molecular/métodos , Microbiologia Industrial/métodos , Kluyveromyces/genética , Proteínas de Plantas/genética , Edulcorantes/metabolismo , Vetores Genéticos/genética , Humanos , Kluyveromyces/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Edulcorantes/análise , Paladar , Triptofano/análise , Triptofano/genética , Triptofano/metabolismo
7.
Nat Commun ; 10(1): 1668, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971695

RESUMO

Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and autoimmunity. Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8+ T cell-mediated responses to LCMV Clone 13 viral infection. These findings indicate that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo.


Assuntos
Imunidade Adaptativa/genética , Sistemas CRISPR-Cas/genética , Técnicas de Transferência de Genes , Testes Genéticos/métodos , Imunidade Inata/genética , Animais , Transplante de Medula Óssea , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Cercopithecus aethiops , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Vetores Genéticos/genética , Genômica/métodos , Células HEK293 , Humanos , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/imunologia , RNA Guia/genética , Quimeras de Transplante , Células Vero
8.
Nat Commun ; 10(1): 1802, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996254

RESUMO

The primary cause of heart failure is the loss of cardiomyocytes in the diseased adult heart. Previously, we reported that the miR-17-92 cluster plays a key role in cardiomyocyte proliferation. Here, we report that expression of miR-19a/19b, members of the miR-17-92 cluster, is induced in heart failure patients. We show that intra-cardiac injection of miR-19a/19b mimics enhances cardiomyocyte proliferation and stimulates cardiac regeneration in response to myocardial infarction (MI) injury. miR-19a/19b protected the adult heart in two distinctive phases: an early phase immediately after MI and long-term protection. Genome-wide transcriptome analysis demonstrates that genes related to the immune response are repressed by miR-19a/19b. Using an adeno-associated virus approach, we validate that miR-19a/19b reduces MI-induced cardiac damage and protects cardiac function. Finally, we confirm the therapeutic potential of miR-19a/19b in protecting cardiac function by systemically delivering miR-19a/19b into mice post-MI. Our study establishes miR-19a/19b as potential therapeutic targets to treat heart failure.


Assuntos
Terapia Genética/métodos , Insuficiência Cardíaca/patologia , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Infarto do Miocárdio/terapia , Animais , Proliferação de Células/genética , Dependovirus/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Insuficiência Cardíaca/terapia , Ventrículos do Coração/patologia , Humanos , Injeções Intralesionais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Regeneração/genética
9.
Nat Commun ; 10(1): 1662, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971684

RESUMO

Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We introduce chromatic multiphoton serial (ChroMS) microscopy, a method integrating one-shot multicolor multiphoton excitation through wavelength mixing and serial block-face image acquisition. This approach provides organ-scale micrometric imaging of spectrally distinct fluorescent proteins and label-free nonlinear signals with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We demonstrate tridimensional (3D) multicolor imaging over several cubic millimeters as well as brain-wide serial 2D multichannel imaging. We illustrate the strengths of this method through color-based 3D analysis of astrocyte morphology and contacts in the mouse cerebral cortex, tracing of individual pyramidal neurons within densely Brainbow-labeled tissue, and multiplexed whole-brain mapping of axonal projections labeled with spectrally distinct tracers. ChroMS will be an asset for multiscale and system-level studies in neuroscience and beyond.


Assuntos
Córtex Cerebral/diagnóstico por imagem , Imagem Tridimensional/métodos , Proteínas Luminescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neuroimagem/métodos , Animais , Astrócitos/metabolismo , Córtex Cerebral/citologia , Cor , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Nestina/genética , Técnicas de Rastreamento Neuroanatômico/métodos , Parvovirinae/genética , Células Piramidais/metabolismo , Transfecção
10.
Nat Commun ; 10(1): 1634, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967552

RESUMO

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.


Assuntos
DNA Complementar/genética , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Subunidade gama Comum de Receptores de Interleucina/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Animais , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular , Códon de Iniciação/genética , Éxons/genética , Sangue Fetal/citologia , Vetores Genéticos/genética , Voluntários Saudáveis , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Mutação , Parvovirinae/genética , Cultura Primária de Células , Fatores de Tempo , Transdução Genética/métodos , Quimeras de Transplante/genética , Transplante Heterólogo/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
11.
Nat Commun ; 10(1): 1598, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962441

RESUMO

Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR-Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. Here we leverage our TRIP technology to integrate ~ 1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5' overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency.


Assuntos
Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes/métodos , RNA Guia/genética , Animais , Linhagem Celular , Análise Mutacional de DNA , Genes Reporter/genética , Loci Gênicos/genética , Vetores Genéticos/genética , Camundongos , Células-Tronco Embrionárias Murinas , Taxa de Mutação , Plasmídeos/genética
12.
Int J Mol Sci ; 20(9)2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027326

RESUMO

Skin transplantation, especially in burn patients, is still challenging because surgeons are faced with limited disposability of autologous donor side material. The in vitro culture of keratinocytes has become an important reconstructive option. However, only non-immunogenic allogenic keratinocytes offer the opportunity to develop a skin graft that can overcome rejection. The purpose of the study was to develop targeted gene modification of keratinocytes in order to reduce immunogenicity for the use as allogenic transplantable skin graft by decreasing the expression of MHC class I. To reduce MHC class I expression, viral vectors containing the US11 gene of human cytomegalovirus were generated and tested on their functionality using Western blotting, indirect immunofluorescence staining, and flow cytometry. Transfected keratinocytes were seeded on commercially available bovine collagen-elastin matrices and further cultured for histological and cell survival assays. Results showed transient down-regulation of MHC class I after 24 h post-transfection, with recovery of MHC class I expression after 48 h. Histological assessments showed long-term cell survival as well as histological patterns comparable to epidermal layers of healthy human skin. The data postulates the potential application of US11 transfected keratinocytes as an approach towards an immune-privileged skin substitute. Nevertheless, further studies and data are needed.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Queratinócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Pele Artificial , Proteínas Virais/metabolismo , Animais , Bovinos , Citomegalovirus/genética , Vetores Genéticos/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Queratinócitos/imunologia , Proteínas de Ligação a RNA/genética , Proteínas Virais/genética
13.
Prep Biochem Biotechnol ; 49(8): 759-766, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31032734

RESUMO

In recent decades, immunotoxins have attracted significant attention in treatment of a wide range of diseases including cancers due to their natural origins and their role in blocking crucial pathways within the cells. Ribosome inactivating proteins (RIPs) are efficient molecules in blocking protein synthesis through interactions with ribosomal rRNA molecules. cDNA molecule encoding HER2 scFv antibody fragment originated from trastuzumab attached to the mature alpha luffin gene fragment was subcloned into pET28a expression vector and expressed in different E. coli expression hosts. Identity of the expressed recombinant protein was investigated through western blotting and the fusion protein was purified using Ni-NTA affinity chromatography. The biological activity (toxicity) of the protein was investigated on DNA and RNA samples. A 58 kDa protein was expressed in E. coli. The best protein expression level was achieved in 0.2 mM IPTG at 30 °C in TB medium using E. coli BL21 (DE3) host strain. The fusion protein showed RNase and DNA glycosylase activity on tested RNA and DNA samples. DNA glycosylase activity of the recombinant fusion protein showed that alpha luffin part of this protein is active in conjugation to the scFv molecule and the expressed protein can be further studied in targeted biological in vitro assays.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Imunotoxinas/genética , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Anticorpos de Cadeia Única/genética , Trastuzumab/genética , Linhagem Celular , Vetores Genéticos/genética , Humanos , Imunotoxinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Anticorpos de Cadeia Única/farmacologia , Trastuzumab/farmacologia
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.
Neurosci Bull ; 35(3): 378-388, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30888608

RESUMO

Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant virus-like particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21 (baby hamster kidney) cells. Collectively, the mutant virus-like particle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.


Assuntos
Vetores Genéticos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Vírus da Floresta de Semliki/genética , Animais , Células Cultivadas , Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Células de Purkinje/citologia , Células de Purkinje/metabolismo
16.
Methods Mol Biol ; 1953: 105-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912018

RESUMO

Small molecule-induced targeted protein degradation is a powerful approach for drug target validation given its selectivity, high kinetic resolution, dose dependency, and reversibility. Out of the several methods that have been reported so far, the 12 kDa degradation tag (dTAG) system has the advantage of hijacking a degradation machinery that is ubiquitously expressed in all human tissues. Therefore it is independent of expressing additional, exogenous factors and additionally permits target validation in vivo. Here, we describe the protocol for generation and validation of clones harboring knock-in of a selectable dTAG cassette at the endogenous locus of proteins of interest using the near-haploid cell line KBM7.


Assuntos
Descoberta de Drogas/métodos , Técnicas de Introdução de Genes/métodos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular , Clonagem Molecular/métodos , Edição de Genes/métodos , Vetores Genéticos/genética , Haploidia , Humanos , Plasmídeos/genética , Proteínas/genética , Proteínas/metabolismo
17.
Methods Mol Biol ; 1953: 231-240, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912025

RESUMO

Retroviral transduction is commonly used to modulate gene expression and is a powerful approach to understand the role of a gene using gain- or loss-of-function strategies. Retroviral vectors can stably integrate non-viral genes into host genomes, providing long-term modulation of gene expression in infected cells and their progeny. Here we describe the generation of retroviral supernatants and the steps to efficiently transduce genes in innate lymphoid cell (ILC) progenitors for subsequent analysis of ILC populations in vivo.


Assuntos
Vetores Genéticos/genética , Linfócitos/metabolismo , Retroviridae/genética , Células-Tronco/metabolismo , Transdução Genética/métodos , Transferência Adotiva/métodos , Animais , Células Cultivadas , Células HEK293 , Humanos , Imunidade Inata , Linfócitos/citologia , Linfócitos/imunologia , Camundongos , Células-Tronco/citologia , Células-Tronco/imunologia
18.
Methods Mol Biol ; 1951: 15-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825141

RESUMO

Nuclear receptors (NRs) are ligand-activated transcription factors. Class 2 NRs, such as the liver X receptor (LXR)α and (LXR)ß, are typically retained in the nucleus bound to the DNA in both the presence and absence of ligand. Upon binding ligands including hydroxylated cholesterol, LXR releases corepressor proteins in exchange for coactivators resulting in target gene transcription. Activity of the LXRs therefore depends on a combination of the local ligand concentration(s) and cofactor expression, which itself is a function of cell and tissue type, mutation load, and epigenetic regulation. Cross talk with other transcription factors or signaling pathways can also alter LXR activity. The role that LXR plays in both normal physiology and disease progression is becoming increasingly apparent, and a better understanding of how and when LXR is activated or repressed is pressing biological and clinical questions.The complexity of LXR regulation makes identifying novel ligands and determining LXR activity in new cell types challenging. Generating cell lines that contain a stably integrated luciferase reporter gene with an upstream LXR-dependent promoter provides a quick, cheap, robust, efficient, and high-throughput solution to identify novel ligands and assess ligand activity in new cell types. Transplant of these stable cell culture cell lines as xenografts allows reporter activation to be assessed in vivo. Here we describe the generation of stable LXR reporter cell lines, how to confirm transgene insertion and select single cell clones, as well a method to assess transgene activity in vitro.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Receptores X do Fígado/genética , Oxisteróis/farmacologia , Análise de Dados , Dosagem de Genes , Vetores Genéticos/genética , Humanos , Ligantes , Receptores X do Fígado/metabolismo , Transdução Genética
19.
Methods Mol Biol ; 1951: 75-85, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825145

RESUMO

Macrophages are professional phagocytic cells that play key roles in innate and adaptive immunity, metabolism, and tissue homeostasis. Lipid metabolism is tightly controlled at the transcriptional level, and one of the key players of this regulation in macrophages and other cell types is the LXR subfamily of nuclear receptors (LXRα and LXRß). The use of LXR double knockout (LXR-DKO) macrophages in vitro has yielded extensive benefits in metabolism research, but this technique is hindered by primary macrophage cell expansion capability, which diminishes along terminal cell differentiation process. Here we detail a method to immortalize LXR double knockout bone marrow-derived macrophage cells at an early stage of differentiation, using a retroviral delivery of a combination of murine v-myc and v-raf oncogenes. This methodology enables the generation of autonomous self-renewing macrophages bearing an LXR-DKO genetic background, as a valuable tool for research in lipid metabolism and other LXR receptor-mediated effects.


Assuntos
Receptores X do Fígado/deficiência , Macrófagos/metabolismo , Animais , Biomarcadores , Linhagem Celular Transformada , Vetores Genéticos/genética , Receptores X do Fígado/metabolismo , Macrófagos/imunologia , Camundongos , Retroviridae/genética , Transdução Genética , Transgenes
20.
Oncol Rep ; 41(5): 2818-2832, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896879

RESUMO

Autophagy and apoptosis both promote cell death; however, the relationship between them is subtle, and they mutually promote and antagonize each other. Apoptin can induce apoptosis of various tumor cells; however, tumor cell death is not only caused by apoptosis. Whether apoptin affects tumor cell autophagy is poorly understood. Therefore, the present study aimed to explore the potential mechanisms underlying the effects of apoptin using recombinant adenoviruses expressing apoptin. Reverse transcription­quantitative polymerase chain reaction, immunoblotting, flow cytometry, fluorescence microscopy and proteomics analyses revealed that apoptin could induce autophagy in MCF­7 breast cancer cells. The results also suggested that apoptin affected autophagy in a time­ and dose­dependent manner. During the early stage of apoptin stimulation (6 and 12 h), the expression levels of autophagy pathway­associated proteins, including Beclin­1, microtubule­associated protein 1A/1B­light chain 3, autophagy­related 4B cysteine peptidase and autophagy­related 5, were significantly increased, suggesting that apoptin promoted the upregulation of autophagy in MCF­7 cells. Conversely, after 12 h of apoptin stimulation, the expression levels of apoptosis­associated proteins were decreased, thus suggesting that apoptosis may be inhibited. Therefore, it was hypothesized that apoptin may enhance autophagy and inhibit apoptosis in MCF­7 cells at the early stage. In conclusion, apoptin­induced cell death may involve both autophagy and apoptosis. The induction of autophagy may inhibit apoptosis, whereas apoptosis may inhibit autophagy; however, occasionally both pathways operate at the same time and involve apoptin. This apoptin­associated selection between tumor cell survival and death may provide a potential therapeutic strategy for breast cancer.


Assuntos
Autofagia/genética , Neoplasias da Mama/terapia , Proteínas do Capsídeo/genética , Terapia Viral Oncolítica/métodos , Adenoviridae/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Vetores Genéticos/genética , Humanos , Células MCF-7 , Vírus Oncolíticos/genética
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