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2.
Anticancer Res ; 40(2): 733-741, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32014915

RESUMEN

BACKGROUND/AIM: GPR87 is a member of the cell surface molecular G protein-coupled receptors (GPCR) family and suggested to contribute to the viability of human tumor cells. Its tumor-specific expression and cell surface location make it a potential molecule for targeted therapy. In the present study, we aimed to examine the effect of silencing GPR87 expression and explore the possibility of establishing gene therapy against GPR87-overexpressing lung cancer. MATERIALS AND METHODS: Twenty malignant cell lines were investigated and GPR87-overexpressing H358 and PC9 lung cancer cells were subjected to inhibiting experiments. A short hairpin siRNA targeting the GPR87 gene was transformed into an adenoviral vector (Ad-shGPR87). Real-time RT-PCR and western blot analyses were performed to evaluate gene and protein expression. Tumors derived from human H358 cells were subcutaneously implanted in nude mice for in vivo experiments. RESULTS AND CONCLUSION: About 50% (10/20) malignant cells showed GPR87-overexpression, especially for lung cancer cells (70%, 7/10). Ad-shGPR87 effectively down-regulated the GPR87 expression, and significantly inhibited the cell proliferation in GPR87-overexpressing H358 and PC9 cells. Treatment with Ad-shGPR87 exerted a significant antitumor effect against the GPR87-expressing H358 xenografts. In addition, the gene expression of H3.3, a recently proved activator for GPR87 transcription, was positively correlated with GPR87 gene expression. Furthermore, a significant decrease of KRAS and c-Myc expression was observed in both cell lines after Ad-shGPR87 infection. In conclusion, GPR87 may play a critical role in cancer cell proliferation, and indicate its potential as a novel target for lung cancer treatment.


Asunto(s)
Terapia Genética/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , ARN Interferente Pequeño/administración & dosificación , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Adenoviridae/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Interferente Pequeño/genética , Receptores del Ácido Lisofosfatídico/biosíntesis , Receptores del Ácido Lisofosfatídico/genética , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Anticancer Res ; 40(1): 161-168, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31892564

RESUMEN

BACKGROUND: Arming of an oncolytic adenovirus (OAd) by inserting expression cassettes of therapeutic transgenes into the OAd genome is a promising approach to enhance the therapeutic effects of an OAd. Ideally, this approach would simultaneously promote the replication of an OAd in tumor cells and transgene product-mediated antitumor effects by expressing therapeutic transgenes. We previously demonstrated that knockdown of cullin 4A (CUL4A), which is an E3 ubiquitin ligase, significantly promoted adenovirus replication by increasing the c-JUN protein level. In addition, previous studies reported that CUL4A was highly expressed in various types of tumor, and was involved in tumor growth and metastasis. MATERIALS AND METHODS: In this study, we developed a novel OAd expressing a short-hairpin RNA (shRNA) against CUL4A (OAd-shCUL4A). RESULTS: OAd-shCUL4 mediated higher levels of cytotoxic effects on various types of human tumor cell than a conventional OAd. Higher levels of OAd genome copy numbers were found in the tumor cells for OAd-shCUL4A, compared with a conventional OAd. CONCLUSION: OAd-shCUL4A showed efficient antitumor effects by both enhancing OAd replication and inhibiting tumor cell growth.


Asunto(s)
Adenoviridae/genética , Proteínas Cullin/genética , Vectores Genéticos/genética , Virus Oncolíticos/genética , ARN Interferente Pequeño/genética , Animales , Línea Celular Tumoral , Supervivencia Celular/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Viroterapia Oncolítica , Interferencia de ARN , Transducción Genética
4.
Toxicol Lett ; 319: 155-159, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31706005

RESUMEN

Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min-1 x mg-1 protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B1 (EC50 < 100 nM) when compared to parental HepG2 cells (EC50∼5 µM). Thus, newly established cell lines are an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP1A2.


Asunto(s)
Citocromo P-450 CYP1A2/genética , Vectores Genéticos/genética , Hepatoblastoma/enzimología , Hepatoblastoma/genética , Lentivirus/genética , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Aflatoxina B1/toxicidad , Línea Celular Tumoral , Citocromo P-450 CYP1A2/biosíntesis , Dermatoglifia del ADN/métodos , Humanos , Hígado/metabolismo , Mycoplasma/química , Fenacetina/farmacocinética , Plásmidos/genética
5.
Prep Biochem Biotechnol ; 50(1): 47-55, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31478797

RESUMEN

Clostridium perfringens enterotoxin (CPE) has anti-prostate cancer effects and the prostate stem cell antigen (PSCA) has been used as a plasmid-based vaccine. So we expressed both of them in the PC3 cells to evaluate their effects on cell cycling and apoptosis. The PC3 cells were transfected either by the pBudCE4.1-CPE-PSCA or empty plasmid. The expression of the cpe and PSCA genes in transfected PC3 was evaluated. The apoptosis genes (Fas, P53, Bak, and Bax) as well as cell cycling genes (cyclin D1 and E) expression was evaluated by qPCR. Successful expression of cpe and PSCA in PC3 cells was confirmed. The flow cytometry results showed the cellular death rates of 62.6% and 21.8% for PC3 cells transformed with recombinant and empty plasmids respectively. Bak, Fas, Bax and P53 genes were significantly upregulated in PC3 cells transformed with pBudCE4.1-CPE-PSCA, while cyclin D1 and E were downregulated when compared with the pBudCE4.1-transfected PC3 and normal cells (p < .05). The results showed the lethal consequences of cpe and PSCA genes expression on PC3 transfected cells. Expression of the cpe and PSCA genes affects the PC3 cell death so it could be a suitable candidate for further researches in prostate cancer vaccine development.


Asunto(s)
Antígenos de Neoplasias/genética , Apoptosis , Ciclo Celular , Enterotoxinas/genética , Vectores Genéticos/genética , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/terapia , Antígenos de Neoplasias/uso terapéutico , Enterotoxinas/uso terapéutico , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/uso terapéutico , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Humanos , Masculino , Proteínas de Neoplasias/uso terapéutico , Células PC-3 , Neoplasias de la Próstata/genética , Transfección/métodos
6.
Int J Nanomedicine ; 14: 9745-9761, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31849466

RESUMEN

Introduction: Cancer gene therapy requires both effective tumor suppressor genes and safe vectors that express target genes efficiently. Inhibitor of growth 4 (ING4) inhibits tumor growth via multiple pathways. Interleukin-24 (IL-24) also has tumor-suppressive activity against a broad spectrum of human cancers. Adenovirus (Ad) vectors exhibit high infection efficiency, but potential toxicity related to high doses of adenovirus has led to careful reconsideration of their use in human clinical trials. Antheraea pernyi silk fibroin (ASF) is a cytocompatible and biodegradable natural polymer, and it possesses Arg-Gly-Asp sequences exhibiting a high binding affinity and selectivity for αvß3 and αvß5 integrin receptors, which are overexpressed in tumor vessels and most tumor cells. Methods: In this study, an Arg-Gly-Asp peptide-modified Ad vector coexpressing ING4 and IL-24 was constructed by homologous recombination of the dual gene coexpression transfer plasmid and RGD-modified pAdEasy-1 adenoviral backbone plasmid. The cationic ASF (CASF) was prepared by modifying ASF with low-molecular-weight PEI. The negatively charged Ad vector was modified with CASF to form a CASF/Ad complex. Results: Human hepatoma carcinoma SMMC-7721 cells and normal hepatic L-02 cells were infected with the CASF/Ad complex, which showed significantly higher infection efficiency than the naked Ad. The CASF/Ad complex could effectively mediate the expression of the target gene ING4 in SMMC-7721 cells and the secretion of the target gene IL-24 from SMMC-7721 cells, thus inducing apoptosis of hepatoma carcinoma SMMC-7721 cells. The viability of SMMC-7721 and L-02 cells infected with the CASF/Ad complex was further assessed, and it was found that the growth of SMMC-7721 cells was significantly inhibited but that the growth and proliferation of L-02 cells were not affected. Conclusion: The CASF/Ad complex constructed in this study, showing improved infection efficiency and enhanced suppressive effects on human hepatoma carcinoma SMMC-7721 cells, has the potential to reduce the dose of adenovirus and still maintain high infection efficiency and tumor inhibition.


Asunto(s)
Carcinoma Hepatocelular/terapia , Proteínas de Ciclo Celular/genética , Vectores Genéticos/genética , Proteínas de Homeodominio/genética , Interleucinas/genética , Neoplasias Hepáticas/terapia , Mariposas Nocturnas/química , Proteínas Supresoras de Tumor/genética , Adenoviridae/genética , Animales , Apoptosis/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Cationes , Línea Celular Tumoral , Fibroínas/química , Fibroínas/genética , Genes Supresores de Tumor , Terapia Genética/métodos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología
7.
World J Microbiol Biotechnol ; 35(11): 175, 2019 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-31673852

RESUMEN

The important metabolic intermediate 5-aminolevulinic acid (ALA) is useful for cancer treatment or plant growth regulation and has consequently received much attention. In this study, we introduced the HemA1 and pgr7 genes from the higher plant Arabidopsis thaliana into recombinant Escherichia coli to overproduce extracellular 5-aminolevulinic acid via the C5 pathway. In the E. coli BL21 (DE3) strain background, the ALA concentration of the strain expressing both HemA1 and pgr7 was the highest and reached 3080.62 mg/L. Among the 7 tested hosts, ALA production was the highest in E. coli Transetta (DE3). In E. coli Transetta GTR/GBP, the expression levels of zwf, gnd, pgl and RhtA were upregulated. Glutamate induced the expression of the GltJ, GltK, GltL and GltS genes that are in involved in glutamate uptake. The recombinant E. coli Transetta GTR/GBP was able to produce 7642 mg/L ALA in modified minimal medium supplemented with 10 g/L glutamate and 15 g/L glucose after 48 h of fermentation at 22 °C. The results provide persuading evidence for the efficient production of ALA from glucose and glutamate in E. coli expressing A. thaliana HemA1 and pgr7. Further optimization of the fermentation process should be done to improve the ALA production to an industrially relevant level.


Asunto(s)
Aldehído Oxidorreductasas/genética , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Escherichia coli/genética , Ácido Glutámico/metabolismo , Ácidos Levulínicos/metabolismo , Proteínas de la Membrana/genética , Aldehído Oxidorreductasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Escherichia coli/enzimología , Fermentación , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/genética , Glucosa/metabolismo , Proteínas de la Membrana/metabolismo , Vía de Pentosa Fosfato , Proteínas Recombinantes
8.
Nat Protoc ; 14(12): 3538-3553, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31748752

RESUMEN

The cellular machinery regulating microRNA biogenesis and maturation relies on a small number of simple steps and minimal biological requirements and is broadly conserved in all eukaryotic cells. The same holds true in disease. This allows for a substantial degree of freedom in the engineering of transgenes capable of simultaneously expressing multiple microRNAs of choice, allowing a more comprehensive modulation of microRNA landscapes, the study of their functional interaction, and the possibility of using such synergism for gene therapy applications. We have previously engineered a transgenic cluster of functionally associated microRNAs to express a module of suppressed microRNAs in brain cancer for therapeutic purposes. Here, we provide a detailed protocol for the design, cloning, delivery, and utilization of such artificial microRNA clusters for gene therapy purposes. In comparison with other protocols, our strategy effectively decreases the requirements for molecular cloning, because the nucleic acid sequence encoding the combination of the desired microRNAs is designed and validated in silico and then directly synthesized as DNA that is ready for subcloning into appropriate delivery vectors, for both in vitro and in vivo use. Sequence design and engineering require 4-5 h. Synthesis of the resulting DNA sequence requires 4-6 h. This protocol is quick and flexible and does not require special laboratory equipment or techniques, or multiple cloning steps. It can be easily executed by any graduate student or technician with basic molecular biology knowledge.


Asunto(s)
Ingeniería Genética/métodos , Terapia Genética/métodos , MicroARNs/síntesis química , Animales , Clonación Molecular/métodos , Vectores Genéticos/genética , Humanos , MicroARNs/genética , Transgenes/genética
9.
World J Gastroenterol ; 25(39): 5961-5972, 2019 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-31660033

RESUMEN

BACKGROUND: Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research. AIM: To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies. METHODS: We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene. RESULTS: The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity. CONCLUSION: Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.


Asunto(s)
Antivirales/farmacología , Vectores Genéticos/genética , Virus de la Hepatitis B/genética , Luciferasas/genética , Replicación Viral/efectos de los fármacos , Línea Celular , Genes Reporteros/genética , Células Hep G2 , Humanos , Lamivudine/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Plásmidos/genética , ARN/genética , ARN Viral/genética , Transfección/métodos , Transgenes/genética , Replicación Viral/genética
10.
J Immunol Res ; 2019: 3616120, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31565660

RESUMEN

Immune cell therapy has emerged as a promising approach to treat malignancies that were up until recently only treated on a palliative basis. Chimeric antigen receptor- (CAR-) modified T lymphocytes (T cells) in particular have proven to be very effective for certain hematological malignancies. The production of CAR T cells usually involves viral transduction and ex vivo culture of T cells. The aim of this study was to explore the use of human platelet lysate (HPL) compared to two commonly used supplements, human AB serum (ABS) and fetal bovine serum (FBS), for modified T cell production. For studying transduction, activated T cells were transduced with lentivirus to deliver GFP transgenes with three different promoters. Transduction efficiency (percent GFP) was similar among the supplements, and a modest increase in the transgene product (mean fluorescence intensity) was observed when HPL was used as a supplement compared to ABS. To study the effect of supplements on expansion, peripheral blood mononuclear cells (PBMCs) were activated and expanded in the presence of interleukin 2 (IL2) for fourteen days. T cell expansions using HPL and ABS were comparable and slightly less than the expansion obtained with FBS. Interestingly, cells expanded in media supplemented with HPL showed a higher percentage of T cells with a central memory phenotype compared to those expanded in ABS or FBS. Protein profiling revealed that the phenotypic differences may be explained by elevated levels of several cytokines in HPL, including IL7. The results suggest that the use of HPL as a cell culture supplement during the production of modified T cells is a reasonable alternative to ABS. Furthermore, the use of HPL may enhance in vivo performance of the final product by enriching for central memory T cells that are associated with long-term persistence following adoptive transfer.


Asunto(s)
Plaquetas/metabolismo , Medios de Cultivo Condicionados/farmacología , Memoria Inmunológica/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Técnicas de Cultivo de Célula , Biología Computacional/métodos , Citocinas/metabolismo , Expresión Génica , Ontología de Genes , Genes Reporteros , Vectores Genéticos/genética , Humanos , Lentivirus/genética , Proteoma , Proteómica , Linfocitos T/metabolismo , Transducción Genética , Transgenes
11.
Nat Commun ; 10(1): 4543, 2019 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-31586074

RESUMEN

Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models.


Asunto(s)
Linfocitos B/patología , Linfoma de Células B Grandes Difuso/genética , Cultivo Primario de Células/métodos , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular/genética , Técnicas de Cocultivo/métodos , Vectores Genéticos/genética , Centro Germinal/citología , Ensayos Analíticos de Alto Rendimiento , Humanos , Linfoma de Células B Grandes Difuso/patología , Ratones , Clasificación del Tumor , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-myc/genética , Retroviridae/genética , Transducción Genética , Ensayos Antitumor por Modelo de Xenoinjerto
12.
Nat Commun ; 10(1): 4524, 2019 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-31586094

RESUMEN

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generate cone photoreceptors from human induced pluripotent stem cells, expressing the chloride pump Jaws. After transplantation into blind mice, we observe light-driven responses at the photoreceptor and ganglion cell levels. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.


Asunto(s)
Ingeniería Celular/métodos , Optogenética/métodos , Células Fotorreceptoras de Vertebrados/trasplante , Degeneración Retiniana/terapia , Animales , Animales Recién Nacidos , Técnicas de Cultivo de Célula/métodos , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/genética , Células HEK293 , Halorrodopsinas/genética , Humanos , Células Madre Pluripotentes Inducidas , Masculino , Ratones , Ratones Noqueados , Degeneración Retiniana/genética , Rodopsina/genética , Transfección , Resultado del Tratamiento
13.
Nat Commun ; 10(1): 4537, 2019 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-31586095

RESUMEN

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis.


Asunto(s)
Distrofina/genética , Terapia Genética/métodos , Microscopía Intravital/métodos , Músculo Esquelético/diagnóstico por imagen , Distrofia Muscular de Duchenne/terapia , Animales , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Distrofina/metabolismo , Exones/genética , Edición Génica/métodos , Genes Reporteros/genética , Vectores Genéticos/química , Vectores Genéticos/genética , Humanos , Luciferasas/química , Luciferasas/genética , Mediciones Luminiscentes , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/diagnóstico por imagen , Distrofia Muscular de Duchenne/genética , Mutación , Resultado del Tratamiento
14.
Int J Mol Sci ; 20(19)2019 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-31590403

RESUMEN

G-protein-coupled receptors associate into dimers/oligomers whose function is not well understood. One approach to investigate this issue is to challenge oligomerization by peptides replicating transmembrane domains and to study their effect on receptor functionality. The disruptor peptides are typically delivered by means of cell-penetrating vectors such as the human immunodeficiency virus (HIV) transcription trans-activation protein Tat. In this paper we report a cyclic, Tat-like peptide that significantly improves its linear analogue in targeting interreceptor sequences in the transmembrane space. The same cyclic Tat-like vector fused to a transmembrane region not involved in receptor oligomerization was totally ineffective. Besides higher efficacy, the cyclic version has enhanced proteolytic stability, as shown by trypsin digestion experiments.


Asunto(s)
Productos del Gen tat/metabolismo , Péptidos Cíclicos/metabolismo , Receptor de Adenosina A2A/metabolismo , Productos del Gen tat/genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Péptidos Cíclicos/genética , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica
15.
Nat Commun ; 10(1): 4479, 2019 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-31578323

RESUMEN

Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level ß-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented ß-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and ß-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders.


Asunto(s)
Anemia de Células Falciformes/terapia , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Lentivirus/genética , Globinas beta/genética , Anemia de Células Falciformes/genética , Animales , Antígenos CD34/metabolismo , Vectores Genéticos/genética , Humanos , Macaca mulatta , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Reproducibilidad de los Resultados , Trasplante Heterólogo , Globinas beta/metabolismo
16.
Nat Protoc ; 14(11): 3205-3219, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31628446

RESUMEN

To understand and control complex tissues, the ability to genetically manipulate single cells is required. However, current delivery methods for the genetic engineering of single cells, including viral transduction, suffer from limitations that restrict their application. Here we present a protocol that describes a versatile technique that can be used for the targeted viral infection of single cells or small groups of cells in any tissue that is optically accessible. First, cells of interest are selected using optical microscopy. Second, a micropipette-loaded with magnetic nanoparticles to which viral particles are bound-is brought into proximity of the cell of interest, and a magnetic field is applied to guide the viral nanoparticles into cellular contact, leading to transduction. The protocol, exemplified here by stamping cultured neurons with adeno-associated viruses (AAVs), is completed in a few minutes and allows stable transgene expression within a few days, at success rates that approach 80%. We outline how this strategy is applied to single-cell infection in complex tissues, and is feasible both in organoids and in vivo.


Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Magnetismo/métodos , Nanopartículas de Magnetita , Animales , Células Cultivadas , Vectores Genéticos/administración & dosificación , Campos Magnéticos , Nanopartículas de Magnetita/administración & dosificación , Neuronas/metabolismo , Ratas , Transducción Genética , Transgenes
17.
Int J Mol Sci ; 20(19)2019 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-31623314

RESUMEN

We aimed to immortalize primarily isolated human deciduous tooth-derived dental pulp cells (HDDPCs) by transfection with piggyBac (PB)-based transposon vectors carrying E7 from human papilloma virus 16 or complementary DNA (cDNA) encoding human telomerase reverse transcriptase (hTERT). HDDPCs were co-transfected with pTrans (conferring PB transposase expression) + pT-pac (conferring puromycin acetyltransferase expression) + pT-tdTomato (conferring tdTomato cDNA expression) and pT-E7 (conferring E7 expression) or pTrans + pT-pac + pT-EGFP (conferring enhanced green fluorescent protein cDNA expression) + pT-hTERT (conferring hTERT expression). After six days, these cells were selected in medium containing 5 µg/mL puromycin for one day, and then cultured in normal medium allowing cell survival. All resultant colonies were harvested and propagated as a pool. Stemness and tumorigenic properties of the established cell lines ("MT_E7" for E7 and "MT_hTERT" for hTERT) with untransfected parental cells (MT) were examined. Both lines exhibited proliferation similar to that of MT, with alkaline phosphatase activity and stemness-specific factor expression. They displayed differentiation potential into multi-lineage cells with no tumorigenic property. Overall, we successfully obtained HDDPC-derived immortalized cell lines using a PB-based transfection system. The resultant and parental cells were indistinguishable. Thus, E7 and hTERT could immortalize HDDPCs without causing cancer-associated changes or altering phenotypic properties.


Asunto(s)
Diferenciación Celular , Elementos Transponibles de ADN , Pulpa Dental/citología , Células Madre/citología , Células Madre/metabolismo , Diferenciación Celular/genética , Línea Celular Transformada , Transformación Celular Neoplásica , Femenino , Vectores Genéticos/genética , Humanos , Proteínas Oncogénicas Virales/genética , Células Madre/patología , Telomerasa/genética , Telomerasa/metabolismo , Diente Primario , Transfección
18.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-31569601

RESUMEN

BACKGROUND: The goal of this study was to determine if adenovirus-delivered LOXL2 protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action; and determine if the overexpression of LOXL2 in transgenic mice can protect against the development of OA-related cartilage damage and joint disability. METHODS: Four-month-old Cho/+ male and female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes were examined by immunostaining and qRT-PCR. The effect of LOXL2 expression on signaling was tested via the pro-inflammatory cytokine IL1ß in the cartilage cell line ATDC5. Finally; the OA by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and assessment of allodynia. RESULTS: The adenovirus treatment upregulated LOXL2; Sox9; Acan and Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection; but not the empty vector injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression. LOXL2 attenuated IL-1ß-induced phospho-NF-κB/p65 and rescued chondrogenic lineage-related genes in ATDC5 cells; demonstrating one potential protective mechanism. LOXL2 attenuated phospho-NF-κB independent of its enzymatic activity. Finally; LOXL2-overexpressing transgenic mice were protected from MIA-induced OA-related functional changes; including the time and distance traveled on the treadmill and allodynia. CONCLUSION: Our study demonstrates that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.


Asunto(s)
Envejecimiento/genética , Aminoácido Oxidorreductasas/genética , Osteoartritis de la Rodilla/etiología , Osteoartritis de la Rodilla/patología , Adenoviridae/genética , Aminoácido Oxidorreductasas/metabolismo , Animales , Artritis Experimental , Cartílago Articular/metabolismo , Cartílago Articular/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Interleucina-1beta/metabolismo , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Osteoartritis de la Rodilla/metabolismo , Transducción Genética
19.
Molecules ; 24(19)2019 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-31569777

RESUMEN

Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0-9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s-1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.


Asunto(s)
Codón , Endo-1,4-beta Xilanasas/genética , Regulación de la Expresión Génica , Penicillium/enzimología , Penicillium/genética , Pichia/genética , Proteínas Recombinantes , Secuencia de Bases , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/metabolismo , Activación Enzimática , Vectores Genéticos/genética , Concentración de Iones de Hidrógeno , Temperatura Ambiental , Termodinámica
20.
Nat Med ; 25(9): 1396-1401, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31501599

RESUMEN

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.


Asunto(s)
Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Adolescente , Adulto , Células de la Médula Ósea/citología , Niño , Preescolar , Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatología , Femenino , Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Lactante , Lentivirus/genética , Masculino , Mutación/genética , España/epidemiología , Reparación del Gen Blanco , Transducción Genética , Adulto Joven
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