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1.
Anticancer Res ; 40(1): 161-168, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892564

RESUMO

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.


Assuntos
Adenoviridae/genética , Proteínas Culina/genética , Vetores Genéticos/genética , Vírus Oncolíticos/genética , RNA Interferente Pequeno/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Terapia Viral Oncolítica , Interferência de RNA , Transdução Genética
2.
Gene ; 724: 144157, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629820

RESUMO

Cellular microRNAs are known to modulate the life-cycle of different viruses. Surprisingly, very little data exists on AAV-induced changes to the cellular microRNAome in general and in hepatic and retinal cells, in particular. We reasoned that inducible microRNA in response to recombinant AAV infection may regulate immediate and long-lived cellular responses necessary for the cell's own survival as well as its ability to control several aspects of viral life-cycle. To study this, we performed a global small RNA sequencing analysis in Adeno-associated virus (AAV) serotypes 2 and 3 infected hepatic and retinal cell models. This screen identified multiple differentially expressed microRNAs, in AAV infected Huh-7 and ARPE-19 cells. Among these, one microRNA (miR-4488) was found to be significantly down regulated (-2.24 fold for AAV2 and -3.32 fold for ARPE-19) in AAV infected cells. An enrichment and pathway analysis of miR-4488 predicted its possible effects on gene targets involved in multiple biological processes including cell-cycle regulation, endoplasmic reticulum stress response and lipid-signalling pathways. Moreover, validation studies in miR-4488 mimic or sponge transfected cells revealed modulation of these target pathways in a cell-specific manner. Further studies demonstrated that overexpression of miR-4488, modestly increased gene expression (126-128%) from AAV2 and AAV3 vectors in Huh-7 cells whereas miR-4488 inhibition in ARPE-19 cells had a similar increase (142-158%) on AAV2 or AAV3 transduction. Our results highlight that recombinant AAV mediated microRNA expression is cell-type and serotype-specific and can target specific host cellular biological pathways.


Assuntos
Dependovirus/genética , MicroRNAs/genética , Infecções por Parvoviridae/genética , Epitélio Pigmentado da Retina/virologia , Transdução Genética/métodos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Parvovirinae/genética , Reprodutibilidade dos Testes , Epitélio Pigmentado da Retina/citologia , Transgenes
3.
Adv Exp Med Biol ; 1185: 109-112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884597

RESUMO

Mutations in more than 80 genes lead to photoreceptor degeneration. Although subretinal delivery of genes to photoreceptor neurons using AAV vectors has proven itself as an efficient therapeutic and investigative tool in various mouse models, the surgical procedure itself could lead to loss of retinal function even in healthy animals, complicating the interpretation of experimental studies and requiring thoroughly designed controls. A noninvasive approach, such as a systemic delivery of genes with AAV through the bloodstream, may serve as a promising direction in tool development. Previous studies have established that AAV9 is capable of crossing the blood-brain and blood-retina barrier and even has a limited capacity to transduce photoreceptors. AAV-PHP.eB is a novel AAV9-based mutant capsid that crosses the blood-brain barrier and efficiently transduces central nervous system in the adult mice. Here, we investigated its ability to cross the blood-retina barrier and transduce retinal neurons. Control experiments demonstrated virtually nonexisting ability of this capsid to transduce retinal cells via intravitreal administration but high efficiency to transduce photoreceptors via subretinal route. Systemic delivery of AAV-PHP.eB in adult mice robustly transduced horizontal cells throughout the entire retina, but not photoreceptors. Our study suggests that AAV-PHP.eB crosses the intra-retinal blood-retinal barrier (IR-BRB), efficiently transduces horizontal cells located adjacent to IR-BRB, but has very limited ability to further penetrate retina and reach photoreceptors.


Assuntos
Barreira Hematorretiniana , Dependovirus , Técnicas de Transferência de Genes , Vetores Genéticos , Retina/citologia , Animais , Capsídeo , Camundongos , Células Fotorreceptoras , Transdução Genética
4.
Int J Nanomedicine ; 14: 8469-8481, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31695375

RESUMO

Background: A pandemic influenza viral strain, influenza A/California/07/2009 (pdmH1N1), has been considered to be a potential issue that needs to be controlled to avoid the seasonal emergence of mutated strains. Materials and methods: In this study, aptamer-antibody complementation was implemented on a multiwalled carbon nanotube-gold conjugated sensing surface with a dielectrode to detect pandemic pdmH1N1. Preliminary biomolecular and dielectrode surface analyses were performed by molecular and microscopic methods. A stable anti-pdmH1N1 aptamer sequence interacted with hemagglutinin (HA) and was compared with the antibody interaction. Both aptamer and antibody attachments on the surface as the basic molecule attained the saturation at nanomolar levels. Results: Aptamers were found to have higher affinity and electric response than antibodies against HA of pdmH1N1. Linear regression with aptamer-HA interaction displays sensitivity in the range of 10 fM, whereas antibody-HA interaction shows a 100-fold lower level (1 pM). When sandwich-based detection of aptamer-HA-antibody and antibody-HA-aptamer was performed, a higher response of current was observed in both cases. Moreover, the detection strategy with aptamer clearly discriminated the closely related HA of influenza B/Tokyo/53/99 and influenza A/Panama/2007/1999 (H3N2). Conclusion: The high performance of the abovementioned detection methods was supported by the apparent specificity and reproducibility by the demonstrated sensing system.


Assuntos
Anticorpos Antivirais/imunologia , Aptâmeros de Nucleotídeos/química , Ouro/química , Vírus da Influenza A Subtipo H1N1/imunologia , Nanotubos de Carbono/química , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pandemias , Suínos/virologia , Animais , Proteínas do Sistema Complemento/metabolismo , Eletrodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Limite de Detecção , Nanopartículas Metálicas/química , Reprodutibilidade dos Testes , Transdução Genética
5.
Anticancer Res ; 39(9): 4643-4652, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519562

RESUMO

BACKGROUND/AIM: Adenoviral-mediated expression of CD40 ligand (CD40L) on dendritic cells (DCs) activates immune check point CD40/CD40L, enhancing the immunostimulation of DCs and effector cells against human renal carcinoma cells (RCC) and inducing tumor cell apoptosis in vitro. MATERIALS AND METHODS: DCs, isolated from buffy coats from healthy donors, were transduced with adenoviruses carrying human CD40L (Ad-hCD40L). Subsequently maturation marker and cytokine expression were analyzed by fluorescence-activated cell sorting and enzyme-linked immunosorbent assay. RESULTS: Adenoviral transduction induced high expression of soluble CD40L and membrane-bound CD40L, leading to a strong CD40-CD40L interaction in DCs. Interestingly, a T-helper cell type 1 shift of expressed cytokines/chemokines was observed due to the expression of membrane-bound CD40L rather than due to soIuble CD40L alone, which significantly reduced immunoactivation of DCs. However, supernatants of Ad-hCD40L-transduced DCs induced apoptosis of RCC cells. Co-culture of Ad-hCD40L DCs with cytokine-induced killer cells led to a significant stimulation of tumor-specific cytokine-induced killer cells, with increased proliferation and cytotoxicity. CONCLUSION: Use of Ad-hCD40L-transduced DCs is a promising approach to treating RCC.


Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Carcinoma de Células Renais/metabolismo , Células Dendríticas/metabolismo , Imunomodulação , Neoplasias Renais/metabolismo , Adenoviridae/genética , Biomarcadores , Ligante de CD40/genética , Carcinoma de Células Renais/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Neoplasias Renais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética
6.
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
7.
Int J Mol Sci ; 20(18)2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31491876

RESUMO

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.


Assuntos
Complexo I de Transporte de Elétrons/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , alfa-Galactosidase/biossíntese , Linhagem Celular , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Doença de Fabry/genética , Doença de Fabry/metabolismo , Dosagem de Genes , Expressão Gênica , Humanos , Células Jurkat , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Transdução Genética , Transgenes , alfa-Galactosidase/genética
8.
EBioMedicine ; 46: 236-247, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31401194

RESUMO

BACKGROUND: Myocardial infarction (MI) is a life-threatening disease, often leading to heart failure. Defining therapeutic targets at an early time point is important to prevent heart failure. METHODS: MicroRNA screening was performed at early time points after MI using paired samples isolated from the infarcted and remote myocardium of pigs. We also examined the microRNA expression in plasma of MI patients and pigs. For mechanistic studies, AAV9-mediated microRNA knockdown and overexpression were administrated in mice undergoing MI. FINDINGS: MicroRNAs let-7a and let-7f were significantly downregulated in the infarct area within 24 h post-MI in pigs. We also observed a reduction of let-7a and let-7f in plasma of MI patients and pigs. Inhibition of let-7 exacerbated cardiomyocyte apoptosis, induced a cardiac hypertrophic phenotype, and resulted in worsened left ventricular ejection fraction. In contrast, ectopic let-7 overexpression significantly reduced those phenotypes and improved heart function. We then identified TGFBR3 as a target of let-7, and found that induction of Tgfbr3 in cardiomyocytes caused apoptosis, likely through p38 MAPK activation. Finally, we showed that the plasma TGFBR3 level was elevated after MI in plasma of MI patients and pigs. INTERPRETATION: Together, we conclude that the let-7-Tgfbr3-p38 MAPK signalling plays an important role in cardiomyocyte apoptosis after MI. Furthermore, microRNA let-7 and Tgfbr3 may serve as therapeutic targets and biomarkers for myocardial damage. FUND: Ministry of Science and Technology, National Health Research Institutes, Academia Sinica Program for Translational Innovation of Biopharmaceutical Development-Technology Supporting Platform Axis, Thematic Research Program and the Summit Research Program, Taiwan.


Assuntos
Apoptose/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Modelos Animais de Doenças , Ecocardiografia , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Camundongos , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/terapia , Miócitos Cardíacos/metabolismo , Suínos , Fatores de Tempo , Transdução Genética , Remodelação Ventricular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
PLoS Pathog ; 15(8): e1007988, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31386698

RESUMO

Adeno-associated viruses (AAV) are Dependoparvoviruses that have shown promise as recombinant vectors for gene therapy. While infectious pathways of AAV are well studied, gaps remain in our understanding of host factors affecting vector genome expression. Here, we map the role of ring finger protein 121 (RNF121), an E3 ubiquitin ligase, as a key regulator of AAV genome transcription. CRISPR-mediated knockout of RNF121 (RNF121 KO) in different cells markedly decreased AAV transduction regardless of capsid serotype or vector dose. Recombinant AAV transduction is partially rescued by overexpressing RNF121, but not by co-infection with helper Adenovirus. Major steps in the AAV infectious pathway including cell surface binding, cellular uptake, nuclear entry, capsid uncoating and second strand synthesis are unaffected. While gene expression from transfected plasmids or AAV genomes is unaffected, mRNA synthesis from AAV capsid-associated genomes is markedly decreased in RNF121 KO cells. These observations were attributed to transcriptional arrest as corroborated by RNAPol-ChIP and mRNA half-life measurements. Although AAV capsid proteins do not appear to be direct substrates of RNF121, the catalytic domain of the E3 ligase appears essential. Inhibition of ubiquitin-proteasome pathways revealed that blocking Valosin Containing Protein (VCP/p97), which targets substrates to the proteasome, can selectively and completely restore AAV-mediated transgene expression in RNF121 KO cells. Expanding on this finding, transcriptomic and proteomic analysis revealed that the catalytic subunit of DNA PK (DNAPK-Cs), a known activator of VCP, is upregulated in RNF121 KO cells and that the DNA damage machinery is enriched at sites of stalled AAV genome transcription. We postulate that a network of RNF121, VCP and DNA damage response elements function together to regulate transcriptional silencing and/or activation of AAV vector genomes.


Assuntos
Carcinoma Hepatocelular/virologia , Proteína Quinase Ativada por DNA/metabolismo , Dependovirus/genética , Genoma Viral , Proteínas de Membrana/metabolismo , Transdução Genética , Proteína com Valosina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteína Quinase Ativada por DNA/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteoma , Transcriptoma , Células Tumorais Cultivadas , Ubiquitina/metabolismo , Ubiquitinação , Proteína com Valosina/genética , Internalização do Vírus
11.
Nat Commun ; 10(1): 3760, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434885

RESUMO

Adeno-associated virus (AAV) receptor (AAVR) is an essential receptor for the entry of multiple AAV serotypes with divergent rules; however, the mechanism remains unclear. Here, we determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, revealing the molecular details by which PKD1 recognizes AAV5 and PKD2 is solely engaged with AAV1. PKD2 lies on the plateau region of the AAV1 capsid. However, the AAV5-AAVR interface is strikingly different, in which PKD1 is bound at the opposite side of the spike of the AAV5 capsid than the PKD2-interacting region of AAV1. Residues in strands F/G and the CD loop of PKD1 interact directly with AAV5, whereas residues in strands B/C/E and the BC loop of PKD2 make contact with AAV1. These findings further the understanding of the distinct mechanisms by which AAVR recognizes various AAV serotypes and provide an example of a single receptor engaging multiple viral serotypes with divergent rules.


Assuntos
Capsídeo/metabolismo , Dependovirus/fisiologia , Receptores de Superfície Celular/metabolismo , Internalização do Vírus , Capsídeo/ultraestrutura , Proteínas do Capsídeo/classificação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Dependovirus/classificação , Dependovirus/genética , Glicosilação , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador , Ligação Proteica , Conformação Proteica , Receptores de Superfície Celular/ultraestrutura , Sorogrupo , Canais de Cátion TRPP , Transdução Genética
12.
Nat Commun ; 10(1): 3806, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444345

RESUMO

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion.


Assuntos
Engenharia Genética/métodos , Interações Hospedeiro-Parasita , Malária Falciparum/parasitologia , Plasmodium falciparum/patogenicidade , Reticulócitos/parasitologia , Animais , Basigina/genética , Basigina/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Ciclofilinas/genética , Ciclofilinas/metabolismo , Eritroblastos/fisiologia , Técnicas de Inativação de Genes , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Plasmodium falciparum/metabolismo , Domínios Proteicos/genética , Proteínas de Protozoários/metabolismo , Reticulócitos/fisiologia , Transdução Genética
13.
Nat Commun ; 10(1): 3415, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363095

RESUMO

Conventional methods to discern adeno-associated virus (AAV) vector transduction patterns are based on high, stable expression of a reporter gene. As a consequence, conventionally described tropisms omit cell types that undergo transient transduction, or have low but undetectable levels of reporter expression. This creates a blind spot for AAV-based genome editing applications because only minimal transgene expression is required for activity. Here, we use editing-reporter mice to fill this void. Our approach sensitively captures both high and low transgene expression from AAV vectors. Using AAV8 and other serotypes, we demonstrate the superiority of the approach in a side-by-side comparison with traditional methods, demonstrate numerous, previously unknown sites of AAV targeting, and better predict the gene editing footprint after AAV-CRISPR delivery. We anticipate that this system, which captures the full spectrum of transduction patterns from AAV vectors in vivo, will be foundational to current and emerging AAV technologies.


Assuntos
Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos/genética , Transdução Genética , Animais , Sistemas CRISPR-Cas , Genes Reporter , Rim/virologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/virologia
14.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1307-1316, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328487

RESUMO

Gene therapy is a rapidly developing field. The most widely used technique for foreign gene transfer is lentiviral-mediated gene therapy. Lentiviral vector has been developed from the first generation to the third generation in terms of safety. The preparation of lentiviruses with high titer remains difficult. In this study, a Fibra-Cel sheet carrier was used as an HEK293T cell carrier matrix, and several sterile cell culture spinners were combined and cultured on a roller bottle machine to scale up the adherent cells. The virus titer was maximized by screening the factors to optimize the lentivirus titer in the third-generation lentivirus packaging process one by one. Fibra-Cel sheet vector was successfully used as the matrix of HEK293T cell adhesion to culture adherent cells at large scale. The optimal conditions for large-scale preparation of the third-generation lentivirus by bottle roller were screened and three batches of lentiviruses were produced on pilot scale. The production time of lentivirus was shortened from 120 hours to 54 hours from plasmid transfection to virus collection; in terms of cost, a rolling bottle machine was used instead of a bioreactor, leading to lower cost and no need for repeated sterilization during the whole process. The safe, effective and low-cost operation of successful production will provide a technical base for the large-scale preparation of lentivirus and thus lay a firm foundation for its clinical application.


Assuntos
Vetores Genéticos , Lentivirus , Células HEK293 , Humanos , Transdução Genética , Transfecção
15.
BMC Cancer ; 19(1): 658, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31272418

RESUMO

BACKGROUND: Expression of Bcr-Abl in hematopoietic stem cells is sufficient to cause chronic myeloid leukemia (CML) and tyrosine kinase inhibitors (TKI) induce molecular remission in the majority of CML patients. However, the disease driving stem cell population is not fully targeted by TKI therapy, and leukemic stem cells (LSC) capable of re-inducing the disease can persist. Single-cell RNA-sequencing technology recently identified an enriched inflammatory gene signature with TNFα and TGFß being activated in TKI persisting quiescent LSC. Here, we studied the effects of human TNFα antibody infliximab (IFX), which has been shown to induce anti-inflammatory effects in mice, combined with TKI treatment on LSC function. METHODS: We first performed GSEA-pathway analysis using our microarray data of murine LSK cells (lin-; Sca-1+; c-kit+) from the SCLtTA/Bcr-Abl CML transgenic mouse model. Bcr-Abl positive cell lines were generated by retroviral transduction. Clonogenic potential was assessed by CFU (colony forming unit). CML mice were treated with nilotinib or nilotinib plus infliximab, and serial transplantation experiments were performed. RESULTS: Likewise to human CML, TNFα signaling was specifically active in murine CML stem cells, and ectopic expression of Bcr-Abl in murine and human progenitor cell lines induced TNFα expression. In vitro exposure to human (IFX) or murine (MP6-XT22) TNFα antibody reduced clonogenic growth of CML cells. Interestingly, TNFα antibody treatment enhanced TKI-induced effects on immature cells in vitro. Additionally, in transplant and serial transplant experiments, using our transgenic CML mouse model, we could subsequently show that IFX therapy boosted TKI-induced effects and further reduced the proportion of malignant stem cells in vivo. CONCLUSION: TNFα signaling is induced in CML stem cells, and anti-inflammatory therapy enhances TKI-induced decline of LSC, confirming that successful targeting of persisting CML stem cells can be enhanced by addressing their malignant microenvironment simultaneously.


Assuntos
Anti-Inflamatórios/uso terapêutico , Infliximab/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Infliximab/farmacologia , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução Genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
PLoS Pathog ; 15(7): e1007888, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31276485

RESUMO

Temperate phages are bacterial viruses that as part of their life cycle reside in the bacterial genome as prophages. They are found in many species including most clinical strains of the human pathogens, Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Previously, temperate phages were considered as only bacterial predators, but mounting evidence point to both antagonistic and mutualistic interactions with for example some temperate phages contributing to virulence by encoding virulence factors. Here we show that generalized transduction, one type of bacterial DNA transfer by phages, can create conditions where not only the recipient host but also the transducing phage benefit. With antibiotic resistance as a model trait we used individual-based models and experimental approaches to show that antibiotic susceptible cells become resistant to both antibiotics and phage by i) integrating the generalized transducing temperate phages and ii) acquiring transducing phage particles carrying antibiotic resistance genes obtained from resistant cells in the environment. This is not observed for non-generalized transducing temperate phages, which are unable to package bacterial DNA, nor for generalized transducing virulent phages that do not form lysogens. Once established, the lysogenic host and the prophage benefit from the existence of transducing particles that can shuffle bacterial genes between lysogens and for example disseminate resistance to antibiotics, a trait not encoded by the phage. This facilitates bacterial survival and leads to phage population growth. We propose that generalized transduction can function as a mutualistic trait where temperate phages cooperate with their hosts to survive in rapidly-changing environments. This implies that generalized transduction is not just an error in DNA packaging but is selected for by phages to ensure their survival.


Assuntos
Bacteriófagos/genética , Bacteriófagos/patogenicidade , Transdução Genética , Bacteriófagos/fisiologia , Simulação por Computador , Empacotamento do DNA/genética , Farmacorresistência Bacteriana/genética , Evolução Molecular , Humanos , Lisogenia/genética , Modelos Biológicos , Prófagos/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/virologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/virologia , Virulência/genética
17.
Anticancer Res ; 39(7): 3719-3725, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262898

RESUMO

BACKGROUND: Hormone therapy and chemotherapy are not effective for castrate-resistant prostate cancer, thus development of novel treatment strategies is required. Gene therapy involving transient high-copy transfection of interleukin (IL)-24 with an adenoviral vector can exert antitumor activity; however, the effects of stable IL-24 transfection are not fully understood. The aim of this study was to investigate the effects of IL-24 overexpression in prostate cancer cells, in vitro. MATERIALS AND METHODS: DU145 cells were transfected the IL-24 gene using a retroviral vector. Apoptosis induction was investigated by the cell death detection ELISA, and the gene expression was analyzed by real time RT-PCR. RESULTS: IL-24 transduction suppressed the growth of prostate cancer and induced tumor cell apoptosis. In addition, up-regulation of epithelial markers and down-regulation of mesenchymal markers were noted, suggesting that tumor aggressiveness was reduced. CONCLUSION: Introduction of IL-24 displays antitumor activity both by induction of apoptosis and regulation of anchorage dependence.


Assuntos
Interleucinas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Apoptose , Proliferação de Células , Humanos , Masculino , Transdução Genética
18.
BMC Bioinformatics ; 20(1): 398, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315557

RESUMO

BACKGROUND: Utilization of quantitative proteomics data on the network level is still a challenge in proteomics data analysis. Currently existing models use sophisticated, sometimes hard to implement analysis techniques. Our aim was to generate a relatively simple strategy for quantitative proteomics data analysis in order to utilize as much of the data generated in a proteomics experiment as possible. RESULTS: In this study, we applied label-free proteomics, and generated a network model utilizing both qualitative, and quantitative data, in order to examine the early host response to Human Immunodeficiency Virus type 1 (HIV-1). A weighted network model was generated based on the amount of proteins measured by mass spectrometry, and analysis of weighted networks and functional sub-networks revealed upregulation of proteins involved in translation, transcription, and DNA condensation in the early phase of the viral life-cycle. CONCLUSION: A relatively simple strategy for network analysis was created and applied to examine the effect of HIV-1 on host cellular proteome. We believe that our model may prove beneficial in creating algorithms, allowing for both quantitative and qualitative studies of proteome change in various biological and pathological processes by quantitative mass spectrometry.


Assuntos
HIV-1/fisiologia , Proteômica/métodos , HIV-1/genética , Humanos , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Transdução Genética
19.
BMC Med Genet ; 20(1): 117, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262253

RESUMO

BACKGROUND: Mesenchymal stem cells (MSCs) are attractive choices in regenerative medicine and can be genetically modified to obtain better results in therapeutics. Bone development and metabolism are controlled by various factors including microRNAs (miRs) interference, which are small non-coding endogenous RNAs. METHODS: In the current study, the effects of forced miR-148b expression was evaluated on osteogenic activity. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with bicistronic lentiviral vector encoding hsa-miR-148b-3p or -5p and the enhanced green fluorescent protein. Fourteen days post-transduction, immunostaining as well as Western blotting were used to analyze osteogenesis. RESULTS: Overexpression of miR-148b-3p increased the osteogenic differentiation of human BM-MSCs as demonstrated by anenhancement of mineralized nodular formation and an increase in the levels of osteoblastic differentiation biomarkers, alkaline phosphatase and collagen type I. CONCLUSIONS: Since lentivirally overexpressed miR-148b-3p increased osteogenic differentiation capability of BM-MSCs, this miR could be applied as a therapeutic modulator to optimize bone function.


Assuntos
Medula Óssea/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Fosfatase Alcalina , Sequência de Bases , Biomarcadores , Medula Óssea/crescimento & desenvolvimento , Medula Óssea/patologia , Diferenciação Celular , Colágeno Tipo I , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Células-Tronco Mesenquimais/citologia , Transdução Genética
20.
Nucleic Acids Res ; 47(19): e114, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31361892

RESUMO

Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging-uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.


Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Lisina/análogos & derivados , Transdução Genética , Azidas/efeitos da radiação , Linhagem Celular , Terapia Genética/métodos , Vetores Genéticos/efeitos da radiação , HIV-1/genética , Humanos , Lentivirus/efeitos da radiação , Lisina/genética , Lisina/efeitos da radiação , Raios Ultravioleta , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/efeitos da radiação
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