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1.
Viruses ; 12(9)2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32927639

RESUMO

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Lentivirus/genética , Testes de Neutralização/métodos , Pandemias , Pneumonia Viral/virologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Linhagem Celular , Infecções por Coronavirus/epidemiologia , Humanos , Soros Imunes/imunologia , Itália/epidemiologia , Plasmídeos/genética , Pneumonia Viral/epidemiologia , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/biossíntese , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/fisiologia , Transfecção , Vesiculovirus/genética , Carga Viral
2.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1395-1404, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748597

RESUMO

By inserting microRNAs into the intron of EF1α promoter, we constructed a novel lentiviral vector knocking down PD-1 gene via microRNA and applied it to CAR-T cells. Lentiviral transduction efficiency and PD-1-silencing efficiency were detected by flow cytometry. PD-1 expression was detected by Western blotting. Relative expression of microRNA was measured by Q-PCR. Cytotoxicity of CAR-T cells based on this vector was tested by luciferase bioluminescence and flow cytometry. Compared with lentiviral vector with microRNA transcribed by U6 promotor, the transduction efficiency of lentiviral vector with microRNA which was inserted into the intron of EF1α promoter was more significant, and the knockdown rate of PD-1 was more than 90%, which was validated by flow cytometry and Western blotting. And the relative expression level of microRNA in Jurkat cells transduced with this novel lentiviral vector was shown by Q-PCR. Compared with normal CAR-T cells, CAR-T cells based on this vector showed stronger cytotoxicity against PD-L1 positive Raji cells. We successfully constructed a novel lentiviral vector that knocked down PD-1 via microRNA and verified the superiority of its transduction efficiency and knockdown efficiency of PD-1. CAR-T cells based on this vector can exert a more powerful cytotoxicity, thus providing theoretical support for the subsequent treatment of PD-L1 positive tumors.


Assuntos
Vetores Genéticos , Lentivirus , MicroRNAs , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Humanos , Lentivirus/genética , MicroRNAs/metabolismo , Receptor de Morte Celular Programada 1 , Regiões Promotoras Genéticas/genética
3.
Wei Sheng Yan Jiu ; 49(3): 467-472, 2020 May.
Artigo em Chinês | MEDLINE | ID: mdl-32693899

RESUMO

OBJECTIVE: To investigate the role of peroxisome proliferator-activated receptor alpha(PPARα) in perfluorododecanoic acid(PFDoA)-induced liver oxidative damage in rats by observing lentivirus-mediated shRNA targeting and down-regulating PPARα expression in rat hepatocytes BRL 3 A. METHODS: A PPARα lentivirus-compatible shRNA interference vector Lenti-iPα and a negative control vector Lenti-NC were constructed, and co-transfected with lentivirus packaging helper plasmids into 293 FT cells for lentivirus packaging. The lentivirus stock solution was collected, concentrated and the virus titer was determined. The experimental grouping was as follows, NC-group(infected with negative control lentivirus, without PFDoA exposure), NC+ group(infected with negative control lentivirus, 75 µmol/L PFDoA exposure), iPα-group(infected with interference lentivirus, without PFDoA exposure), iPα+ group(infected with interference lentivirus, 75 µmol/L PFDoA exposure). Rat hepatocytes BRL 3 A cells were treated with lentivirus for 96 h, and then exposed with 75 µmol/L PFDoA in the NC+ group and iPα+ group in the last 24 h. The interference of PPARα in BRL 3 A cells and the role of PPARα in reactive oxygen species(ROS) changes caused by PFDoA were observed. RESULTS: Lentivirus-mediated shRNA successfully achieved targeted downregulation of PPARα expression in BRL 3 A cells. Compared with the NC-group, the mean fluorescence intensity of ROS in rat hepatocytes BRL 3 A in the iPα-group was 12043. 42±808. 58, significantly increased(P<0. 05); The transcription levels of acyl-CoA thioesterases(Acot) 1 gene and its protein expression levels were 0. 43±0. 04 and 0. 34±0. 08, respectively, both significantly decreased(P<0. 05). After PFDoA treatment, compared with NC+ group, the mean fluorescence intensity of ROS in iPα+ group was 12386. 25±356. 36, which also increased significantly(P<0. 05). The transcription levels of Acot1 gene and its protein expression levels were 0. 85±0. 10 and 0. 33±0. 04, respectively, which also decreased significantly(P<0. 05). CONCLUSION: PPARα and its downstream target protein Acot1 may play a role in scavenging ROS in rat hepatocytes BRL 3 A, keeping hepatocytes from oxidative damage caused by foreign substances to the liver.


Assuntos
Lentivirus/genética , PPAR alfa , Animais , Regulação para Baixo , Hepatócitos , Ácidos Láuricos , Estresse Oxidativo , RNA Interferente Pequeno , Ratos
4.
PLoS One ; 15(7): e0232915, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706785

RESUMO

Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for B cell malignancies, with emerging potential for the treatment of other hematologic cancers and solid tumors. The strength of the promoter within the CAR cassette will alter CAR-polypeptide levels on the cell surface of the T cell-impacting on the kinetics of activation, survival and memory cell formation in T cells. In addition to the CAR, promoters can be used to drive other genes of interest to enhance CAR T cell function. Expressing multiple genes from a single RNA transcript can be effectively achieved by linking the genes via a ribosomal skip site. However, promoters may differ in their ability to transcribe longer RNAs, or could interfere with lentiviral production, or transduction frequencies. In this study we compared the ability of the strong well-characterized promoters CMV, EF-1, hPGK and RPBSA to drive functional expression of a single RNA encoding three products: GFP, CAR, plus an additional cell-survival gene, Mcl-1. Although the four promoters produced similarly high lentiviral titres, EF-1 gave the best transduction efficacy of primary T cells. Major differences were found in the ability of the promoters to drive expression of long RNA encoding GFP, CAR and Mcl-1, highlighting promoter choice as an important consideration for gene therapy applications requiring the expression of long and complex mRNA.


Assuntos
Engenharia Genética/métodos , Regiões Promotoras Genéticas/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Lentivirus/genética , Células MCF-7 , RNA Mensageiro/genética , Transgenes/genética
5.
Nat Protoc ; 15(8): 2387-2412, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641802

RESUMO

Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.


Assuntos
Lentivirus/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA/métodos , Fluxo de Trabalho , Sequência de Bases
6.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 979-991, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567281

RESUMO

Adoptive immunotherapy based on chimeric antigen receptor-modified T cells (CAR-T) is one of the most promising strategies to treat malignant tumors, but its application in solid tumors is still limited. Glypican-3 (GPC3) is a meaningful diagnostic, therapeutic, and prognostic biomarker for hepatocellular carcinoma (HCC). The second/third generation GPC3-targeted CAR-T cells are generated to treat HCC. In order to improve the therapeutic effect, we constructed a fourth-generation lentiviral vector to express GPC3 CAR, human interleukin-7 (IL-7) and CCL19. Then the lentiviral vector and packaging plasmids were co-transfected into HEK293T cells to generate CAR lentiviral particles. Human T lymphocyte cells were transduced with CAR lentiviral to develop the fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19). In vitro, we used cell counting, transwell assay, luciferase bioluminescence assay and flow cytometry to compare the proliferation, chemotaxis, cytotoxicity and subtype distribution between GPC3-BBZ-7×19 CAR-T cells and the second generation GPC3-targeted CAR-T cells (GPC3-BBZ). In vivo, we established GPC3-positive HCC xenograft model in immunodeficient mice, then untransduced T cells (non-CAR-T) or GPC3-BBZ-7×19 CAR-T cells were injected. Tumor growth in mice was observed by bioluminescence imaging. Results showed that compared with GPC3-BBZ CAR-T, GPC3-BBZ-7×19 CAR-T cells had stronger proliferation, chemotactic ability, and higher composition of memory stem T cells (Tscm) (P values<0.05). However, there were no significant difference in cytotoxicity and cytokine secretion between them. In addition, GPC3-BBZ-7×19 CAR-T cells could significantly eliminate GPC3-positive HCC xenografts established in immunodeficient mice. Therefore, the fourth-generation GPC3-targeted CAR-T cells (secreting IL-7 and CCL19) are expected to be more durable and effective against HCC and produce tumor-specific memory, to provide a preclinical research basis for future clinical trials.


Assuntos
Carcinoma Hepatocelular , Quimiocina CCL19 , Interleucina-7 , Neoplasias Hepáticas , Linfócitos T , Animais , Linhagem Celular Tumoral , Quimiocina CCL19/metabolismo , Glipicanas/metabolismo , Células HEK293 , Humanos , Interleucina-7/metabolismo , Lentivirus/genética , Camundongos , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
PLoS Comput Biol ; 16(6): e1007810, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32598363

RESUMO

A large group of biopharmaceuticals is produced in cell lines. The yield of such products can be increased by genetic engineering of the corresponding cell lines. The prediction of promising genetic modifications by mathematical modeling is a valuable tool to facilitate experimental screening. Besides information on the intracellular kinetics and genetic modifications the mathematical model has to account for ubiquitous cell-to-cell variability. In this contribution, we establish a novel model-based methodology for influenza vaccine production in cell lines with overexpressed genes. The manipulation of the expression level of genes coding for host cell factors relevant for virus replication is achieved by lentiviral transduction. Since lentiviral transduction causes increased cell-to-cell variability due to different copy numbers and integration sites of the gene constructs we use a population balance modeling approach to account for this heterogeneity in terms of intracellular viral components and distributed kinetic parameters. The latter are estimated from experimental data of intracellular viral RNA levels and virus titers of infection experiments using cells overexpressing a single host cell gene. For experiments with cells overexpressing multiple host cell genes, only final virus titers were measured and thus, no direct estimation of the parameter distributions was possible. Instead, we evaluate four different computational strategies to infer these from single gene parameter sets. Finally, the best computational strategy is used to predict the most promising candidates for future modifications that show the highest potential for an increased virus yield in a combinatorial study. As expected, there is a trend to higher yields the more modifications are included.


Assuntos
Vacinas contra Influenza , Influenza Humana/prevenção & controle , Cultura de Vírus/métodos , Replicação Viral/genética , Células A549 , Apoptose , Sítios de Ligação , Linhagem Celular , Citoplasma/metabolismo , Endossomos/metabolismo , Edição de Genes , Humanos , Cinética , Lentivirus/genética , Modelos Teóricos , Distribuição Normal , RNA Viral , Proteínas Recombinantes/química
8.
J Vis Exp ; (159)2020 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-32478714

RESUMO

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.


Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Animais , Camundongos , Ratos , Ratos Transgênicos
9.
Invest Ophthalmol Vis Sci ; 61(5): 33, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32428234

RESUMO

Purpose: This study aimed to investigate the role and pathophysiological mechanism of ATP binding cassette transporter A1 (ABCA1) in regulating the IOP and aqueous humor outflow. Methods: ABCA1 expression was measured in trabecular meshwork samples obtained from patients with POAG and human donor eyes by Western blot. To further evaluate the functional significance of ABCA1, porcine angular aqueous plexus (AAP) cells, which are equivalent to human Schlemm's canal endothelial cells, were either treated with ABCA1 agonist GW3965 or transduced with lentivirus expressing ABCA1-shRNA. Transendothelial electrical resistance, protein expression, and nitric oxide (NO) concentration were measured. GW3965 was administered by intracameral injection. IOP and aqueous humor outflow facility were also measured. Results: ABCA1 expression was significantly higher in the trabecular meshwork tissue of patients with POAG compared with controls. ABCA1 upregulation in angular aqueous plexus cells decreased the transendothelial electrical resistance in the angular aqueous plexus monolayers accompanied by a 0.56-fold decrease in caveolin-1 expression and a 2.85-fold and 1.17-fold increase in endothelial NO synthase expression and NO concentration, respectively (n = 3, P < 0.05). Conversely, ABCA1 downregulation increased transendothelial electrical resistance and caveolin-1 expression and decreased endothelial NO synthase expression and NO production (n = 3, P < 0.05). GW3965 decreased IOP and significantly increased conventional outflow facility (P < 0.05). Conclusions: Regulation of aqueous humor outflow via the caveolin-1/endothelial NO synthase/NO pathway is a newly defined function of ABCA1 that is different from its traditional role in mediating cholesterol efflux. ABCA1 is a compelling, novel therapeutic candidate for the treatment of glaucoma and ocular hypertension.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/fisiologia , Caveolina 1/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Pressão Intraocular/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Animais , Humor Aquoso/fisiologia , Benzoatos/farmacologia , Benzilaminas/farmacologia , Western Blotting , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Glaucoma de Ângulo Aberto/cirurgia , Humanos , Lentivirus/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Malha Trabecular/metabolismo , Trabeculectomia , Transfecção
10.
Life Sci ; 253: 117660, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32294474

RESUMO

AIMS: Osteoporosis has been known to generally result from an imbalance between bone formation and resorption. Osteogenesis is the process of differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Sirtuin6 (SIRT6) has been reported to mediate osteogenic differentiation (OD) in rat bone MSCs (rBMSCs). The present study aimed to assess the influence of microRNA miR-186 on the proliferation and OD potential of rBMSCs. MAIN METHODS: OD was performed and evaluated through Alizarin red S staining, alkaline phosphatase (ALP) activity, and specific marker expression. KEY FINDINGS: miR-186 downregulation was observed during OD. rBMSCs with miR-186 overexpression were generated via transfection. Compared with vehicle negative controls, miR-186 upregulation significantly repressed rBMSCs' OD, as evidenced by a reduced ALP activity and decreased mRNA levels of osteogenic markers [osteocalcin, Runx2, BSP, and ALP]. Furthermore, bioinformatic prediction and dual-luciferase reporter assay demonstrated that miR-186 targeted SIRT6 3'-UTR for silencing. SIRT6 overexpression reversed the inhibitory effect of miR-186 on the OD of rBMSCs. Additionally, further examination showed that the activation of nuclear factor-kappa B (NFκB) pathway was involved in the miR-186/SIRT6 signal axis, and phorbol 12-myristate 13-acetate, a NFκB activator, also inhibited the OD of rBMSCs. SIGNIFICANCE: The present study results may demonstrate a novel mechanism of rBMSCs OD via miR-186-SIRT6 interaction.


Assuntos
MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Sirtuínas/genética , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Regeneração Óssea/genética , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Lentivirus/genética , Células-Tronco Mesenquimais/citologia , NF-kappa B/metabolismo , Osteocalcina/metabolismo , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Sirtuínas/metabolismo , Transfecção
11.
PLoS One ; 15(4): e0228511, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348309

RESUMO

OBJECTIVE: To evaluate intraperitoneal (IP) tumor engraftment, metastasis and growth in a pre-clinical murine epithelial ovarian cancer (EOC) model using both transabdominal ultrasound (TAUS) and bioluminescence in vivo imaging system (IVIS). METHODS: Ten female C57Bl/6J mice at six weeks of age were included in this study. Five mice underwent IP injection of 5x106 ID8-luc cells (+ D- luciferin) and the remaining five mice underwent IP injection of ID8-VEGF cells. Monitoring of tumor growth and ascites was performed weekly starting at seven days post-injection until study endpoint. ID8-luc mice were monitored using both TAUS and IVIS, and ID8-VEGF mice underwent TAUS monitoring only. Individual tumor implant dimension and total tumor volume were calculated. Average luminescent intensity was calculated and reported per mouse abdomen. Tumor detection was confirmed by gross evaluation and histopathology. All data are presented as mean +/- standard deviation. RESULTS: Overall, tumors were successfully detected in all ten mice using TAUS and IVIS, and tumor detection correlated with terminal endpoint histology/ H&E staining. For TAUS, the smallest confirmed tumor measurements were at seven days post-injection with mean long axis of 2.23mm and mean tumor volume of 4.17mm3. However, IVIS imaging was able to detect tumor growth at 14 days post-injection. Ascites formation was detected in mice at 21 days post-injection. CONCLUSIONS: TAUS is highly discriminatory for monitoring EOC in pre-clinical murine model, allowing for detection of tumor dimension as small as 2 mm and as early as seven days post-injection compared to IVIS. In addition, TAUS provides relevant information for ascites development and detection of multiple small metastatic tumor implants. TAUS provides an accurate and reliable method to detect and monitor IP EOC growth in mouse xenografts.


Assuntos
Abdome/diagnóstico por imagem , Carcinoma Epitelial do Ovário/diagnóstico por imagem , Carcinoma Epitelial do Ovário/patologia , Transplante de Neoplasias , Neoplasias Peritoneais/diagnóstico por imagem , Neoplasias Peritoneais/patologia , Ultrassonografia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Vetores Genéticos/metabolismo , Lentivirus/genética , Luciferases/genética , Camundongos Endogâmicos C57BL , Necrose , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Curr Opin Ophthalmol ; 31(3): 147-154, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32175942

RESUMO

PURPOSE OF REVIEW: Gene therapy offers, for the first time, the possibility to cure diseases such as retinitis pigmentosa. The positive outcomes that led to the U.S. Food and Drug Administration (FDA) approval of Luxturna to treat Leber congenital amaurosis caused by RPE65 mutations created an optimistic atmosphere in the research, clinical and patient community. Despite this first success, we must understand that this is not a 'one treatment for all'. This review aims to explain the basic concepts of gene therapy and how they translate in different approaches that are utilized in ongoing clinical trials here reviewed. RECENT FINDINGS: In 2017, the FDA approved the first gene therapy treatment. In parallel, other approaches have gained attention. Different delivery methods (adeno-associated virus, lentivirus), injection sites (subretinal, intravitreal, suprachoroidal) and methodologies (gene replacement, silencing, editing) are currently being tested. SUMMARY: Gene therapy is an evolving field in medicine and ophthalmology. Its success and application depends on several factors that are specific to the disease to treat. For now, we know it's a relatively safe approach and we look forward to the continued advancements of current ongoing clinical trials.


Assuntos
Edição de Genes , Inativação Gênica , Terapia Genética/métodos , Vetores Genéticos/genética , Degeneração Retiniana/terapia , Dependovirus/genética , Humanos , Lentivirus/genética , Degeneração Retiniana/genética
13.
Arch Virol ; 165(5): 1109-1120, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32189084

RESUMO

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease with a mortality rate of up to 50% in humans. To avoid safety concerns associated with the use of live virus in virus neutralization assays and to detect human serum neutralizing antibodies, we prepared lentiviral particles containing the CCHF glycoprotein (lenti-CCHFV-GP). Incorporation of the GP into the lentiviral particle was confirmed by electron microscopy and Western blotting. Lenti-CCHFV-GP was found to be able to infect a wide range of cell lines, including BHK-21, HeLa, HepG2, and AsPC-1 cells. In addition, lenti-CCHFV-GP was successfully used as an alternative to CCHFV for the detection of neutralizing antibodies. Sera collected from CCHF survivors neutralized lenti-CCHFV-GP particles in a dose-dependent manner. Our results suggest that the lenti-CCHFV-GP pseudovirus can be used as a safe tool for neutralization assays in low-containment laboratories.


Assuntos
Técnicas de Visualização da Superfície Celular , Glicoproteínas/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Lentivirus/crescimento & desenvolvimento , Testes de Neutralização/métodos , Proteínas Virais/imunologia , Internalização do Vírus , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular , Vetores Genéticos , Glicoproteínas/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Especificidade de Hospedeiro , Humanos , Lentivirus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Virais/genética
14.
Mol Biol (Mosk) ; 54(1): 95-102, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32163393

RESUMO

Cancer stem cells (CSCs) are the most malignant subpopulation of tumor cells that possess a tumorigenic potential and resistantance to chemotherapy. These properties make CSCs a promising target for the development of targeted antitumor therapy which is especially in demand in highly aggressive cancers. However, the correct identification of cancer cells with stem properties remains a challenge. A newly developed lentivirus-based reporter SORE6 allows to directly identify CSCs by measuring gene expression of the embryonic stem cell factors SOX2 and OCT4. In the current study the reporter was modified to enable isolation of SOX2^(+)/OCT4^(+) cells by immunomagnetic separation and then was used to transduce HCC1806 and MDA-MB-453 triple-negative breast cancer (TNBC) cell lines. To validate the modified reporter, SOX2^(+)/OCT4^(+) populations were isolated and analyzed for the content of NANOG, a key transcription factor of pluropotency which expression is regulated by SOX2/OCT4. The percentage of SOX2^(+)/OCT4^(+) cells was assessed for each cell line. An increased content of NANOG protein was found in isolated SOX2^(+)/OCT4^(+) cell fractions indicating that the modified reporter is suitable for further studying the CSC subset.


Assuntos
Genes Reporter , Separação Imunomagnética/métodos , Lentivirus/genética , Células-Tronco Neoplásicas/citologia , Linhagem Celular Tumoral , Humanos , Proteína Homeobox Nanog/análise , Células-Tronco Neoplásicas/metabolismo
15.
Nat Protoc ; 15(4): 1436-1458, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132718

RESUMO

Embedded viral barcoding in combination with high-throughput sequencing is a powerful technology with which to track single-cell clones. It can provide clonal-level insights into cellular proliferation, development, differentiation, migration, and treatment efficacy. Here, we present a detailed protocol for a viral barcoding procedure that includes the creation of barcode libraries, the viral delivery of barcodes, the recovery of barcodes, and the computational analysis of barcode sequencing data. The entire procedure can be completed within a few weeks. This barcoding method requires cells to be susceptible to viral transduction. It provides high sensitivity and throughput, and enables precise quantification of cellular progeny. It is cost efficient and does not require any advanced skills. It can also be easily adapted to many types of applications, including both in vitro and in vivo experiments.


Assuntos
Rastreamento de Células/métodos , Células Clonais/citologia , Código de Barras de DNA Taxonômico/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Proliferação de Células/genética , DNA/genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Camundongos
16.
PLoS One ; 15(2): e0228910, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053639

RESUMO

A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.


Assuntos
Técnicas de Inativação de Genes/métodos , Engenharia Genética/métodos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Corantes Fluorescentes , Edição de Genes , Vetores Genéticos , Humanos , Lentivirus/genética , Plasmídeos , RNA Guia/genética
17.
PLoS One ; 15(2): e0229085, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084174

RESUMO

The sodium iodide symporter (NIS) transports iodide, which is necessary for thyroid hormone production. NIS also transports other monovalent anions such as tetrafluoroborate (BF4-), pertechnetate (TcO4-), and thiocyanate (SCN-), and is competitively inhibited by perchlorate (ClO4-). However, the mechanisms of substrate selectivity and inhibitor sensitivity are poorly understood. Here, a comparative approach was taken to determine whether naturally evolved NIS proteins exhibit variability in their substrate transport properties. The NIS proteins of thirteen animal species were initially assessed, and three species from environments with differing iodide availability, freshwater species Danio rerio (zebrafish), saltwater species Balaenoptera acutorostrata scammoni (minke whale), and non-aquatic mammalian species Homo sapiens (human) were studied in detail. NIS genes from each of these species were lentivirally transduced into HeLa cells, which were then characterized using radioisotope uptake assays, 125I- competitive substrate uptake assays, and kinetic assays. Homology models of human, minke whale and zebrafish NIS were used to evaluate sequence-dependent impact on the organization of Na+ and I- binding pockets. Whereas each of the three proteins that were analyzed in detail concentrated iodide to a similar degree, their sensitivity to perchlorate inhibition varied significantly: minke whale NIS was the least impacted by perchlorate inhibition (IC50 = 4.599 µM), zebrafish NIS was highly sensitive (IC50 = 0.081 µM), and human NIS showed intermediate sensitivity (IC50 = 1.566 µM). Further studies with fifteen additional substrates and inhibitors revealed similar patterns of iodide uptake inhibition, though the degree of 125I- uptake inhibition varied with each compound. Kinetic analysis revealed whale NIS had the lowest Km-I and the highest Vmax-I. Conversely, zebrafish NIS had the highest Km and lowest Vmax. Again, human NIS was intermediate. Molecular modeling revealed a high degree of conservation in the putative ion binding pockets of NIS proteins from different species, which suggests the residues responsible for the observed differences in substrate selectivity lie elsewhere in the protein. Ongoing studies are focusing on residues in the extracellular loops of NIS as determinants of anion specificity. These data demonstrate significant transport differences between the NIS proteins of different species, which may be influenced by the unique physiological needs of each organism. Our results also identify naturally-existing NIS proteins with significant variability in substrate transport kinetics and inhibitor sensitivity, which suggest that the affinity and selectivity of NIS for certain substrates can be altered for biotechnological and clinical applications. Further examination of interspecies differences may improve understanding of the substrate transport mechanism.


Assuntos
Boratos/metabolismo , Animais , Linhagem Celular , Células HeLa , Humanos , Cinética , Lentivirus/genética , Percloratos/metabolismo , Simportadores/metabolismo , Tiocianatos/metabolismo , Baleias , Peixe-Zebra
18.
Nat Med ; 26(2): 200-206, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31988463

RESUMO

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.


Assuntos
Cromossomos Humanos X , Terapia Genética/métodos , Doença Granulomatosa Crônica/genética , Lentivirus/genética , Adolescente , Antígenos CD34/genética , Criança , Pré-Escolar , Comorbidade , Inativação Gênica , Genes Reguladores , Vetores Genéticos , Doença Granulomatosa Crônica/terapia , Células-Tronco Hematopoéticas/citologia , Humanos , Masculino , NADPH Oxidases/genética , Neutrófilos/metabolismo , Segurança do Paciente , Regiões Promotoras Genéticas , Condicionamento Pré-Transplante , Resultado do Tratamento , Reino Unido , Estados Unidos , Adulto Jovem
19.
mBio ; 11(1)2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964730

RESUMO

Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2-) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy.IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.


Assuntos
Anticorpos Biespecíficos/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Lentivirus/genética , Mutação , Anticorpos Biespecíficos/imunologia , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Antígenos/imunologia , Biomarcadores Tumorais , Linhagem Celular Tumoral , Terapia Genética , Humanos , Ligação Proteica , Transdução Genética
20.
Biochem Biophys Res Commun ; 524(1): 96-102, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980173

RESUMO

Mesothelin (MSLN) has been reported to be overexpressed in ovarian cancer and may be an ideal target for immunotherapy. Recent studies have suggested that natural killer (NK) cells may be better chimeric antigen receptor (CAR) drivers because of their favorable innate characteristics, such as directly recognizing and killing tumor cells, resulting in a graft-versus-tumor effect but irresponsible for graft-versus-host disease (GVHD). The therapeutic effects of CAR-engineered NK cells targeting MSLN in ovarian cancer have not been evaluated. In this study, MSLN- and CD19-targeted CAR NK-92 (MSLN- and CD19-CAR NK) cells were constructed. Both MSLN- and CD19-CAR molecules were highly expressed on the surface of NK-92 cells following lentiviral gene transduction. MSLN-CAR NK cells specifically killed MSLN-positive ovarian cancer cells (OVCAR-3 and SK-OV-3), rather than MSLN-negative cells (SK-HEP-1), in vitro. Moreover, compared with parental NK-92 cells and CD19-CAR NK cells, stronger cytokine secretion was detected in MSLN-CAR NK cells cocultured with OVCAR-3 and SK-OV-3. Furthermore, MSLN-CAR NK cells effectively eliminated ovarian cancer cells in both subcutaneous and intraperitoneal tumor models; these cells also significantly prolonged the survival of intraperitoneally tumor-bearing mice. These results demonstrate that MSLN-CAR NK cells have robust specific antitumor activity, both in vitro and in vivo, suggesting that mesothelin could be a potential target for CAR NK cells and could be applied in the treatment of ovarian cancer.


Assuntos
Carcinoma Epitelial do Ovário/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Neoplasias Ovarianas/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Antígenos CD19/metabolismo , Apoptose , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Humanos , Imunoterapia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/metabolismo , Lentivirus/genética , Camundongos , Modelos Biológicos , Neoplasias Experimentais , Transfecção
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