Academic literature on the topic 'Lentivirali'
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Journal articles on the topic "Lentivirali"
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Breckpot, Karine, David Escors, Frederick Arce, Lucienne Lopes, Katarzyna Karwacz, Sandra Van Lint, Marleen Keyaerts, and Mary Collins. "HIV-1 Lentiviral Vector Immunogenicity Is Mediated by Toll-Like Receptor 3 (TLR3) and TLR7." Journal of Virology 84, no. 11 (March 17, 2010): 5627–36. http://dx.doi.org/10.1128/jvi.00014-10.
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ABSTRACT Lentiviral vectors are promising vaccine vector candidates that have been tested extensively in preclinical models of infectious disease and cancer immunotherapy. They are also used in gene therapy clinical trials both for the ex vivo modification of cells and for direct in vivo injection. It is therefore critical to understand the mechanism(s) by which such vectors might stimulate the immune system. We evaluated the effect of lentiviral vectors on myeloid dendritic cells (DC), the main target of lentiviral transduction following subcutaneous immunization. The activation of DC cultures was independent of the lentiviral pseudotype but dependent on cell entry and reverse transcription. In vivo-transduced DC also displayed a mature phenotype, produced tumor necrosis factor alpha (TNF-α), and stimulated naive CD8+ T cells. The lentiviral activation of DC was Toll-like receptor (TLR) dependent, as it was inhibited in TRIF/MyD88 knockout (TRIF/MyD88−/−) DC. TLR3−/− or TLR7−/− DC were less activated, and reverse transcription was important for the activation of TLR7−/− DC. Moreover, lentivirally transduced DC lacking TLR3 or TLR7 had an impaired capacity to induce antigen-specific CD8+ T-cell responses. In conclusion, we demonstrated TLR-dependent DC activation by lentiviral vectors, explaining their immunogenicity. These data allow the rational development of strategies to manipulate the host's immune response to the transgene.
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Nguyen, Tuan Huy, Tatiana Khakhoulina, Andrew Simmons, Philippe Morel, and Didier Trono. "A Simple and Highly Effective Method for the Stable Transduction of Uncultured Porcine Hepatocytes Using Lentiviral Vector." Cell Transplantation 14, no. 7 (August 2005): 489–96. http://dx.doi.org/10.3727/000000005783982828.
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Gene therapy is an attractive approach for the treatment of a wide spectrum of liver diseases. Lentiviral vectors allow the stable integration of transgenes into the genome of nondividing differentiated cells including hepatocytes and could provide long-lasting expression of a therapeutic gene. To develop such approaches, preclinical studies in large animal models such as pigs are necessary to evaluate the feasibility and safety of stable lentiviral integration and long-term vector expression. In addition, effective lentivector-mediated gene transfer onto porcine hepatocytes could advance in cell-based therapies for acute liver failure. To investigate this issue, porcine hepatocytes were transduced in suspension immediately after their isolation in University of Wisconsin (UW) solution containing vitamin E. Up to 80% of hepatocytes stably expressed a GFP transgene after a single exposure to lentiviral vector coding for GFP under the control of either liver-specific or ubiquitous promoters. Moreover, porcine hepatocytes cryopreserved in UW solution containing fetal bovine serum, dimethyl sulfoxide, and vitamin E remained highly transducible with lentiviral vector after thawing. When thawed, transduced in suspension, and immediately transplanted into the spleen of immunodeficient mice, ex vivo lentivirally transgene marked xenogeneic hepatocytes were detected in murine liver. We demonstrated that porcine hepatocytes are highly susceptible to lentiviral vector and describe an easy methodology to efficiently, rapidly, and stably introduce transgenes into uncultured porcine hepatocytes.
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Chinn, Harrison K., Jennifer L. Gardell, Lisa R. Matsumoto, Kevin P. Labadie, Tara N. Mihailovic, Nicole A. P. Lieberman, Amira Davis, Venu G. Pillarisetty, and Courtney A. Crane. "Hypoxia-inducible lentiviral gene expression in engineered human macrophages." Journal for ImmunoTherapy of Cancer 10, no. 6 (June 2022): e003770. http://dx.doi.org/10.1136/jitc-2021-003770.
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BackgroundHuman immune cells, including monocyte-derived macrophages, can be engineered to deliver proinflammatory cytokines, bispecific antibodies, and chimeric antigen receptors to support immune responses in different disease settings. When gene expression is regulated by constitutively active promoters, lentiviral payload gene expression is unregulated, and can result in potentially toxic quantities of proteins. Regulated delivery of lentivirally encoded proteins may allow localized or conditional therapeutic protein expression to support safe delivery of adoptively transferred, genetically modified cells with reduced capacity for systemic toxicities.MethodsIn this study, we engineered human macrophages to express genes regulated by hypoxia responsive elements included in the lentiviral promoter region to drive conditional lentiviral gene expression only under hypoxic conditions. We tested transduced macrophages cultured in hypoxic conditions for the transient induced expression of reporter genes and the secreted cytokine, interleukin-12. Expression of hypoxia-regulated genes was investigated both transcriptionally and translationally, and in the presence of human tumor cells in a slice culture system. Finally, hypoxia-regulated gene expression was evaluated in a subcutaneous humanized-mouse cancer model.ResultsEngineered macrophages were shown to conditionally and tranisently express lentivirally encoded gene protein products, including IL-12 in hypoxic conditions in vitro. On return to normoxic conditions, lentiviral payload expression returned to basal levels. Reporter genes under the control of hypoxia response elements were upregulated under hypoxic conditions in the presence of human colorectal carcinoma cells and in the hypoxic xenograft model of glioblastoma, suggesting utility for systemic engineered cell delivery capable of localized gene delivery in cancer.ConclusionsMacrophages engineered to express hypoxia-regulated payloads have the potential to be administered systemically and conditionally express proteins in tissues with hypoxic conditions. In contrast to immune cells that function or survive poorly in hypoxic conditions, macrophages maintain a proinflammatory phenotype that may support continued gene and protein expression when regulated by conditional hypoxia responsive elements and naturally traffic to hypoxic microenvironments, making them ideal vehicles for therapeutic payloads to hypoxic tissues, such as solid tumors. With the ability to fine-tune delivery of potent proteins in response to endogenous microenvironments, macrophage-based cellular therapies may therefore be designed for different disease settings.
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Grund, Nadja, Patrick Maier, Uwe Appelt, Heike Allgayer, Frederik Wenz, W. Jens Zeller, Stefan Fruehauf, and Stefanie Laufs. "Impact of Chemoselective Pressure on Integration Site Patterns of Lentivirally Transduced Human Hematopoietic Stem Cells." Blood 112, no. 11 (November 16, 2008): 4622. http://dx.doi.org/10.1182/blood.v112.11.4622.4622.
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Abstract Hematologic side effects of cancer chemotherapy like myelosuppression are frequently dose-limiting. Lentiviral gene therapy with cytostatic drug resistance gene transfer to human hematopoietic stem cells (CD34+) is a promising approach to overcome this problem. In this context it is of interest if chemotherapy mediated selection has an impact on lentiviral integration site patterns of transduced hematopoietic stem cells (CD34+). Concerning this issue, human CD34+ cells transduced with a lentiviral self-inactivating (SIN) vector encoding MGMTP140K (the O6-BG resistant mutant of O6-methylguanine- DNA methyltransferase) were in vitro treated with the alkylating agent BCNU. For integration site analysis LM-PCR was performed and integration patterns of the treated and untreated CD34+ cells were analyzed and compared with an in silico set of 106 random integrations. We found different integration preferences of the lentiviral vector between either the treated (82 integrations) or the untreated (30 integrations) CD34+ cells and the in silico set: both groups showed chromosomal preferences, a significant bias for integrations in genes (74,4% in the treated, respectively 70% in the untreated to 40% in the in silico group), especially by favouring introns, a random integration distribution regarding transcription start sites (TSS), and most importantly no significant differences concerning the number of integrations in or near cancer genes. Concerning all integration characteristics we could not find significant differences when comparing the untreated with the treated group. In conclusion, the general distribution of lentiviral integrations in either untreated or treated human CD34+ cells showed no distinct differences between both groups but significant differences compared to the in silico integration set. These results suggest that chemoselection of cells lentivirally overexpressing a specific chemoresistence gene might not influence the integration pattern. Therefore chemotherapy pressure seems not to hamper the safety of lentiviral vectors in gene transfer studies.
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Morris, Julia C., Melissa Conerly, Bobbie Thomasson, Jan Storek, Stanley R. Riddell, and Hans-Peter Kiem. "Induction of cytotoxic T-lymphocyte responses to enhanced green and yellow fluorescent proteins after myeloablative conditioning." Blood 103, no. 2 (January 15, 2004): 492–99. http://dx.doi.org/10.1182/blood-2003-07-2324.
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Abstract Lentiviral vectors are increasingly being used for transferring genes into hematopoietic stem cells (HSCs) due to their ability to transduce nondividing cells. Whereas results in in vitro studies and the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) model have been highly encourgaging, studies in large animals have not confirmed the superior transduction of HSCs using lentiviral vectors versus oncoretroviral vectors. In contrast to the stable gene marking we have consistently achieved with oncoretroviral vectors in animals that received myeloablative conditioning, we observed the complete disappearance of genetically modified enhanced green or yellow fluorescent protein–expressing cells in 5 baboons that received transplants of HSCs transduced with lentiviral vectors alone or in combination with oncoretroviral vectors. Immune responses to transgene products have been found to be involved in the disappearance of gene-modified cells after nonmyeloablative conditioning. Thus, we examined whether the disappearance of genemodified cells after ablative conditioning may be due to an immune response. In 4 of 5 animals, cytotoxic T lymphocytes specific for the transgene protein were readily detected, demonstrating that immune reactions were responsible for the disappearance of the gene-marked cells in the animals. In summary, we report the induction of transgene-specific immune responses after transplantation of lentivirally transduced repopulating cells in a myeloablative setting.
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Delgado, Oscar de Jesus Reyes, and Bibiana Moreno Carranza. "Caracterización de un modelo murino de enterocolitis necrotizante." South Florida Journal of Development 2, no. 5 (October 15, 2021): 6793–800. http://dx.doi.org/10.46932/sfjdv2n5-034.
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Introducción: La enterocolitis necrotizante (ECN) es la patología gastrointestinal de las más comunes y devastadoras en recién nacido con muy bajo peso al nacer (rango entre 500-1500g) y se caracteriza por inflamación y necrosis intestinal. Los objetivos de este estudio fueron desarrollar un modelo murino de ECN así como un modelo de sobreexpresión de proteínas en el intestino mediante la administración enteral mediante sonda de vectores lentivirales.
Métodos: Para el modelo de ECN se utilizaron cepas de ratón C57BL6 y CD1 a los cuales se les trató por 6 veces cada dos horas con una dosis de anoxia con CO2 al 100% durante 10 o 7.5 minutos seguida una reoxigenación mediante hiperoxia al 95% por 5 minutos. Además, para activar el sistema inmune se administró LPS en las primeras dos dosis. Para la sobreexpresión de prolactina (PRL) en el intestino se administraron vectores lentivirales que sobreexpresan GFP (como control) o PRL por vía enteral a ratones CD1 en edades postnatales P2 y P3. Posteriormente se analizó la presencia de GFP y prolactina de las muestras de intestino mediante visualización por microscopia de fluorescencia y Western blot, respectivamente.
Resultados: Se obtuvo una mortalidad del 45% y una eficiencia de desarrollo de ECN entre los animales vivos del 100% en ratones CD1 de edad postnatal P1, en contraste con la mortalidad de 85% y la eficiencia de desarrollo de ECN entre los animales vivos del 0% en ratones C57Bl6 de P1. En relación al modelo de sobreexpresión de proteínas en el intestino, se detectó GFP en el intestino de ratones administrados con 106 TU/ml vectores lentivirales para la sobreexpresión de GFP en el día P2 y evaluados 24 horas después. No se observó la sobreexpresión de PRL en el intestino de ratones administrados con 106 y 108 TU/ml vectores lentivirales para la sobreexpresión de PRL en los días P2 y P3 y evaluados 48 horas después.
Conclusión: El modelo de ECN en ratones CD1 de P1 tuvo una efectividad del 100% a pesar de una mortalidad elevada. Además, se logró estandarizar el método para la sobreexpresión de proteínas en el intestino de ratones en P2 24 horas después de la administración de vectores lentivirales por la via enteral. La determinación de sobreexpresión de PRL en el intestino no fue conclusiva.
Background: Necrotizating enterocolitis (NEC) is one of the most common and devastating gastrointestinal disease in newborns with very low weight birth (range among 500 -1500 g). NEC is characterized by intestinal inflammation and necrosis. Our aims of this study were to develop a NEC murine model and a intestinal protein expression model by means of enteral administration of lentiviral vectors.
Method: For the NEC model were used C57BL6 and CD1 mice which were treated with anoxia with 100% CO2 for 10 or 7.5 minutes followed by 95% O2 for 5 minutes. This treatment was repeated six times with a 2 hours interval. Moreover, to activate the immune system, LPS was administrated orally in the first two doses. For the overexpression of prolactin (PRL) in the intestine, lentiviral vectors that overexpress GFP (as a control) or PRL were administered by orally to CD1 mice at postnatal ages P2 and P3. Then, the presence of GFP and prolactin in the intestine samples was analyzed by fluorescence microscopy and Western blot, respectively.
Result: Mortality of 45% and a NEC development efficiency of 100% was obtained among live animals in CD1 mice of P1 postnatal age, in contrast to the mortality of 85% and development efficiency of 0% among live animals in C57BL6 mice of P1 age. In relation to the protein overexpression model in the intestine, GFP was detected in the mice gut administrated with 106 lentiviral vectors for the GFP overexpression on P2 evaluated 24 hours later. PRL overexpression was not observed in mice that received on day P1 postnatal 106 and 108 TU/ml of lentiviral vectors for the overexpression of PRL and evaluated on days P2 and P3.
Conclusion: NEC model had an effectiveness of 100% in CD1 mice of 1 day of life, despite the high mortality. Moreover, a method for protein overexpression in the intestine was standardized. Lenviral vectors were orally administered 24 hours after birth and the expression of the protein was detected 24 hours later. Prolactin overexpression determination was not conclusive.
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Smolina, Natalia, Anna Kostareva, Joseph Bruton, Alexey Karpushev, Gunnar Sjoberg, and Thomas Sejersen. "Primary Murine Myotubes as a Model for Investigating Muscular Dystrophy." BioMed Research International 2015 (2015): 1–12. http://dx.doi.org/10.1155/2015/594751.
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Muscular dystrophies caused by defects in various genes are often associated with impairment of calcium homeostasis. Studies of calcium currents are hampered because of the lack of a robust cellular model. Primary murine myotubes, formed upon satellite cell fusion, were examined for their utilization as a model of adult skeletal muscle. We enzymatically isolated satellite cells and induced them to differentiation to myotubes. Myotubes displayed morphological and physiological properties resembling adult muscle fibers. Desmin and myosin heavy chain immunoreactivity in the differentiated myotubes were similar to the mature muscle cross-striated pattern. The myotubes responded to electrical and chemical stimulations with sarcoplasmic reticulum calcium release. Presence of L-type calcium channels in the myotubes sarcolemma was confirmed via whole-cell patch-clamp technique. To assess the use of myotubes for studying functional mutation effects lentiviral transduction was applied. Satellite cells easily underwent transduction and were able to retain a positive expression of lentivirally encoded GFP up to and after the formation of myotubes, without changes in their physiological and morphological properties. Thus, we conclude that murine myotubes may serve as a fruitful cell model for investigating calcium homeostasis in muscular dystrophy and the effects of gene modifications can be assessed due to lentiviral transduction.
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Curcio, Alinne G., Fabiana F. Bressan, Carla S. Paes De Carvalho, Célia R. Quirino, Flavio V. Meirelles, and Angelo J. B. Dias. "Efficiency of transgene expression in bovine cells varies according to cell type and gene transfer method." Revista Colombiana de Ciencias Pecuarias 32, no. 1 (March 27, 2019): 34–42. http://dx.doi.org/10.17533/udea.rccp.v32n1a04.
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Background: Production of transgenic animals is still a low-efficiency biotechnology, and simple alternatives should be used to improve the rate of transgenic bovine production by nuclear transfer. One such alternative is selecting the appropriate donor cell type and transfection method. Objective: To investigate the effect of cell type (fetal or adult fibroblasts, and cumulus cells), and gene transfer method (lipofection and lentiviral transduction) on the incorporation, expression, and fluorescence intensity of transgene on bovine cells analyzed by flow cytometry. Methods: Fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) were transfected using lipofection, or transduced using lentiviral particles produced with Green Fluorescent Protein (GFP) expressing plasmids, and analyzed by flow cytometry. Results: Lentiviral transduction resulted in higher transgene expression rates for all cell types (FF: 88.8 ± 0.98; AF: 91.6 ± 2.96; CC: 60.7% ± 14.7) compared to lipofection (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65; CC: 3.9% ± 1.97). Cumulus cells showed lower transgene expression rates than the other cell types. Regarding fluorescence intensity, there was no significant difference between lipofection and lentiviral transduction; in both treatments, higher fluorescence intensity was obtained when adult cells were used instead of fetal cells. Conclusion: Gene transfer efficiency varies according to cell type, and gene transfer method, with lentiviral transduction achieving higher transgene expression rate, and adult fibroblasts showing better transgene expression.Keywords: cloning, epigenetics, lipofection, lentiviral transduction, nuclear reprogramming. Resumen Antecedentes: La producción de animalestransgénicossigue siendo una biotecnología de baja eficiencia, y se deberían utilizar alternativas sencillas para mejorar la tasa de producción de bovinos transgénicos mediante transferencia nuclear. Una de estas alternativas es la selección del tipo mas apropiado de célula donante y método de transferencia génica. Objetivo: Investigar el efecto del tipo celular (fibroblastos fetales o adultos, y celulas del cumulus), y el método de transferencia génica (lipofección y transducción lentiviral) en la incorporación, expresión génica, y la intensidad de fluorescencia del transgén en células bovinas analizadas por citometría de flujo. Métodos: Fibroblastos fetales (FF), fibroblastos adultos (AF), y células del cúmulo (CC) fueron transfectados a través de lipofección o transducidos utilizando partículas lentivirales producidas con plásmidos que expresan la proteína verde fluorescente (GFP). Resultados: La transducción lentiviral dio lugar a mayores tasas de expresión del transgen en todos los tipos de células (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96, CC: 60,7% ± 14,7) en comparación con la lipofección (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). Las células del cúmulus mostraron menores tasas de expresión del transgen que los otros tipos celulares. En cuanto a la intensidad de fluorescencia, no hubo diferencias significativas entre lipofección y transducción lentiviral; en ambos tratamientos, se obtuvo una mayor intensidad de fluorescencia cuando se usaron células adultas en lugar de células fetales. Conclusión: La eficiencia de la transferencia de genes varía según el tipo de célula y el método de transferencia génica, con la transducción lentiviral se logra una mayor tasa de transfección, y los fibroblastos adultos muestran una mejor expresión transgénica.Palabras clave: clonación, epigenética, lipofección, reprogramación nuclear, transducción lentiviral. Resumo Antecedentes: A produção de animais transgênicos é uma biotecnologia que ainda apresenta baixa eficiência e alternativas simples devem ser usadas para o aumento da produção de bovinos transgênicos por transferência nuclear. Uma destas alternativas compreende a seleção do tipo apropriado de célula doadora de núcleo e do método de transferência gênica. Objetivo: Investigar a influência do tipo celular (fibroblastos fetais ou adultos, e células do cumulus), e do método de transferência gênica (transfecção por lipofecção ou transdução lentiviral) na incorporação, expressão, e na intensidade de fluorescência do transgene em células bovinas analisadas por citometria de fluxo. Métodos: Fibroblastos fetais (FF), fibroblastos adultos (AF), e células do cumulus (CC) foram submetidas à lipofecção ou à transfecção lentiviral utilizando plasmídeos expressando a Proteína Fluorescente Verde – GFP). Resultados: A transdução lentiviral resultou em maiores taxas de expressão do transgene em todos os tipos celulares (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96; CC: 60,7% ± 14.7) quando comparada com a lipofeccção (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). As células do cumulus apresentaram menores taxas de expressão quando comparadas aos outros tipos celulares. Com relação à intensidade de fluorescência, não houve diferença significativa entre a lipofecção e a transdução lentiviral e em ambos os tratamentos as células adultas apresentaram maior intensidade de fluorescência do que as células fetais. Conclusão: A eficiência de transferência gênica varia de acordo com o tipo celular, e com o método de transferência gênica, sendo que a transdução lentiviral resultou em maiores taxas, e que os fibroblastos adultos mostraram melhor expressão do transgene.Palavras-chave: clonagem, epigenética, lipofecção, reprogramação nuclear, transdução lentiviral.
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Kreso, Antonija, Catherine A. O'Brien, Peter van Galen, Olga I. Gan, Faiyaz Notta, Andrew M. K. Brown, Karen Ng, et al. "Variable Clonal Repopulation Dynamics Influence Chemotherapy Response in Colorectal Cancer." Science 339, no. 6119 (December 13, 2012): 543–48. http://dx.doi.org/10.1126/science.1227670.
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Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.
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Rappoldt, Liam, Adrienne Weeks, Rodney Ouellete, Jeremy Roy, Catherine Taylor, Craig McCormick, Kathleen Attwood, and Inhwa Kim. "TMOD-26. ESTABLISHING A PATIENT-DERIVED, IN-VITRO ORGANOTYPIC SLICE CULTURE MODEL OF GBM." Neuro-Oncology 22, Supplement_2 (November 2020): ii233. http://dx.doi.org/10.1093/neuonc/noaa215.976.
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Abstract Glioblastoma Multiforme (GBM) is the most common primary malignant brain tumour. This tumour is universally fatal with a median survival of 15 months. Driving this pathology is an extremely heterogeneic tumour and complex tumour microenvironment. GBM research is primarily conducted using immortalized or primary cell lines due to their practicality and reproducibility. However, these cell lines do not effectively recapitulate the tumour microenvironment. Mouse models address these shortcomings but are laborious and expensive. We propose to utilize a patient derived organotypic culture model of GBM as an intermediary. We have utilized this model to test genetic manipulation via lentiviral transduction and the feasibility of utilizing this model to understand patient derived extracellular vesicles (EVs). We have sectioned and cultured patient derived organotypic models for 14 days without loss of viability. To determine if these organotypic cultures are amenable to lentiviral manipulation, tissue sections were transduced with far-red fluorescent lentivirus and efficiency determined by confocal laser scanning microscopy (CLSM) and flow cytometry (FC). To determine feasibility as a model for EVs, media obtained from patient-derived organotypic cultures was analyzed by western blot, nanoparticle tracking analysis (NTA), and nanoFlow Cytometry (nFC). In the future these EVs will be compared to those found in patient serum. The model of GBM has been lentivirally transduced to express a far-red fluorescent vector in approximately 15% of the slice, quantified by CLSM and FC. EV-sized particles positive for canonical EV markers have been identified in the media by NTA, nFC and western blot. Using lentiviral-mediated genetic engineering and emerging EV science, this organotypic slice culture models yields exciting utility in GBM research. The established organotypic slice culture model will likely be a valuable tool in the study of GBM biology and EV dynamics.
Dissertations / Theses on the topic "Lentivirali"
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FIRRITO, CLAUDIA. "Targeted Gene Correction and Reprogramming of SCID-X1 Fibroblasts to Rescue IL2RG Expression in iPSC-derived Hematopoietic Cells." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/94656.
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La terapia genica basata sull’utilizzo di vettori integranti è stata già applicata con successo per la cura di varie malattie genetiche come le malattie da accumulo lisosomiale (LSD), la beta-talassemia (β-Thal) e le immunodeficienze primarie (PID). L’immunodeficienza combinata grave legata al cromosoma X (SCID-X1) è una malattia monogenica letale causata da mutazioni del gene codificante la catena comune gamma del recettore per l’interleuchina 2 (IL2RG). I primi studi clinici per la SCID-X1 hanno mostrato il potenziale terapeutico della terapia genica basata su vettori integranti, risultando nella ricostituzione del compartimento linfoide grazie al vantaggio selettivo delle cellule geneticamente modificate. D’altra parte, tali studi hanno evidenziato il rischio di mutagenesi inserzionale dovuto all’integrazione casuale del virus nel genoma della cellula ospite e all’espressione non regolata del transgene, sottolineando la necessità di sviluppare nuove strategie di terapia genica più sicure.
In questo lavoro, sfruttando la tecnologia delle Zinc-Finger Nucleasi (ZFN) per indurre una rottura del doppio filamento del DNA in maniera sito specifica e dei vettori lentivirali difettivi per l’integrazione (IDLV) per l’introduzione di un templato donatore, abbiamo impiegato il processo di riparazione del DNA guidata dall’omologia per la correzione delle mutazioni che causano la SCID-X1, ripristinando così la funzione genica e l’espressione fisiologica del gene IL2RG.
Mediante l’integrazione di un cDNA correttivo del gene IL2RG a valle del promotore endogeno sia in cellule B linfoblastodi, che esprimono costitutivamente la catena gamma comune, sia in linfociti T da donatori sani, che richiedono IL2RG per la loro sopravvivenza, abbiamo dimostrato la funzionalità e l’attività fisiologica del gene modificato. Abbiamo quindi accoppiato la correzione genica con la selezione delle cellule mediante l’inclusione di una cassetta excidibile di espressione della GFP o della resistenza alla puromicina (PuroR) a valle del cDNA correttivo, al fine di correggere fibroblasti, che normalmente non esprimono IL2R, derivati da pazienti SCID-X1. Abbiamo quindi ottenuto una popolazione di fibroblasti corretti che abbiamo “ riprogrammato” mediante un nuovo vettore di reprogramming che esprime i fattori di trascrizione (SOX2, OCT4, KLf4) e il microRNA cluster 367, generando così una fonte illimitata di cellule staminali pluripotenti indotte (iPSC) geneticamente corrette di interesse terapeutico.
L’espressione transiente della Cre-ricombinasi mediante IDLV ha inoltre permesso l’excisione del vettore di reprogramming e della cassetta di selezione, permettendo così l’ottenimento di cellule iPSC corrette, prive di vettore e con un normale cariotipo. Infine, attraverso il differenziamento delle cellule iPSC in progenitori T-linfoidi, un tipo cellulare assente nei pazienti SCID-X1, e l’osservazione di un vantaggio selettivo delle cellule linfoidi derivate dalle iPSC corrette, abbiamo dimostrato la correzione funzionale dell’allele IL2RG mutato.
In conclusione questi dati dimostrano la validità della nostra strategia di integrazione sito-specifica che, mediante la correzione e la riprogrammazione cellulare, consente di ottenere cellule iPSC geneticamente corrette, aprendo la strada a nuove opportunità terapeutiche più sicure per il trattamento della SCID-X1.Gene replacement by integrating vectors has been successfully used to treat several inherited diseases, such as Lysosomal Storage Disorders (LSD), Thalassemia and Primary Immunodeficiencies (PIDs). X-linked Combined Immunodeficiency (SCID-X1) is a fatal monogenic disorder, caused by mutation of the Interleukin 2 Receptor common γ-chain (IL2RG) gene. For SCID-X1, the early clinical studies have clearly shown the therapeutic potential of integrating vector based gene replacement therapy, which achieved efficient lymphoid reconstitution thanks to the selective growth advantage of the genetically modified cells. However, these studies also highlighted the potential risk of insertional mutagenesis due to random integration of the vector into the host cell genome and to unregulated transgene expression, thus calling for the development of safer gene therapy approaches. Here, by combining the Zinc Finger Nuclease (ZFNs) technology to induce site-specific DNA double-strand breaks (DSB) and of Integrase-Defective Lentiviral Vector (IDLV) to deliver a corrective donor template, we exploited Homology Driven Repair (HDR) to correct SCID-X1 mutation in situ, restoring both physiological expression and function of the IL2RG gene . By knocking-in a corrective IL2RG cDNA transgene downstream of its endogenous promoter in B-lymphoblastoid cells, which constitutively express IL2RG, and in primary T-lymphocytes, which requires IL2RG for their survival and growth, we provide evidence of physiologic activity of the gene-edited IL2RG gene. By including an excisable GFP- or a Puromycin Resistance (PuroR) expression cassette downstream of the corrective cDNA, we coupled correction with exogenous selection of corrected SCID-X1 primary fibroblasts, which do not physiologically express IL2RG, and obtained an enriched population of gene-corrected cells. We then reverted this population to pluripotency by using a novel reprogramming vector that expresses OCT4, SOX2, KLF4 and microRNA cluster 302-367 to obtain a potentially unlimited source of gene-corrected induced pluripotent stem cells (iPSC). We thus generated several gene-corrected bona-fide iPSCs, as confirmed by molecular analyses for targeted integration, which were characterized for their pluripotent state. IDLV-mediated transient delivery of the Cre-recombinase resulted in the co-excision of the reprogramming vector together with the selector cassette, thus allowing the generation of several gene-corrected, reprogramming-factor free iPSCs with normal karyotypes. Finally, by differentiating corrected iPSC to T-lymphoid progenitor cells, which are lacking in SCID-X1 patients, and showing a selective growth advantage of those derived from corrected iPSCs, we provide evidence of the functional correction of the IL2RG mutant allele. Overall these data demonstrate the feasibility of our targeted gene editing strategy, which couples gene correction with cell reprogramming to generate disease-free IPSC, thus paving the way for the development of novel and safer therapeutic approaches for SCID-X1.
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CATTANEO, STEFANO. "Combinatorial gene therapy for epilepsy." Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/128275.
Full textAbstract:
Epilepsy is a neurological disease characterized by a persistent predisposition to generate seizures, that affects about 1% of the world population. About 30% of epileptic patients are drug-resistant, thus refractory to currently available anti-epileptic drugs (AEDs). Less than 10% of these drug-resistant patients are eligible for resective brain surgery, often due to generalized or multiple epileptic foci, or due to proximity of the epileptic focus to eloquent brain areas. Therefore, gene therapy may represent a doable approach for the unmet medical need of these patients.
Neuropeptide Y (NPY) can act as an endogenous anticonvulsant. NPY expression is increased both in rodent and human hippocampal sections from temporal lobe epilepsy surgical samples, despite the strong loss of hilar GABAergic interneurons. Therefore, NPY-based gene therapy may represent a novel approach for the treatment of focal epilepsies. Ideally, however, such vectors should contain multiple elements (at least NPY and Y2Rs driven by appropriate promoters).
In the past, great advancements in the field of viral vectors based on HSV-1 have been made by our laboratory. We therefore aimed at combining the potential of HSV vectors to accommodate large payloads with the complexity of the NPY system to create an “ideal” combinatorial therapeutic cassette. However, residual concerns on the safety and translatability of our new generation HSV-1 based vectors (named J∆NI8) let us first characterize their electrophysiological properties in primary neuronal culture, to assess both safety and efficacy profiles. Surprisingly and disappointingly, we show that mutations in the envelope glycoprotein B (gB), which is responsible for viral entry and cell fusion, might arise during viral vector production. In turn, mutated gB can increase firing frequency while reducing both input resistance and resting membrane potential of transduced neurons. Altogether, these data suggest that careful evaluation of envelope glycoproteins is needed to develop safe HSV-1 replication-defective vectors for the treatment of CNS disorders. We, therefore, decided to move to LV vectors, a more robustly characterized platform despite a more limited packaging capacity compared with HSV vectors.
To potentiate the protective effect of NPY, we developed a combinatorial gene therapy approach based on the expression of NPY together with its receptor (Y2). Since Y2 receptors act mainly pre-synaptically to reduce glutamate release by lowering Ca2+
influx, transgenes expression was driven by the minimal CamKII promoter, thereby biasing their expression in excitatory neurons. We characterized the ability of our lentiviral vectors to express NPY and its functional Y2 receptor in hippocampal neurons and mouse brains. Telemetry video-EEG monitoring was then used to assess the effect of the therapeutic genes on the epileptic phenotype of a genetic mouse model of epilepsy.
We found that the combined expression of NPY and Y2 is sufficient to reduce both the frequency and duration of seizures in the Synapsin triple-KO epilepsy model. These data further strengthen the hypothesis that strategies aimed at the delivery of NPY and Y2 may be successful for the treatment of epilepsy, particularly for pharmaco-resistant and genetic forms of the disease.L'epilessia è una malattia neurologica caratterizzata da una persistente predisposizione a generare crisi, che colpisce circa l'1% della popolazione mondiale. Circa il 30% dei pazienti epilettici sono resistenti ai farmaci, quindi refrattari ai farmaci antiepilettici attualmente disponibili (AED). Meno del 10% di questi pazienti resistenti ai farmaci sono eleggibili per la chirurgia, spesso a causa di foci epilettici generalizzati o multipli, o a causa della vicinanza del focus epilettico alle aree cerebrali eloquenti. Pertanto, la terapia genica può rappresentare un approccio fattibile. Il neuropeptide Y (NPY) può agire come un anticonvulsivo endogeno. L'espressione di NPY è aumentata sia nelle sezioni ippocampali di roditori che in quelle di campioni chirurgici umani di epilessia del lobo temporale, nonostante la forte perdita di interneuroni GABAergici a livello dell’ilo. Pertanto, la terapia genica basata su NPY può rappresentare un nuovo approccio per il trattamento delle epilessie focali. Idealmente, tuttavia, tali vettori dovrebbero contenere più elementi (almeno NPY e Y2R guidati da promotori appropriati). In passato, il nostro laboratorio ha fatto grandi progressi nel campo dei vettori virali basati su HSV-1. Abbiamo quindi mirato a combinare il potenziale dei vettori HSV di ospitare DNA di grandi dimensioni, e la complessità del sistema NPY, per creare una cassetta terapeutica combinatoria "ideale". Tuttavia, le preoccupazioni residue in merito alla sicurezza della nostra nuova generazione di vettori basati su HSV-1 (chiamati J∆NI8) ci hanno spinto a valutare i profili di sicurezza ed efficacia in vitro per valutare l’effetto dell’infezione sulle proprietà elettrofisiologiche in neuroni primari. Sorprendentemente e in maniera deludente, abbiamo dimostrato che mutazioni nella glicoproteina B dell'involucro (gB), che è responsabile dell'entrata virale e della fusione cellulare, potrebbero sorgere durante la produzione del vettore virale. A livello elettrofisiologico, abbiamo inoltre visto che la gB mutata può aumentare la frequenza di potenziali d’azione e contemporaneamente ridurre sia la resistenza di ingresso che il potenziale di riposo neuroni trasdotti. Complessivamente, questi dati suggeriscono che un'attenta valutazione delle glicoproteine dell'involucro è necessaria per sviluppare vettori sicuri non replicativi basati su HSV-1 per il trattamento dei disturbi del SNC. Abbiamo quindi deciso di passare ai vettori Lentivirali (LV), una piattaforma più robusta e caratterizzata nonostante una capacità di carico più limitata rispetto ai vettori HSV. Per potenziare l'effetto protettivo di NPY, abbiamo sviluppato un approccio combinatorio di terapia genica basato sull'espressione di NPY insieme al suo recettore (Y2). Poiché i recettori Y2 agiscono principalmente a livello pre-sinaptico per diminuire il rilascio di glutammato riducendo l’ingresso di Ca2+, l'espressione dei transgeni è stata guidata dal promotore minimal CamKII, orientando così la loro espressione selettivamente nei neuroni eccitatori. Abbiamo successivamente caratterizzato la capacità dei nostri vettori LV di esprimere NPY e il suo recettore funzionale Y2 nei neuroni ippocampali e nel cervello dei topi. In seguito, abbiamo utilizzato un sistema di monitoraggio video-EEG mediante telemetria per valutare l'effetto dei geni terapeutici sul fenotipo epilettico in un modello genetico di epilessia. Abbiamo scoperto che l'espressione combinata di NPY e Y2 è sufficiente a ridurre sia la frequenza che la durata delle crisi nel modello di epilessia Synapsin triple-KO. Questi dati rafforzano ulteriormente l'ipotesi che le strategie mirate all’utilizzo di NPY e Y2 possono avere successo per il trattamento dell'epilessia, in particolare per le forme resistenti ai farmaci ma anche per forme genetiche della malattia.
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STARINIERI, FRANCESCO. "Investigating liver tissue dynamics to improve in vivo gene therapy." Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/122896.
Full textAbstract:
The liver is a relevant target organ for in vivo gene therapy, being involved in several coagulation disorders and metabolic diseases. Adeno-associated viral (AAV) vectors have been extensively used for liver gene therapy, obtaining successful results in clinical trials. Despite AAV vector genomes remain mostly episomal, the transgene has been shown to be maintained for several years in the adult liver. However, active hepatocyte proliferation during liver growth currently challenges application of AAV-vector gene therapy to young individuals. We have previously developed lentiviral vectors (LV) that integrate their genome into host DNA and achieve stable transgene expression in adult mice, dogs, and non-human primates. Here we performed and in-depth analysis of maintenance of LV-transduced hepatocytes following post-natal liver growth and homeostasis and of the age-dependent impact on liver-directed LV gene therapy in mice. We observed a high hepatocyte proliferation rate in newborn mice that decreased over time, with only 25% of hepatocytes contributing to liver growth, generating the vast majority of the adult liver. No major differences have been observed between proliferation of LV-transduced and non-transduced hepatocytes. We then observed a higher hepatocyte transduction efficiency in young (newborn and juvenile) mice compared to adults, paralleled by a lower uptake of LV by non-parenchymal cells. By intravenously (i.v.) administering LV expressing a human coagulation factor IX (FIX) transgene we observed the highest FIX output in mice treated as juvenile, which substantially dropped when mice were treated from the 4th week of age. Newborn-treated mice showed an intermediate level of transgene output. Young mice also showed a higher percentage of multiple-transduced hepatocytes, that might contribute to the differences in transgene output. We then investigated the distribution of transduced hepatocytes in the liver lobule and observed a preferential transduction in the peri-central area in young-treated mice and in the peri-portal area in adult-treated mice. We observed that also Kupffer cells shift from the central to the portal area during growth, however their depletion did not reduce the peri-portal transduction bias in adult mice, indicating that they are not determining the LV transduction bias. Overall, our work showed that i.v. LV administration to young mice results in higher hepatocytes transduction and transgene output than in adult-treated mice, with maintenance of the transgene following cell proliferation during liver growth. These findings inform further development of liver-directed LV gene therapy towards application to pediatric patients and shed light on mechanisms of post-natal liver growth in mice, which are relevant also for genome editing strategies.Il fegato è un importante organo bersaglio per la terapia genica in vivo, a causa del suo ruolo in diversi disordini della coagulazione e malattie metaboliche. I vettori adeno-associati (AAV) sono stati ampiamente usati per la terapia genica diretta al fegato, ottenendo significativi risultati terapeutici in diversi trial clinici. Nonostante il genoma dei vettori AAV rimane prevalentemente episomale, il transgene è mantenuto per diversi anni nel fegato adulto. Tuttavia, la proliferazione degli epatociti durante la crescita ostcola l’utilizzo dei vettori AAV in individui giovani. In passato abbiamo sviluppato vettori lentivirali (LV) che integrano il loro genoma in quello della cellula, e hanno mostrato espressione stabile in topi, cani e primati non umani adulti. Abbiamo effettuato un’analisi approfondita del mantenimento degli epatociti trasdotti con LV in seguito a crescita post-natale e omeostasi, e dell’impatto dell’età sulla terapiagenica con LV diretta al fegato nel topo. Abbiamo osservato una maggiore proliferazione degli epatociti in topi neonati, che decresce nel tempo fino, con solo il 25% degli epatociti che contribuisce alla crescita, generando la grande maggioranza del fegato adulto. Non abbiamo osservato differenze rilevanti tra la proliferazione di epatociti trasdotti e non trasdotti. Abbiamo poi osservato una maggiore efficienza di trasduzione degli epatociti in topi neonati o giovani rispetto agli adulti, in parallelo a un minor indirizzamento nelle cellule non parenchimali. Somministrando intravena (i.v.) un LV che esprime il fattore IX della coaglazione (FIX) umano abbiamo osservato la maggior produzione di FIX in topi trattai da giovani, che decresce sostanzialmente in quelli trattati dalla 4° settimana di vita. I topi neonati hanno mostrato invece un livello intermedio. I topi giovani hanno mostrato inoltre una percentuale più alta di epatociti trasdotti a multipla copia, che può contribuire alla differenza di secrezione. Abbiamo poi esaminato la distribuzione degli epatociti trasdotti nel lobulo epatico e abbiamo una preferenza di trasduzione per gli epatociti nell’area peri-centrale nei topi giovani e in quella peri-portale nei topi adulti. Queste differenze sono mantenute nel tempo, indicando che sono dovute a differenze di trasduzione e non causate da silenziamento del transgene. Abbiamo osservato che anche le cellule di Kupffer si spostano dalla zona centrale a quella portale durante la crescita, ma la loro deplezione aumenta la trasduzione della zona portale nei topi adulti, suggerendo che non determinano la distribuzine del LV nel lobulo. In conclusione, il nostro lavoro mostra che la somministrazione i.v. di LV in topi giovani porta a una maggiore trasduzione degli epatociti e secrezione del prodotto del transgene rispetto ai topi adulti, con mantenimento del transgene in seguito a proliferazione cellulare durante la crescita del fegato. Queste osservazioni forniscono informazioni sullo sviluppo della terapia genica con LV diretta al fegato verso l’applicazione in pazienti pediatrici, e gettano luce sui meccanismi di crescita post-natale del fegato nei topi, che sono rilevanti anche per strategie di modificazione sito-specifica del genoma.
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Bobisse, Sara. "Ridirezionamento dell'immunità anti-tumorale in terapia cellulare adottiva: trasferimento genico del T-Cell Receptor mediato da vettori lentivirali." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425463.
Full textAbstract:
Genetic modification of T cells with genes encoding a tumor-specific T-cell Receptor (TCR) represents a novel strategy to obtain large quantities of tumor-reactive T lymphocytes to be employed in adoptive cell therapy protocols. As a transfer method, lentiviral vectors might represent an appealing alternative to the most widely used oncoretroviral vectors, because they do not require cell division for nuclear uptake.
We have characterized the TCR of a highly cytotoxic T lymphocyte (CTL) clone recognizing the HLA-A2-restricted Melan-A/MART-1 melanocyte differentiation antigen on both pulsed T2 cells and SK-23 MEL melanoma tumor cells. The ? (V?2.2) e ? (V?14) chains of the TCR were cloned and used to construct a lentiviral vector carrying a bidirectional promoter and capable of a robust and coordinated expression of the two transgenes.
Transduction of Jurkat T leukemia cells showed that more than 60% of cells could be transduced without selection at a low MOI, as assessed by antigen-specific tetramer staining. High expression Jurkat clones disclosed that the new assembled TCR was at high intensity, very stable over time, and fully functional, as demonstrated by intracellular signaling upon TCR triggering. Transduction of activated PBMC produced a highly expressed transgenic TCR in around 5-10% of cells. This initial low, but clearly detectable, fraction of transduced lymphocytes could be quickly expanded (4-6 weeks) upon subsequent antigen-specific in vitro restimulation. The resulting population had a 50-70% of transgenic TCR expression and mainly a CD8+ phenotype, specifically recognized antigen-expressing melanoma cells (cytokine production and cytotoxic activity), and exerted relevant therapeutic effects in vitro upon adoptive transfer in SK-23 MEL-bearing mice.
Our results indicate that LV constitute a valid tool for stable and high-intensity expression of transgenic TCR, and support further studies to address potential feasibility of this approach for clinical application.
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CABRIOLU, ANNALISA. "Sviluppo di vettori virali per la terapia genica della β−Talassemia." Doctoral thesis, Università degli Studi di Cagliari, 2012. http://hdl.handle.net/11584/266135.
Full textAbstract:
Beta−thalassemia major is a severe congenital anemi for which there is presently no curative therapy other than allogeneic hematopoietic stem cell transplantation. This therapeutic option, however, applies only to the minority of thalassemia patients who have an HLA−matched bone marrow donor. Gene therapy by the delivery of a regulated globin gene to autologous hematopoietic stem cells is an attractive alternative approach as it is in principle applicable to all thalassemic subjects. Current vectors, althougheffective in correcting thalassemia in murine models still suffer some drawbacks in terms of safety and also in terms of low titer and expression. The aim of this study was to assemble globin vectors improved in both these aspects. Modifications of the globin cassette in the intron2 and in the LCR of the beta-‐globin gene can increase the expression of the globin gene without reducing the vector titer. We also examine the variegation in the expression among the different pools of transduce cells and we suppose that the presence of sequences with chromatin opening activity among the segments of b-‐globin IVS2
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CASTELLANI, STEFANO. "Valutazione dell'efficienza, efficacia e sicurezza di vettori lentivirali nel trasferimento del gene CFTR in sistemi modello di epitelio respiratorio in fibrosi cistica." Doctoral thesis, Università degli Studi di Cagliari, 2007. http://hdl.handle.net/11584/265953.
Full textAbstract:
One of the possible strategies for therapy of Cystic Fibrosis is based on gene therapy. Gene therapy goal is to provide a normal copy of CFTR gene to defective tissues by using different gene transfer agents. HIV-1 derived vectors allow a prolonged expression of therapeutic gene and they are able to infect quiescent cells like respiratory epithelium cells.
Primary goal of the project is concerned with the realization of a lentivirus working live agent, transferring CFTR WT gene, or SHRNA molecules directed against ENAC subunits; ENAC is a sodium channel hyperactive in cystic fibrosis.
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Martelli, Francesco. "Sviluppo di un approccio terapeutico basato su piccoli rna diretti verso i segmenti genomici codificanti la polimerasi del virus dell'influenza." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3423954.
Full textAbstract:
Influenza A viruses, capable of infecting humans and birds, are associated not only to seasonal epidemics, but also to pandemics, with an high impact on Public Health. The emergence of influenza viruses to the currently available drugs and the lack of universal vaccination strategies are main human and veterinarian concerns. Thus, the research of innovative targets for the development of therapeutic anti-influenza A virus strategies is still required. The aim of this study was to develop an innovative antiviral approach based on the delivery into eukaryotic cells susceptible to influenza virus infection of small RNA molecules targeting the highly conserved regions mapping at the 5’ ends of the 3 viral genomic segments encoding for the polymerase (PA, PB1, PB2), an enzyme essential for viral replication (Giannecchini S. et al., 2009- 2011). The target sequences represent the packaging signals of these 3 genomic segments and play an important role during the assembly and budding of the newly formed particles (Qinshan Gao et al., 2012).
To this end, we developed different lentiviral vectors expressing miRNAs [pLenti6/V5-GW/EmGFP-miR] or antisense-RNAs [pLentilox 3.7 GFP] targeting PA, PB1 and PB2 segments. Recombinant lentiviral particles, produced by transfecting human embryonic kidney cells (293T) with both vectors in combination with a packaging systems, were harvested and titrated on human alveolar adenocarcinoma cell line (A549) by FACS analysis. The lentiviral vectors transduction efficiency on different cell lines was evaluated by FACS assay and by quantitative Real-Time PCR. The transduced cells were infected with epidemiologically relevant human influenza viruses type A and B and avian viruses type A. The viral inhibitory activity was assessed by employing an infectivity assay.
We showed that recombinant lentiviral particles expressing miRNA or antisense-RNAs efficiently transduced A549 cell lines that are highly susceptible to influenza virus replication. More importantly, a reduction from 1 to 3 logatims of the viral titre was obtained in all tested cell lines infected with human and avian subtypes of influenza type A viruses. By contrast, no inhibition of influenza type B virus was observed. Furthermore, mutations within the target sequences abolished the antiviral effects of the developed vectors, thus confirming the specificity of the approach. Our study contributes to the demonstration that the expression of small RNAs targeting the packaging signal of the polymerases gene might represent an efficient strategy against the influenza A virus infection in human (especially in pandemic events) and birds.I virus dell'influenza di tipo A sono in grado di infettare gli esseri umani e gli uccelli ed essendo responsabili non solo di di epidemie stagionali, ma anche di pandemie, hanno un impatto importante per la Salute Pubblica. L' insorgenza di virus influenzali resistenti ai farmaci attualmente disponibili e la mancanza di una terapia vaccinale universale sono una fonte di costante preoccupazione tanto per la popolazione umana quanto per quella animale. Pertanto, la ricerca di nuovi bersagli per lo sviluppo di approcci terapeutici / preventivi nei confronti del virus influenzale di tipo A è ancora necessaria. Lo scopo di questo studio è stato quello di sviluppare e valutare una strategia antivirale innovativa basata sul delivery in cellule eucariotiche suscettibili d’infezione da parte del virus dell’influenza di tipo A di piccoli RNA. Questi ultimi hanno come bersaglio le regioni conservate presenti al 5’ dei 3 segmenti del genoma virale codificanti la polimerasi (PA, PB1, PB2), enzima essenziale perché il virus possa replicarsi (Giannecchini S. et al., 2009- 2011). Nelle regioni bersaglio si trovano i segnali di packaging, che svolgono un ruolo importante durante le fasi di assemblaggio e gemmazione delle particelle virali nascenti (Qinshan Gao et al., 2012). A tale scopo, abbiamo sviluppato diversi vettori lentivirali esprimenti miRNA [pLenti6/V5-GW/EmGFP-miR] o antisenso-RNA [pLentilox 3.7 GFP] diretti verso i segmenti genomici PA, PB1 e PB2. Le particelle lentivirali ricombinanti, sono state prodotte trasfettando cellule umane embrionali renali (293T) con entrambi i vettori insieme ad un appropriato sistema di packaging. Leparticelle lentivirali ricombinanti sono state utilizzate per trasdurre le cellule umane di derivazione alveolare (A549) che rappresentano un ottimo modello per lo studio di molecole in grado di interferire con la replicazione del virus dell’influenza di tipo A. Le cellule trasdotte sono state quindi infettate con virus dell'influenza di tipo A di origine umana e aviaria e con un virus di tipo B. L’effetto di inibizione sul titolo della progenie virale è stato valutato mediante un test di infettività. Abbiamo dimostrato che le particelle lentivirali ricombinanti che esprimono miRNA o antisenso-RNA, trasducendo in modo efficiente la linea cellulare A549, determinano una riduzione del titolo virale che varia dagli 1 a 3 logaritmi, a seconda del virus e del bersaglio molecolare preso in esame. Al contrario, non è stata osservato nessun effetto nel caso del virus dell'influenza di tipo B. Inoltre, mutazioni all'interno delle sequenze bersaglio fanno sì che non vi siano più effetti antivirali dei vettori sviluppati, confermando la specificità dell’approccio sviluppato. I nostri dati contribuiscono alla dimostrazione che il delivery intracellualre di piccoli RNA specifici per i segnali di packaging dei geni virali codificanti la polimerasi, possono rappresentare una valida strategia terapeutica nei confronti dei virus dell’influenza umani, specialmente nell’eventualità di insorgenza di virus pandemici, e aviari.
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Reis, Luiza Cunha Junqueira. "Geração de células de pluripotência induzida (iPS) humanas utilizando vetores lentivirais e determinação do perfil de integração lentiviral." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-26022013-092913/.
Full textAbstract:
As células iPS surgiram com a promessa de contornar as limitações das células-tronco embrionárias, como questões éticas, segurança, compatibilidade e disponibilidade. Essas células podem ser obtidas a partir de células somáticas de indivíduos normais ou de pacientes com doenças genéticas, fazendo destas uma importante ferramenta para o screening de drogas, modelos de doenças e testes toxicológicos. Grandes avanços ocorreram na reprogramação de células diferenciadas pela expressão forçada de fatores de transcrição (FT), principalmente, através de vetores lentivirais (VL), que proporcionam uma reprogramação eficiente. Entretanto, a inserção lentiviral no genoma humano e sua influência na reprogramação é pouco conhecida. Neste trabalho, avaliamos o perfil de inserção dos VL utilizados na geração de iPS. As iPS foram geradas e caracterizadas por nosso grupo a partir de fibroblastos humanos transduzidos com VL contendo 3 FT [SOX2, TCL-1A e C-MYC (célula TSM)], e de células mesenquimais derivadas de tecido adiposo com um vetor lentiviral policistrônico contendo 4 FT [OCT4, SOX2, KLF4 e C-MYC (iPS 4FT)]. Cinco colônias isoladas de cada iPS foram mapeadas e analisadas quanto aos sítios de inserção pela técnica de LM-PCR. O DNA genômico digerido foi amplificado com um primer específico para o LTR viral e outro para um linker sintético. Os produtos foram clonados, sequenciados, e analisados em bancos de dados para identificar similaridades com o genoma humano, entre outras análises. Na célula TSM, 176 sequências, obtidas com a técnica de LM-PCR, apresentaram identidade com o genoma humano, sendo que cerca de 50% ocorreram em regiões gênicas com 94% destas em introns. Já nas iPS 4FT, 251 sequências apresentaram identidade, com cerca de 45% atingindo genes, 92% destas em introns. As inserções distribuíram-se por todos os cromossomos, com preferência pelos cromossomos 16, 17 e 20 para a TSM e pelos cromossomos 11, 15 e 17 para a iPS 4FT. Analisamos a distância da inserção ao sítio de início de transcrição (TSS), e inserções próximas a ilhas CpG, que em geral correspondem a regiões regulatórias. A maior proporção de inserção ocorreu a partir de ±30Kb de distância desses sítios. Os sítios frágeis e as regiões repetitivas do genoma foram atingidas, mas com uma frequência baixa. Os resultados mostraram uma preferência de inserção lentiviral por regiões gênicas nas iPS, indicando a possível participação de proteínas como LEDGF/p75 na integração nas células estudadas. Este trabalho mostrou que o local da integração pode contribuir para a reprogramação e, apesar de possíveis efeitos negativos das integrações, estas as células iPS ainda são uma ferramenta importante para estudos in vitro. E identificar fatores que influenciem a seleção do sítio de inserção é importante para determinar regiões cromossômicas \"seguras\" para a integração, aumentando a segurança no uso clínico.The induced pluripotent stem (iPS) cells came with the promise of circumvent some of the limitations in the use of embryonic stem cells, like ethical issues, biological safety, immune compatibility and availability. This cells can be generated from somatic cells of normal individuals or from patients with some genetic disease, making then an important tool for drug screening, construction of disease models and toxicological trials. Great advances have happened in reprogramming differentiated cells through the forced exogenous expression of transcription factors (TF), mostly by lentiviral vectors (LV), which provide an efficient reprogramming. However, the lentiviral insertion in the human genome and its influence in reprogramming is not well known. In this work, we evaluate the insertion profile of LV used to generate human iPS cells. The iPS cells were generated, by our group, from human fibroblasts transduced by LV containing 3 TF [SOX2, TCL-1A and C-MYC (TSM reprogrammed cell)], and from mesenchymal cells derived from human adipose tissue transduced by a polycistronic LV containing 4 TF [OCT4, SOX2, KLF4 and C-MYC (iPS 4TF)]. Five isolated colonies of each iPS cell were mapped and analyzed for the insertion sites through LM-PCR technique. The digested genomic DNA was amplified with a primer for the viral LTR e another for a synthetic linker. The products were cloned, sequenced and analyzed in database to identify similarities with the human genome, among other analyzes. In TSM cell, 176 sequences, derived from the LM-PCR technique, presented identity with the human genome, and about 50% of those occurred in genic regions with 94% in introns. In iPS 4TF, 251 sequences showed identity, with about 45% reaching genes, 92% of these in introns. The insertions were distributed on all chromosomes, with preference for the 16, 17 and 20 for the TSM cell, and for the 11, 15 and 17 for the iPS 4TF. We analyzed the distance of the insertion from de transcription start site, and insertions near CpG islands, which, overall, correspond to regulatory regions. The highest proportion of insertion occurred starting ±30Kb distance from these sites. The fragile sites and the repetitive regions of the genome were also reached, but with low frequency. The results showed a preference of lentiviral insertion for genic regions in iPS, indicating the potential participation of proteins like LEDGF/p75 in integration in the cells of this work. This work shows that the integration site may contribute to the reprogramming, and, despite possible negative effects of integration, these iPS cells are still an important tool for in vitro studies. Identify factors that influence the selection of insertion site is important for determination of \"safe\" chromosomal regions for the integration, increasing the safe in clinical use.
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Ngom, Mor. "Small molecule stimulators for enhanced yield of human hematopoietic stem cells." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC320.
Full textAbstract:
Une transduction efficace des cellules souches hematopoïetiques est un préalable pour la thérapie génique des maladies génétiques comme la β‐thalassemie, l’Adrenoleucodystrophie et le Déficit Immunitaire Combiné Sévère. La petite molécule UM171 à été décrite comme étant une molécule capable de stimuler l’expansion in vitro des cellules souches hématopoïétiques humaines, permettant ainsi une plus large application des thérapies basées sur les cellules souches. Nous avons aussi conduit des études supplémetaires pour confirmer la capacité de UM171 à expandre les souches hématopoïétiques. Durant ce travail, nous avons découvert que UM171 pouvait aussi augmenter de maniére significative, l’efficience de la transduction lentivirale des cellules hématopoïetiques primitives dérivées de sang de cordon. En plus, nous avons montré que UM171 augmentait la transduction des cellules hématopoïeques ayant les phénotypes les plus immatures. Des études plus approfondies ont aussi révélé que UM171 pouvait aussi augmenter la transduction des cellules souches hématopoïétiques avec des lentivirus ayant diffèrent pseudotypes. Au total ces découvertes ont pour conséquence, une nette amélioration des protocoles d’expansion et de transduction des cellules souches hématopoïétiques à travers un meilleur rendement en cellules souches et des taux élevés de transfert de gène en utilisant des quantités réduites de particules viralesEfficient lentiviral gene transfer to hematopoietic stem cells is a prerequisite for theultimate goal of gene therapy for a range of major genetic diseases such as β‐thalassemia, Adrenoleucodystrophy and severe combined immnodeficiency. The small molecule UM171 was recently described as having potent ability to stimulate ex vivo expansion of human hematopoietic stem cells, another key to safer and wider application of stem cell mediated therapies. Here we have conducted additional studies to confirm the stem cell expansion properties of UM171 and in the course of this work discovered that it also has the ability to significantly enhance the efficiency of the lentiviral transduction of primitive hematopietic cells in human cord blood. Subsequent work confirmed that this enhancing effect extends importantly to the most primitive hematopoietic subset as assessed phenotypically and by functional readout in immunodeficient mouse xenografts. Further detailed characterization ofthis phenomenom revealed that UM171’s effects are manifest rapidly and extend to a range of lentiviral pseudotypes. Together these findingsprovide an avenue for improved protocols for hematopoietic stem cell transduction that achieve higher gene efficiency and stem cell recovery coupled with the potential for reduced viral titer requirements
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Cordeil, Stéphanie. "Etude de la différence de susceptibilité des lentivirus de primates aux interférons de type I." Thesis, Lyon, École normale supérieure, 2012. http://www.theses.fr/2012ENSL0781.
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Les IFN-I (interférons de type I), principalement IFN et , constituent un mécanisme de défense primordial de l’hôte contre les pathogènes. Pourtant, dans le cas du VIH-1 (virus de l’immunodéficience humaine), la relation entre les IFN-I et la réplication virale apparaît plus complexe. En effet, si les IFN-I inhibent la réplication du VIH-1 ex vivo, un état d’hyperactivation permanent de la réponse IFN-I a été récemment associé à la progression vers le SIDA ainsi qu’à une forte virémie chez les patients infectés par le VIH-1. De même, la dérégulation de la réponse IFN-I est un critère déterminant dans l’issue pathogénique de certains modèles d’infection virale chez le singe. Si l’hypothèse du rôle pathogénique des IFN-I s’avère correcte, le VIH-1 pourrait avoir évolué afin de se répliquer même en présence d’une telle réponse, qui semble être au final, plus délétère pour l’hôte que pour le virus. L’objectif de ce travail a été d’évaluer la résistance du VIH-1 aux IFN. Dans ce contexte, le VIH-1 a été comparé au VIH-2 et au SIVmac (virus de l’immunodéficience simienne), virus phylogénétiquement proches mais peu ou pas pathogènes pour l’homme, lors de l’infection de plusieurs types cellulaires tels que des lymphocytes, des macrophages et des cellules dendritiques. En accord avec l’hypothèse initiale de travail, les expériences réalisées ont montré que le VIH-1 est capable de se répliquer dans les cellules primaires prétraitées avec des doses d’IFN comparables à celles mesurées in vivo, alors que la réplication des virus VIH-2/SIVmac est complètement bloquée, même à des concentrations très faibles d’IFN. Ce travail a permis de démontrer que le blocage induit par l’IFN s’exerce au niveau des phases précoces de l’infection et plus précisément à l’étape de la transcription inverse. En effet, les données obtenues suggèrent que l’IFN induit l’expression d’un effecteur cellulaire qui affecte différentiellement la stabilité des complexes viraux, ce qui se traduit par un défaut d’accumulation de l’ADN viral plus important pour le VIH-2 et le SIVmac, que pour le VIH-1. La différence de susceptibilité des lentivirus de primates aux IFN-I pourrait ainsi expliquer en partie, les différents niveaux de réplication de ces virus, associés à leurs degrés de pathogénicité in vivoType I Interferons (IFN-α/β, herein IFNs) provide an important mechanism of defense against pathogens and regulate in a paracrine and autocrine manner both intrinsic and adaptive immune responses. In the case of HIV-1 however, the relationship between IFNs and viral replication appears more complex. Indeed, if IFNs have been described to interfere with HIV-1 at basically all phases of its life cycle ex vivo, an IFN-induced state is linked to AIDS progression and to high viral loads in HIV-1 infected individuals. Similarly, a deregulated and prolonged IFN production/state seems one of the main distinguishing features between pathogenic and non-pathogenic SIV infection in primate animal models, suggesting that a deregulated IFN-state may be more detrimental to the host than to the virus itself in vivo.If this hypothesis is correct and if HIV-1 plays an active role in the perpetration of this antiviral state, it is possible that HIV-1 may have overall evolved to cope with this environment, remaining able to replicate despite it.To determine whether HIV-1 was better armed to replicate in the presence of an IFN-state environment than other primate lentiviruses, we compared HIV-1 to SIVmac and more importantly to HIV-2 that albeit capable of inducing AIDS in humans does so in a much less aggressive manner. In agreement with the initial hypothesis, our results indicate that HIV-1 is better fit to replicate in primary cells in the presence of amounts of IFN comparable to the ones measured in vivo, while the replication of HIV-2/SIVmac viruses is completely blocked even in the presence of low levels of IFN. By decorticating the effects of IFNs on the early and late phases of the viral life cycle in primary macrophages, we show here that the main target of the differential action of IFNs are the early phases of infection. More specifically, with time kinetics that we determine herein, IFNs induce cellular factor/s that differentially affect the stability of pre-reverse transcription complexes of HIV-2, but not of HIV-1. Our results could underlie a different evolutionary adaptation of primate lentiviruses to interferons that might be responsible for their different pathogenicity in vivo
Books on the topic "Lentivirali"
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Trono, Didier, ed. Lentiviral Vectors. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-56114-6.
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Escors, David, Karine Breckpot, Frederick Arce, Grazyna Kochan, and Holly Stephenson. Lentiviral Vectors and Gene Therapy. Basel: Springer Basel, 2012. http://dx.doi.org/10.1007/978-3-0348-0402-8.
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Escors, David. Lentiviral Vectors and Gene Therapy. Basel: Springer Basel, 2012.
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Lentiviral vector systems for gene transfer. Georgetown, TX: Eurekah.com, 2003.
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Federico, Maurizio, ed. Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3753-0.
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Trono, Didier. Lentiviral Vectors. Springer, 2012.
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Trono, Didier. Lentiviral Vectors. Springer London, Limited, 2012.
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Lentiviral Vectors. Springer, 2002.
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Stephenson, Holly, David Escors, Karine Breckpot, Frederick Arce, and Grazyna Kochan. Lentiviral Vectors and Gene Therapy. Springer, 2012.
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Escors, David, Karine Breckpot, and Frederick Arce. Lentiviral Vectors and Gene Therapy. Springer, 2012.
Find full textBook chapters on the topic "Lentivirali"
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Pfeifer, Alexander, and Andreas Hofmann. "Lentiviral Transgenesis." In Methods in Molecular Biology, 391–405. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-59745-471-1_21.
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Nakagawa, Terunaga, and Casper C. Hoogenraad. "Lentiviral Transgenesis." In Methods in Molecular Biology, 117–42. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60761-974-1_8.
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Giry-Laterrière, Marc, Els Verhoeyen, and Patrick Salmon. "Lentiviral Vectors." In Methods in Molecular Biology, 183–209. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-095-9_8.
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Chang, Lung-Ji, Yingchi Chen, and Chienling Chen. "Lentiviral Vectors." In Encyclopedia of Cancer, 1–5. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-27841-9_7088-3.
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Chang, Lung-Ji, Yingchi Chen, and Chienling Chen. "Lentiviral Vectors." In Encyclopedia of Cancer, 2466–69. Berlin, Heidelberg: Springer Berlin Heidelberg, 2017. http://dx.doi.org/10.1007/978-3-662-46875-3_7088.
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Curran, M. A., and G. P. Nolan. "Nonprimate Lentiviral Vectors." In Current Topics in Microbiology and Immunology, 75–105. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-56114-6_4.
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Larochelle, A., K. W. Peng, and S. J. Russell. "Lentiviral Vector Targeting." In Current Topics in Microbiology and Immunology, 143–63. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-56114-6_7.
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Zufferey, R. "Production of Lentiviral Vectors." In Current Topics in Microbiology and Immunology, 107–21. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-56114-6_5.
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Markusic, David, and Jurgen Seppen. "Doxycycline Regulated Lentiviral Vectors." In Lentivirus Gene Engineering Protocols, 69–76. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-60761-533-0_4.
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Liu, Shuang. "Production of Lentiviral Particles." In Methods in Molecular Biology, 123–28. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-8802-0_12.
Full textConference papers on the topic "Lentivirali"
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Heckl, BC, M. Carlet, M. Grunert, B. Vick, R. Marschalek, D. Schewe, K. Spiekermann, W. Hiddemann, and I. Jeremias. "Genetic characteristics determine lentiviral transduction rates in patient-derived ALL cells." In 30. Jahrestagung der Kind-Philipp-Stiftung für pädiatrisch-onkologische Forschung. Georg Thieme Verlag KG, 2017. http://dx.doi.org/10.1055/s-0037-1602185.
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Xia, Yifeng, Narayana Yeddula, Eugene Ke, Mathias Leblanc, and Inder Verma. "Abstract 2973: Mouse models of lung cancer mediated by lentiviral gene delivery." In Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA. American Association for Cancer Research, 2014. http://dx.doi.org/10.1158/1538-7445.am2014-2973.
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Byrne, William L., Patrick Forde, and Gerald O'Sullivan. "Abstract 5645: Lentiviral-mediated cell-specific RNA interference in regulatory T cells." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-5645.
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Yin, Yinfei, Richard H. Argent, Rajendra Kumari, Susan A. Watson, Peter King, Brett M. Hall, and Anna M. Grabowska. "Abstract 752:In vivopharmaceutical targets screening using lentiviral inducible-knockdown shRNA system." In Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC. American Association for Cancer Research, 2010. http://dx.doi.org/10.1158/1538-7445.am10-752.
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Chang, Edmund C., and Jeffrey M. Rosen. "Abstract 5334: Novel lentiviral barcoding strategy for lineage tracing of cancer stem cells." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-5334.
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Yin, Yinfei, Rajendra Kumari, Siddharth Subramaniam, Peter King, Sue Watson, Philip Mallinder, and Anna Grabowska. "Abstract 3205:In vivooncology target screening using a lentiviral inducible- shRNA knockdown system." In Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.am2013-3205.
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Lago, Bárbara, Nayhanne Paula, Giovana Angelice, Francisco Mello, Vanessa Paula, Maryrose Lavatori, Francisco Aragão, and Elisabeth Lampe. "Development of lentiviral vectors for inhibition of hepatitis B virus, via small interfering RNAi." In IV International Symposium on Immunobiologicals & VII Seminário Anual Científico e Tecnológico. Instituto de Tecnologia em Imunobiológicos, 2019. http://dx.doi.org/10.35259/isi.sact.2019_32729.
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Ranzani, Marco, Daniela Cesana, Cynthia C. Bartholomä, Francesca Sanvito, Michela Riba, Mauro Pala, Fabrizio Benedicenti, et al. "Abstract 3169: Lentiviral vector-based insertional mutagenesis identifies new clinically relevant liver cancer genes." In Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.am2013-3169.
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Wilson, Andrew A., George J. Murphy, Hiroshi Hamakawa, Letty W. Kwok, Sreedevi Srinivasan, Avi-Hai Hovav, Richard Mulligan, Salomon Amar, Bela Suki, and Darrell N. Kotton. "Amelioration Of Emphysema In Mice Via Lentiviral Transduction Of Long-lived Pulmonary Alveolar Macrophages." In American Thoracic Society 2010 International Conference, May 14-19, 2010 • New Orleans. American Thoracic Society, 2010. http://dx.doi.org/10.1164/ajrccm-conference.2010.181.1_meetingabstracts.a5291.
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Smith, Blake, Stephanie Dougan, and Michael Birnbaum. "1232 Engineering a pseudotyped lentiviral platform to enable lineage-specific transduction of immune cells." In SITC 37th Annual Meeting (SITC 2022) Abstracts. BMJ Publishing Group Ltd, 2022. http://dx.doi.org/10.1136/jitc-2022-sitc2022.1232.
Full textReports on the topic "Lentivirali"
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Gardner, Murray B. Genetic Immunization for Lentiviral Immunodeficiency Virus Infection and Disease. Fort Belvoir, VA: Defense Technical Information Center, October 1998. http://dx.doi.org/10.21236/ada361721.
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Gardner, Murray B. Genetic Immunization for Lentiviral Immunodeficiency Virus Infection and Disease. Fort Belvoir, VA: Defense Technical Information Center, November 1997. http://dx.doi.org/10.21236/ada338248.
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Yes, Jiing-Kuan. Anti-Angiogenic Gene Therapy of Prostate Cancer with Lentiviral Vectors. Fort Belvoir, VA: Defense Technical Information Center, July 2004. http://dx.doi.org/10.21236/ada428533.
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Yee, Jiing-Kuan. Anti-Angiogenic Gene Therapy of Prostate Cancer With Lentiviral Vectors. Fort Belvoir, VA: Defense Technical Information Center, July 2002. http://dx.doi.org/10.21236/ada410315.
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Yee, Jiing-Kuan. Anti-Angiogenic Gene Therapy of Prostate Cancer with Lentiviral Vectors. Fort Belvoir, VA: Defense Technical Information Center, July 2003. http://dx.doi.org/10.21236/ada418264.
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Chang, Edmund. Novel Strategy for Lineage Tracing of Cancer Stem Cells by Lentiviral Barcoding. Fort Belvoir, VA: Defense Technical Information Center, October 2012. http://dx.doi.org/10.21236/ada583773.
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Chang, Edmund C. Novel Strategy for Lineage Tracing of Cancer Stem Cells by Lentiviral Barcoding. Fort Belvoir, VA: Defense Technical Information Center, October 2010. http://dx.doi.org/10.21236/ada549081.
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