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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.
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Kavalerchik, Edward, Charlene Barroga, Larissa Balaian, Jennifer M. Black, and Catriona H. M. Jamieson. "A Novel Mouse Xenograft Model for Generation of JAK2 V617F Driven Leukemic Stem Cells From Human Embryonic Stem Cells." Blood 114, no. 22 (November 20, 2009): 1433. http://dx.doi.org/10.1182/blood.v114.22.1433.1433.
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Abstract Abstract 1433 Poster Board I-456 Introduction Many myeloproliferative neoplasms (MPN) arise in the hematopoietic stem cell (HSC) population upon acquisition of a somatic mutation leading to an aberrantly activated tyrosine kinase, JAK2 V617F. Identification of cell type and context specific genetic and epigenetic events driving leukemic transformation of MPNs has been hampered by the limited supply of the MPN patient blood and marrow samples. Recent publications demonstrate that human embryonic stem cells (hESC) can be differentiated to HSCs efficiently upon coculture with stromal cell lines derived from the murine aorta-gonad-mesonephros (AGM) region. These methods can provide a potentially limitless supply of self-renewing stem cells. We have utilized two hES cell lines, H9 and Hues9 cells, to differentiate toward HSC. We developed novel in vitro and in vivo model systems involving lentivirally enforced expression of genes, such as JAK2V617F, wild type JAK2 and/or activated beta-catenin, in an attempt to generate MPN stem cells. Methods and Results To generate MPN stem cells in initial experiments (n=4), karyotypically normal Hues9 cells were lentivirally transduced with luciferase-GFP and constitutively active beta-catenin (Beta-Cat) alone or in combination with 1) JAK2 V617F, 2) wild-type JAK2, or 3) backbone lentiviral vector. The engraftment capacity of these genetically modified Hues9 cells was compared following intrahepatic transplantation (105-106 cells/mouse) of neonatal RAG2-/-gamma c-/- mice. Lentiviral transduction efficiency was 50-60%. Bioluminescent imaging demonstrated human engraftment at 6 and 11 weeks in all subgroups. The greatest proportion of human cells was noted in the thymus at week 6 and 11, and in the spleens at week 6. There was a 2.4 fold increase in thymic engraftment of CD34+CD45+ cells derived from JAK2 V617F transduced Hues9 cells and a 1.7-fold increase in CD38+CD45+ cells in the JAK2-wild type subset of mice compared with backbone controls. However, overall engraftment rates were low. To determine if hESC differentiation into CD34+ cells improved engraftment, both H9 and Hues9 hES cells were plated in 6 well plates onto mitomycin treated stromal cell lines AM20.1B4 (AM) and UG26.1B6 (UG) that were derived from aorta/mesenchyme and the urogenital ridge of the AGM region of 10 dpc murine embryos respectively. Peak CD34 expression was observed on day 10-12 (n=4) with a sharp decline in CD34 expression by day 18 (n=2). Notably, CD34+ cells began to express CD31 by day 18 with over 50% of CD34+ cells expressing CD31 by day 21 (n=2). Both undifferentiated H9 and Hues9 hES cells as well as CD34+ derivatives expressed CD90. RUNX-1 and GATA-1 expression was higher on day 12 compared to day 18 by Q-RT-PCR in H9 cultured on UG stroma. Plating of 100,000 hESC from day 10-12 cultures with UG stroma generated colonies in hematopoietic progenitor assays with in vitro replating potential while day 18-24 hESC cultures did not. In vitro derived CD34+ cells from day 13 H9 cultures with UG stroma were double selected for CD34 using immunomagnetic beads. The cells were transduced for 6 hr with lentiviral luciferase GFP, Beta-Cat alone (N=4), or Beta-Cat and lentiviral backbone vector (LV) (N=4), wild-type JAK2 (N=4) or JAK2 V617F. The cells were transplanted intrahepatically (5×104 cells/mouse) as above and monitored for engraftment by bioluminescent imaging. Human engraftment was detected at 6 weeks in 2 out of 3 JAK2 V617F/Beta-Cat, 1 of 4 wild-type JAK2/ Beta-Cat, and 0 of 4 Beta-Cat transplanted mice. Conclusions Co-culturing of hES cell lines, H9 and Hues9, on AGM stromal cell lines appears to generate replatable colonies in hematopoietic progenitor assays. Furthermore, engraftment of lentiviral luciferase transduced hESC in RAG2-/-gamma c-/- mouse hematopoietic organs can be monitored by non-invasive bioluminescent imaging. Although enhanced by lentivirally enforced expression of genes that promote hematopoietic differentiation, hematopoietic engraftment is not robust suggesting a relative dearth of environmental cues that promote human hematopoietic differentiation. Transplantation of genetically engineered hESC together with supportive stroma as well as secondary transplantation will be used to monitor changes in hESC derived HSC self-renewal capacity upon introduction of oncogenes in order to provide further insights into MPN stem cell generation. Disclosures Barroga: Wintherix: Employment. Jamieson: Merck: Research Funding; Pfizer: Research Funding; Wintherix: Consultancy; TargeGen: Research Funding; Celgene: Research Funding; Coronado Biosciences: Research Funding.
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Pfeifer, Alexander. "Lentiviral transgenesis." Transgenic Research 13, no. 6 (December 2004): 513–22. http://dx.doi.org/10.1007/s11248-004-2735-5.
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Spirin, P. V., A. E. Vilgelm, and V. S. Prassolov. "Lentiviral vectors." Molecular Biology 42, no. 5 (October 2008): 814–25. http://dx.doi.org/10.1134/s002689330805018x.
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Verma, Inder. "Lentiviral Vectors." Nature Biotechnology 17, S4 (December 1999): 7. http://dx.doi.org/10.1038/70115.
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Lever, Andrew M. L., Padraig M. Strappe, and Jing Zhao. "Lentiviral vectors." Journal of Biomedical Science 11, no. 4 (July 2004): 439–49. http://dx.doi.org/10.1007/bf02256092.
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Yew, Chee-Hong Takahiro, Narmatha Gurumoorthy, Fazlina Nordin, Gee Jun Tye, Wan Safwani Wan Kamarul Zaman, Jun Jie Tan, and Min Hwei Ng. "Integrase deficient lentiviral vector: prospects for safe clinical applications." PeerJ 10 (August 12, 2022): e13704. http://dx.doi.org/10.7717/peerj.13704.
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HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material transduced by lentiviral vector is integrated into the cell genome to introduce new functions, repair defective cell metabolism, and stimulate certain cell functions. Various measures have been administered in different generations of lentiviral vector systems to reduce the vector’s replicating capabilities. Despite numerous demonstrations of an excellent safety profile of integrative lentiviral vectors, the precautionary approach has prompted the development of integrase-deficient versions of these vectors. The generation of integrase-deficient lentiviral vectors by abrogating integrase activity in lentiviral vector systems reduces the rate of transgenes integration into host genomes. With this feature, the integrase-deficient lentiviral vector is advantageous for therapeutic implementation and widens its clinical applications. This short review delineates the biology of HIV-1-erived lentiviral vector, generation of integrase-deficient lentiviral vector, recent studies involving integrase-deficient lentiviral vectors, limitations, and prospects for neoteric clinical use.
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Perry, Christopher, and Andrea C. M. E. Rayat. "Lentiviral Vector Bioprocessing." Viruses 13, no. 2 (February 9, 2021): 268. http://dx.doi.org/10.3390/v13020268.
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Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.
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Kreisberg, Jason. "Safer lentiviral vectors." Nature Biotechnology 30, no. 6 (June 2012): 508. http://dx.doi.org/10.1038/nbt.2276.
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Qasim, Waseem, Conrad A. Vink, and Adrian J. Thrasher. "Hybrid Lentiviral Vectors." Molecular Therapy 18, no. 7 (July 2010): 1263–67. http://dx.doi.org/10.1038/mt.2010.76.
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Fässler, Reinhard. "Lentiviral transgene vectors." EMBO reports 5, no. 1 (January 2004): 28–29. http://dx.doi.org/10.1038/sj.embor.7400053.
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Schmidts, Andrea, Leah C. Marsh, Ambike A. Srivastava, Amanda A. Bouffard, Angela C. Boroughs, Irene Scarfò, Rebecca C. Larson, et al. "Cell-based artificial APC resistant to lentiviral transduction for efficient generation of CAR-T cells from various cell sources." Journal for ImmunoTherapy of Cancer 8, no. 2 (September 2020): e000990. http://dx.doi.org/10.1136/jitc-2020-000990.
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BackgroundAdoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve the care of patients suffering from numerous other cancers in the future. However, the high manufacturing cost of CAR-T cell therapies poses a major barrier to their broader clinical application. Among the key cost drivers of CAR-T production are single-use reagents for T cell activation and clinical-grade viral vector. The presence of variable amounts of contaminating monocytes in the starting material poses an additional challenge to CAR-T manufacturing, since they can impede T cell stimulation and transduction, resulting in manufacturing failure.MethodsWe created K562-based artificial antigen-presenting cells (aAPC) with genetically encoded T cell stimulation and costimulation that represent an inexhaustible source for T cell activation. We additionally disrupted endogenous expression of the low-density lipoprotein receptor (LDLR) on these aAPC (aAPC-ΔLDLR) using CRISPR-Cas9 gene editing nucleases to prevent inadvertent lentiviral transduction and avoid the sink effect on viral vector during transduction. Using various T cell sources, we produced CD19-directed CAR-T cells via aAPC-ΔLDLR-based activation and tested their in vitro and in vivo antitumor potency against B cell malignancies.ResultsWe found that lack of LDLR expression on our aAPC-ΔLDLR conferred resistance to lentiviral transduction during CAR-T production. Using aAPC-ΔLDLR, we achieved efficient expansion of CAR-T cells even from unpurified starting material like peripheral blood mononuclear cells or unmanipulated leukapheresis product, containing substantial proportions of monocytes. CD19-directed CAR-T cells that we produced via aAPC-ΔLDLR-based expansion demonstrated potent antitumor responses in preclinical models of acute lymphoblastic leukemia and B-cell lymphoma.ConclusionsOur aAPC-ΔLDLR represent an attractive approach for manufacturing of lentivirally transduced T cells that may be simpler and more cost efficient than currently available methods.
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Park, Frank. "Lentiviral vectors: are they the future of animal transgenesis?" Physiological Genomics 31, no. 2 (October 2007): 159–73. http://dx.doi.org/10.1152/physiolgenomics.00069.2007.
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Lentiviral vectors have become a promising new tool for the establishment of transgenic animals and the manipulation of the mammalian genome. While conventional microinjection-based methods for transgenesis have been successful in generating small and large transgenic animals, their relatively low transgenic efficiency has opened the door for alternative approaches, including lentiviral vectors. Lentiviral vectors are an appealing tool for transgenesis in part because of their ability to incorporate into genomic DNA with high efficiency, especially in cells that are not actively dividing. Lentiviral vector-mediated transgene expression can also be maintained for long periods of time. Recent studies have documented high efficiencies for lentiviral transgenesis, even in animal species and strains, such as NOD/ scid and C57Bl/6 mouse, that are very difficult to manipulate using the standard transgenic techniques. These advantages of the lentiviral vector system have broadened its use as a gene therapy vector to additional applications that include transgenesis and knockdown functional genetics. This review will address the components of the lentiviral vector system and recent successes in lentiviral transgenesis using both male- and female-derived pluripotent cells. The advantages and disadvantages of lentiviral transgenesis vs. other approaches to produce transgenic animals will be compared with regard to efficiency, the ability to promote persistent transgene expression, and the time necessary to generate a sufficient number of animals for phenotyping.
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Yang, Tao, Bing Zhang, Betty K. Pat, Ming Q. Wei, and Glenda C. Gobe. "Lentiviral-Mediated RNA Interference against TGF-Beta Receptor Type II in Renal Epithelial and Fibroblast Cell Populations In Vitro Demonstrates Regulated Renal Fibrogenesis That Is More Efficient than a Nonlentiviral Vector." Journal of Biomedicine and Biotechnology 2010 (2010): 1–12. http://dx.doi.org/10.1155/2010/859240.
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Background. Lentiviral constructs reportedly can integrate into the genome of non-dividing, terminally differentiated cells and dividing cells, for long-term gene expression. This investigation tested whether a third generation lentiviral-mediated small interfering RNA (siRNA) delivered into renal epithelial and fibroblast cells against type II transforming growth factor-beta receptor (siRNA-TBRII) could better attenuate renal fibrogenesis in comparison with a non-lentiviral construct.Methods. HIV-derived lentiviral and non-lentiviral constructs were used to transfect cells with siRNA-TBRII or siRNA-EGFP control. Human embryonic kidney (HEK-293T), renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) were transfected and gene silencing quantified (fluorescence microscopy, Western blotting, fluorescence-activated cell sorting). Renal fibrogenesis was assessed using extracellular matrix protein synthesis (fibronectin and collagen-III; Western immunoblot), and α-smooth muscle actin (α-SMA) was analysed as a marker of fibroblast activation and epithelial-to-mesenchymal transdifferentiation (EMT).Results. Lentiviral-mediated siRNA-TBRII significantly suppressed TBRII expression in all cell lines, and also significantly suppressed renal fibrogenesis. In comparison with the non-lentiviral construct, lentiviral-mediated siRNA-TBRII produced stronger and more persistent inhibition of collagen-III in NRK-49F cells, fibronectin in all renal cell lines, and α-SMA in renal epithelial cells.Conclusions. Lentiviral vector systems against TBRII can be delivered into renal cells to efficiently limit renal fibrogenesis by sequence-specific gene silencing.
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Zipeto, Maria Anna, Qingfei Jiang, Leslie A. Crews, Angela Court Recart, and Catriona HM Jamieson. "Regulation of Self-Renewal through Modulation of Let-7 Stem Cell Regulatory Family of microRNAs in Chronic Myeloid Leukemia Stem Cells." Blood 124, no. 21 (December 6, 2014): 4527. http://dx.doi.org/10.1182/blood.v124.21.4527.4527.
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Abstract Introduction Chronic myeloid leukemia (CML) was one of the first malignancies shown to be initiated in hematopoietic stem cells by the BCR-ABL1 oncogene and sustained in blast crisis (BC) by progenitor cells that co-opt stem cell properties and behave like leukemia stem cells (BC LSCs). The BCR-ABL fusion oncogene encodes a constitutively active tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitor (TKI) therapy targeting BCR-ABL suppresses CML during the chronic phase (CP), progenitors undergo expansion as a consequence of subsequent genetic and epigenetic alterations that fuel blast crisis transformation, BC LSC generation and TKI resistance. Self-renewing human BC LSCs harbor increased expression of Inflammation responsive adenosine deaminase acting on RNA (ADAR1), which can alter transcript as well as microRNA (miRNA) maturation, splicing and translation by Adenosine (A)-to-Inosine (I) editing of double stranded RNA. miRNAs are a family of small non-coding RNA molecules that regulate gene expression at a post-transcriptional level by inhibiting protein translation and/or reducing mRNA stability. Eukaryotic cells employ miRNAs in diverse biological processes including cell proliferation, differentiation, pluripotency and self-renewal. The stem cell pluripotency RNA binding protein LIN28B plays critical roles in BC transformation of CML. In this study we sought to characterize CML related-oncogenes, such as BCR-ABL, JAK2 and ADAR1, alone or in stromal co-culture in terms of their ability to regulate LSC self-renewal through modulation of let-7 /LIN28B stem cell transcriptional regulatory axis. Methods MiRNAs were extracted from purified CD34+ cells derived from CP and BC CML patient samples as well as cord blood by RNeasy microKit (QIAGEN) and let-7 expression was evaluated by qRT-PCR using miScript Primer assay (QIAGEN). CD34+ cord blood (n=3) were transduced with lentiviral human BCR-ABL, JAK2, let-7a, wild type ADAR1 and ADAR1 mutant, which lacks a functional deaminase domain. Then, 72 hours after transduction, lentivirally transduced cells were plated on irradiated SL/M2 cells. After 5 days of culture, cells were collected for RNA and microRNA extraction. Transduction efficiency and LIN28B levels were evaluated by qRT-PCR and let-7 expression was quantified by qRT-PCR using miScript primer assay. Hematopoietic Progenitor and Replating assaywere performed on lenti-let-7a-overexpressing CB cells to assess differentiation, survival and self-renewal capacity. Results Lentiviral overexpression of human BCR-ABL in CD34+ CB did not induce any significant change in let-7 family members and LIN28B expression in absence of stromal co-culture. However, stromal co-culture of BCR-ABL overexpressing CB led to the significant downregulation of members of the let-7 family as well as to upregulation of their target gene LIN28B, thus suggesting that extrinsic microenvironmental cues are necessary for modulating let-7 family levels in presence of BCR-ABL. Notably, qRT-PCR of CB transduced with JAK2 showed significant upregulation of ADAR1 in the absence of stroma, thus suggesting that JAK2 might be a mediator of inflammatory cytokine-driven ADAR1 activation. Lentiviral overexpression of both human JAK2 and ADAR1 significantly reduced the expression of let-7 family members and induced up-regulation of LIN28B. Interestingly, lentiviral overexpression of ADAR1 mutant did not induce any significant change in most let-7 family members. Finally, lentiviral overexpression of let-7a induced significant reduction in survival and self-renewal. Conclusion These finding suggest that BCR-ABL requires extrinsic signals from the niche to modulate self-renewal of BC LSCs. Conversely, lentiviral JAK2 overexpression induces activation of aberrant RNA editing and subsequent reduction of let-7 family members in the absence of the niche. Interestingly, experiments with ADAR1 mutant, suggest that ADAR1 downregulates most of the let-7 family members in a RNA–editing dependent way manner. In summary these findings suggest a novel mechanism for BC LSC generation that may have utility in prognostication and selective LSCs targeting. Disclosures Jamieson: J&J: Research Funding; Sanofi: Research Funding, Travel Support, Travel Support Other; Roche: Research Funding.
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Takeuchi, Yasuhiro. "Special Issue “Lentiviral Vectors”." Viruses 14, no. 7 (July 8, 2022): 1492. http://dx.doi.org/10.3390/v14071492.
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Debyser, Zeger. "Biosafety of Lentiviral Vectors." Current Gene Therapy 3, no. 6 (December 1, 2003): 517–25. http://dx.doi.org/10.2174/1566523034578177.
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Sarkis, Chamsy, Stephanie Philippe, Jacques Mallet, and Che Serguera. "Non-Integrating Lentiviral Vectors." Current Gene Therapy 8, no. 6 (December 1, 2008): 430–37. http://dx.doi.org/10.2174/156652308786848012.
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Merten, Otto-Wilhelm, Matthias Hebben, and Chiara Bovolenta. "Production of lentiviral vectors." Molecular Therapy - Methods & Clinical Development 3 (2016): 16017. http://dx.doi.org/10.1038/mtm.2016.17.
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Abbas-Terki, Toufik, William Blanco-Bose, Nicole Déglon, William Pralong, and Patrick Aebischer. "Lentiviral-Mediated RNA Interference." Human Gene Therapy 13, no. 18 (December 10, 2002): 2197–201. http://dx.doi.org/10.1089/104303402320987888.
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Lillico, Simon, Douglas Vasey, Tim King, and Bruce Whitelaw. "Lentiviral transgenesis in livestock." Transgenic Research 20, no. 3 (October 7, 2010): 441–42. http://dx.doi.org/10.1007/s11248-010-9448-8.
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Oelze, Beatrice, Kirsten Elger, Patrik Schadzek, Laura Burmeister, Anika Hamm, Sandra Laggies, Virginia Seiffart, Gerhard Gross, and Andrea Hoffmann. "The inflammatory signalling mediator TAK1 mediates lymphocyte recruitment to lipopolysaccharide-activated murine mesenchymal stem cells through interleukin-6." Molecular and Cellular Biochemistry 476, no. 10 (May 29, 2021): 3655–70. http://dx.doi.org/10.1007/s11010-021-04180-8.
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AbstractAs a response to pro-inflammatory signals mesenchymal stem cells (MSCs) secrete agents and factors leading to lymphocyte recruitment, counteracting inflammation, and stimulating immunosuppression. On a molecular level, the signalling mediator TGF-β-activated kinase 1 (TAK1) is activated by many pro-inflammatory signals, plays a critical role in inflammation and regulates innate and adaptive immune responses as well. While the role of TAK1 as a signalling factor promoting inflammation is well documented, we also considered a role for TAK1 in anti-inflammatory actions exerted by activated MSCs. We, therefore, investigated the capacity of lipopolysaccharide (LPS)-treated murine MSCs with lentivirally modulated TAK1 expression levels to recruit lymphocytes. TAK1 downregulated by lentiviral vectors expressing TAK1 shRNA in murine MSCs interfered with the capacity of murine MSCs to chemoattract lymphocytes, indeed. Analysing a pool of 84 secreted factors we found that among 26 secreted cytokines/factors TAK1 regulated expression of one cytokine in LPS-activated murine MSCs in particular: interleukin-6 (IL-6). IL-6 in LPS-treated MSCs was responsible for lymphocyte recruitment as substantiated by neutralizing antibodies. Our studies, therefore, suggest that in LPS-treated murine MSCs the inflammatory signalling mediator TAK1 may exert anti-inflammatory properties via IL-6.
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Gonzalez-Murillo, Africa, M. Luz Lozano, Eugenio Montini, Juan A. Bueren, and Guillermo Guenechea. "Unaltered repopulation properties of mouse hematopoietic stem cells transduced with lentiviral vectors." Blood 112, no. 8 (October 15, 2008): 3138–47. http://dx.doi.org/10.1182/blood-2008-03-142661.
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Abstract Recent studies of retroviral-mediated gene transfer have shown that retroviral integrations themselves may trigger nonmalignant clonal expansion of hematopoietic stem cells (HSCs) in transplant recipients. These observations suggested that previous conclusions of HSC dynamics based on gamma-retroviral gene marking should be confirmed with improved vectors having a more limited capacity to transactivate endogenous genes. Because of the low trans-activation activity of self-inactivating lentiviral vectors (LVs), we have investigated whether the LV marking of mouse HSCs induces a competitive repopulation advantage in recipients of serially transplants. As deduced from analyses conducted in primary and secondary recipients, we concluded that lentivirally transduced HSCs have no competitive repopulation advantages over untransduced HSCs. By linear amplification-mediated polymerase chain reaction (LAM-PCR) analysis, we characterized LV-targeted genes in HSC clones that engrafted up to quaternary recipients. Although 9 clones harbored integrations close to defined retroviral insertion sites, none was characterized as a common integration site, and none was present in HSC clones repopulating quaternary recipients. Taken together, our results show unaltered repopulation properties of HSCs transduced with LVs, and confirm early studies suggesting the natural capacity of a few HSC clones to generate a monoclonal or oligoclonal hematopoiesis in transplant recipients.
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Llano, Manuel, Maria Vanegas, Oliver Fregoso, Dyana Saenz, Susan Chung, Mary Peretz, and Eric M. Poeschla. "LEDGF/p75 Determines Cellular Trafficking of Diverse Lentiviral but Not Murine Oncoretroviral Integrase Proteins and Is a Component of Functional Lentiviral Preintegration Complexes." Journal of Virology 78, no. 17 (September 1, 2004): 9524–37. http://dx.doi.org/10.1128/jvi.78.17.9524-9537.2004.
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ABSTRACT Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegration complexes (PICs) showed that endogenous LEDGF/p75 is a component of functional HIV-1 and FIV PICs. However, HIV-1 and FIV infection and replication in LEDGF/p75-deficient cells was equivalent to that in control cells, whether cells were dividing or growth arrested. Two-long terminal repeat circle accumulation in nondividing cell nuclei was also equivalent to that of LEDGF/p75 wild-type cells. Virions produced in LEDGF/p75-deficient cells had normal infectivity. We conclude that LEDGF/p75 fully accounts for cellular trafficking of diverse lentiviral, but not oncoretroviral, integrases and is the main lentiviral integrase-to-chromatin tethering factor. While lentiviral PIC nuclear import is unaffected by LEDGF/p75 knockdown, this protein is a component of functional lentiviral PICs. A role in HIV-1 integration site distribution merits investigation.
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Arsenijevic, Yvan, Adeline Berger, Florian Udry, and Corinne Kostic. "Lentiviral Vectors for Ocular Gene Therapy." Pharmaceutics 14, no. 8 (July 31, 2022): 1605. http://dx.doi.org/10.3390/pharmaceutics14081605.
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This review offers the basics of lentiviral vector technologies, their advantages and pitfalls, and an overview of their use in the field of ophthalmology. First, the description of the global challenges encountered to develop safe and efficient lentiviral recombinant vectors for clinical application is provided. The risks and the measures taken to minimize secondary effects as well as new strategies using these vectors are also discussed. This review then focuses on lentiviral vectors specifically designed for ocular therapy and goes over preclinical and clinical studies describing their safety and efficacy. A therapeutic approach using lentiviral vector-mediated gene therapy is currently being developed for many ocular diseases, e.g., aged-related macular degeneration, retinopathy of prematurity, inherited retinal dystrophies (Leber congenital amaurosis type 2, Stargardt disease, Usher syndrome), glaucoma, and corneal fibrosis or engraftment rejection. In summary, this review shows how lentiviral vectors offer an interesting alternative for gene therapy in all ocular compartments.
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Marquez Loza, Laura, Eric Yuen, and Paul McCray. "Lentiviral Vectors for the Treatment and Prevention of Cystic Fibrosis Lung Disease." Genes 10, no. 3 (March 14, 2019): 218. http://dx.doi.org/10.3390/genes10030218.
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Despite the continued development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs for the treatment of cystic fibrosis (CF), the need for mutation agnostic treatments remains. In a sub-group of CF individuals with mutations that may not respond to modulators, such as those with nonsense mutations, CFTR gene transfer to airway epithelia offers the potential for an effective treatment. Lentiviral vectors are well-suited for this purpose because they transduce nondividing cells, and provide long-term transgene expression. Studies in primary cultures of human CF airway epithelia and CF animal models demonstrate the long-term correction of CF phenotypes and low immunogenicity using lentiviral vectors. Further development of CF gene therapy requires the investigation of optimal CFTR expression in the airways. Lentiviral vectors with improved safety features have minimized insertional mutagenesis safety concerns raised in early clinical trials for severe combined immunodeficiency using γ-retroviral vectors. Recent clinical trials using improved lentiviral vectors support the feasibility and safety of lentiviral gene therapy for monogenetic diseases. While work remains to be done before CF gene therapy reaches the bedside, recent advances in lentiviral vector development reviewed here are encouraging and suggest it could be tested in clinical studies in the near future.
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Paszkiet, Brian, Andrew Worden, Yajin Ni, Saran Bao, Franck Lemiale, Boro Dropulic, and Laurent Humeau. "CD86 and CD54 Co-Expression on VSV-G Pseudotyped HIV-1 Based Vectors Improves Transduction and Activation of Human Primary CD4+ T Lymphocytes." Blood 104, no. 11 (November 16, 2004): 1754. http://dx.doi.org/10.1182/blood.v104.11.1754.1754.
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Abstract We established the first clinical ex vivo HIV-based vector gene therapy trial in humans with HIV+ CD4+ T-cells. Briefly, this therapy involves modifying patient CD4+ T-cells with our modified lentiviral vector carrying an anti-HIV payload. These cells are then activated and expanded, and re-infused back into the patient. However, cGMP regulations require the use of costly clinical grade reagents (i.e. Retronectin™, CD3/CD28 stimulating paramagnetic beads). In an attempt to reduce ex-vivo processing costs, but not at the expense of transduction levels, we sought to determine a way to directly activate CD4+ T-cells with modified lentiviral vectors. 293FT HEK cell lines, used for producing our lentiviral vectors, were modified to co-express the natural CD28 stimulatory ligand B7.2 (CD86) and ICAM-1 (CD54) proteins on their membrane for co-stimulation and anchoring purposes. When CliniMACS purified normal donor CD4+ T cells were co-cultured with CD54/CD86-expressing cells, in the presence of soluble OKT3 CD3 antibody, CD25 and CD69 activation markers were upregulated, indicating that functional proteins were being expressed at the cell membrane. These CD54 and/or CD86 expressing cells could subsequently be transfected with lentiviral vector plasmid constructs in order to produce host-derived CD54 and/or CD86 bearing HIV-based vectors. EGFP-expressing lentiviral vectors, VRX494, with CD54/CD86-modified envelopes were produced both in these cell lines and by transient transfection of all relevant plasmids, and titers were assayed on Hela-Tat cells by FACS. CD54 modified lentiviral vectors showed increased binding to CD4+ T-cells, as evidenced by significant cell clumping. CD86 (as well as CD54 plus CD86) modified lentiviral vector, with soluble OKT3 CD3 antibody, was shown to activate T-cells, above the levels seen with unmodified lentiviral vectors, as evidenced by the increase in cell surface CD25 and CD69 expression and also the increase in cell size. Cellular expansion of modified lentiviral vector transduced CD4+ T cells reached levels close to CD3:CD28 bead stimulated CD4+ T cell controls over a period of 2 to 3 weeks. The CD3/TCR repertoire was assessed by flow cytometry and, compared to the well-established CD3/CD28 coated M450 Dynabeads stimulatory system as a control, no skewing of the repertoire was observed. CD86 was shown to improve levels of transduction in pre-activated lymphocytes with CD3/CD28 coated M450 Dynabeads. However, CD86 co-expression was crucial for transducing minimally activated CD4+ T cells with only soluble OKT3 CD3 antibody. Levels of transduction and activation were on average 2 to 3 times higher with the modified lentiviral vectors. To our knowledge, we are reporting the first generation of lentiviral particles exhibiting an adhesion property with stimulatory abilities. The development of such a lentiviral vector has valuable implications for clinical application by reducing the number of exogenous reagents in large scale cell processing.
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Wang, Cathy X., Blythe D. Sather, Xuefeng Wang, Jennifer Adair, Iram Khan, Swati Singh, Shanshan Lang, et al. "Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells." Blood 124, no. 6 (August 7, 2014): 913–23. http://dx.doi.org/10.1182/blood-2013-12-546218.
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Key PointsRapamycin significantly enhances lentiviral vector gene delivery to hematopoietic stem cells while preserving engraftment potential. Rapamycin-mediated transduction enhancement is not accompanied by alterations in lentiviral integration profile.
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Fichter, Christina, Anupriya Aggarwal, Andrew Kam Ho Wong, Samantha McAllery, Vennila Mathivanan, Bailey Hao, Hugh MacRae, et al. "Modular Lentiviral Vectors for Highly Efficient Transgene Expression in Resting Immune Cells." Viruses 13, no. 6 (June 18, 2021): 1170. http://dx.doi.org/10.3390/v13061170.
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Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4+ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4+ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing.
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Agosto, Luis M., Jianqing J. Yu, Megan K. Liszewski, Clifford Baytop, Nikolay Korokhov, Laurent M. Humeau, and Una O'Doherty. "The CXCR4-Tropic Human Immunodeficiency Virus Envelope Promotes More-Efficient Gene Delivery to Resting CD4+ T Cells than the Vesicular Stomatitis Virus Glycoprotein G Envelope." Journal of Virology 83, no. 16 (June 3, 2009): 8153–62. http://dx.doi.org/10.1128/jvi.00220-09.
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ABSTRACT Current gene transfer protocols for resting CD4+ T cells include an activation step to enhance transduction efficiency. This step is performed because it is thought that resting cells are resistant to transduction by lentiviral-based gene therapy vectors. However, activating resting cells prior to transduction alters their physiology, with foreseeable and unforeseeable negative consequences. Thus, it would be desirable to transduce resting CD4+ T cells without activation. We recently demonstrated, contrary to the prevailing belief, that wild-type human immunodeficiency virus (HIV) integrates into resting CD4+ T cells. Based on that finding, we investigated whether a commonly used, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped lentiviral gene therapy vector could also integrate into resting CD4+ T cells. To investigate this, we inoculated resting CD4+ T cells with lentiviral particles that were pseudotyped with VSV-G or CXCR4-tropic HIV Env and assayed binding, fusion, reverse transcription, and integration. We found that the VSV-G-pseudotyped lentiviral vector failed to fuse to resting CD4+ T cells while HIV Env-pseudotyped lentiviral vectors fused, reverse transcribed, and integrated in resting cells. Our findings suggest that HIV Env could be used effectively for the delivery of therapeutic genes to resting CD4+ T cells and suggest that fusion may be the critical step restricting transduction of resting CD4+ T cells by lentiviral gene therapy vectors.
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Kuhn, Ulrich, Atsushi Terunuma, Wolfgang Pfutzner, Ruth Ann Foster, and Jonathan C. Vogel. "In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors." Journal of Virology 76, no. 3 (February 1, 2002): 1496–504. http://dx.doi.org/10.1128/jvi.76.3.1496-1504.2002.
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ABSTRACT For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.
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Oldham, Robyn AA, Elliot M. Berinstein, and Jeffrey A. Medin. "Lentiviral vectors in cancer immunotherapy." Immunotherapy 7, no. 3 (March 2015): 271–84. http://dx.doi.org/10.2217/imt.14.108.
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Klimatcheva, Ekaterina. "Lentiviral vectors and gene therapy." Frontiers in Bioscience 4, no. 1-3 (1999): d481. http://dx.doi.org/10.2741/klimatcheva.
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Follenzi, Antonia, Laura Santambrogio, and Andrea Annoni. "Immune Responses to Lentiviral Vectors." Current Gene Therapy 7, no. 5 (October 1, 2007): 306–15. http://dx.doi.org/10.2174/156652307782151515.
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Full textAbstract:
More than two decades have passed since genetically modified HIV was used for gene delivery. Through continuous improvements these early marker gene-carrying HIVs have evolved into safer and more effective lentiviral vectors. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production. Accordingly, lentivector technologies now have widespread use in basic biology and translational studies for stable transgene overexpression, persistent gene silencing, immunization, in vivo imaging, generating transgenic animals, induction of pluripotent cells, stem cell modification and lineage tracking, or site-directed gene editing. Moreover, in the present high-throughput ‘-omics’ era, the commercial availability of premade lentiviral vectors, which are engineered to express or silence genome-wide genes, accelerates the rapid expansion of this vector technology. In the present review, we assess the advances in lentiviral vector technology, including basic lentivirology, vector designs for improved efficiency and biosafety, protocols for vector production and infection, targeted gene delivery, advanced lentiviral applications and issues associated with the vector system.