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Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector

Fontes, A. M.; Melo, F. U. F.; Greene, L. J.; Faca, V. M.; Lin, Y.; Gerson, S. L.; Covas, D. T.
Fonte: FUNPEC-EDITORA; RIBEIRAO PRETO Publicador: FUNPEC-EDITORA; RIBEIRAO PRETO
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
66.48%
Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1-kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (+/- 931.7)- and 295,400 (+/- 75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (+/- 493,700)- and 308,000 (+/- 139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/mu g protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.; FINEP [01.07.0652.00]; FINEP; FAPESP; FAPESP [1998/14247-6]; CNPq [314458/2009-3...

Expressão do vetor retroviral pCLPG medido em receptores de transplante de medula óssea.; Assessment of pCLpG retroviral vector expression in vivo utilizing serial transplantation of transduced bone marrow cells.

Fratini, Paula
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 05/03/2009 Português
Relevância na Pesquisa
46.59%
O vetor retroviral é uma ferramenta de transferência gênica largamente utilizada em ensaios de laboratório e em protocolos clínicos. Nosso laboratório desenvolveu um novo vetor, chamado pCLPG, com expressão viral sob comando de p53, um supressor de tumor e um ativador indutível de transcrição, com alvo de estabelecer um vetor com alta expressão. O sistema pCLPG demonstrou um nível de expressão superior ao vetor não modificado em ensaios em cultura de células. Neste projeto, nosso objetivo foi caracterizar a expressão do vetor pCLPG in vivo, utilizando um modelo animal de transdução de células da medula óssea (CMO) do camundongo C57BL/6 seguido por transplante em animais recipientes previamente irradiados para abolir o sistema hematopoiético. Visando observar a expressão sustentada do transgene in vivo, padronizamos o transplante de CMO seriado, transdução do vetor retroviral, realizamos análise do gene repórter eGFP por citometria de fluxo e análise por real time PCR, além da observação de outros tecidos como baço, timo, sangue periférico. Realizamos também analises hematológicas nos animais transplantados para observação de possíveis efeitos adversos relacioanados com a presença do retrovírus. Com estes ensaios não foi observado uma diferença significante entre o desempenho do vetor parental pCLeGFP e o pCLPGeGFP. Tanto o número de células eGFP positivas quanto a expressão do gene repórter diminuíram ao longo do processo de transplante seriado. Expressão foi observada em 3-4%...

Construção e caracterização in vitro  de um vetor retroviral bicistrônico codificando endostatina e interleucina-2 para utilização em terapia gênica; Construction and chracterization in vitro of a bicistronic retroviral vector coding endostatin and interleukin-2 for use in gene therapy

Calvo, Fernanda Bernardes
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 09/12/2009 Português
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66.57%
A terapia gênica tem sido empregada em estudos pré-clínicos e clínicos, com o intuito de amenizar ou curar uma doença. Vetores retrovirais são uma ferramenta de transferência gênica largamente utilizada. Vetores bicistrônicos são uma alternativa interessante para o tratamento de doenças complexas. Na construção de um vetor bicistrônico pode-se empregar várias estratégias dentre elas a utilização da sequência IRES. A endostatina, fragmento do colágeno XVIII, tem sido muito utilizada na terapia anti-angiogênica devido sua ação inibitória no crescimento de células endoteliais. A imunoterapia tem sido utilizada como tratamento coadjuvante de tumores. Dentre as citocinas utilizadas, a interleucina-2 promovendo a proliferação de linfócitos T, tem sido utilizada em diversos estudos pré-clínicos e clínicos. O objetivo deste projeto foi construir e caracterizar in vitro um vetor retroviral bicistrônico codificando endostatina e interleucina-2 utlizando a sequência IRES. A construção do vetor foi realizada em três etapas, sendo comprovada a construção final por análise de restrição e seqüenciamento. Células de empacotamento foram transfectadas com o vetor, e posteriormente realizada a transdução na célula alvo. A endostatina e a interleucina-2 foram determinadas por Dot blot...

Caracterização do padrão de integração do retrovírus pMFG-FVIII-P140K em linhagens celulares humanas produtoras de fator VIII recombinante; Characterization of the integration pattern of pMFG-FVIII-P140K retroviral vector in human cell lines producer of recombinant factor VIII

Freitas, Marcela Cristina Corrêa de
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 14/03/2011 Português
Relevância na Pesquisa
66.65%
A hemofilia A é uma doença caracterizada pela deficiência do fator VIII (FVIII) de coagulação sanguínea e atinge 1 em 5000 homens. Países desenvolvidos, utilizam como terapia o FVIII recombinante (rFVIII), por apresentar maior segurança e consistir uma fonte ilimitada. Estudos realizados em nosso laboratório, utilizando o vetor retroviral pMFG-FVIII-P140K, originaram as linhagens celulares humanas, HepG2FVIIIdB/P140K (hepática) e Hek293FVIIIdB/P140K (renal), que foram selecionadas pelo mesmo sistema de seleção, o tratamento in vitro com as drogas Benzilguanina e Temozolomide. O presente trabalho teve por objetivo caracterizar e detalhar o padrão de integração do vetor retroviral pMFG-FVIII-P140K nas duas linhagens celulares humanas, e verificar se o perfil de integração está relacionado ao tipo celular específico ou se este sofre influência da estratégia de seleção a qual as células foram submetidas anteriormente. Foi utilizada a técnica de LM-PCR (ligação-mediada por PCR) que permite localizar o sítio de integração do vetor viral, por meio do sequênciamento do produto de PCR obtido após a digestão com enzimas de restrição e ligação de uma sequência adaptadora (LINKER) ao DNA genômico. Após o seqüenciamento as sequências foram submetidas a análises em bancos de dados de genomas (Human BLAT...

Autonomous growth of BALB/MK keratinocytes transfected with a retroviral vector carrying the human epidermal growth factor gene

Choudhuri,Jomuna V.; Mathor,Monica B.; Silva,Flávia H.; Han,Sang W.
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2008 Português
Relevância na Pesquisa
66.43%
Epidermal growth factor (EGF), which promotes epidermal regeneration and wound closure, is important for the proliferation and differentiation of epidermal and epithelial tissues in animals. Exogenous EGF is a promising therapeutic agent for wound healing, but its general use is restricted by the limited availability of this protein. In this work, we show that the transfection of mouse BALB/MK keratinocytes, which are totally dependent on EGF for growth and migration, with mature cDNA for human EGF via a retroviral vector abolished the cells requirement for exogenous EGF. The transformed cells had normal morphology and a growth rate that varied according to the source of the retroviral vector used. Keratinocyte transfection with EGF cDNA provides a time- and cost-efficient means of culturing keratinocytes and yields cells that may be useful for skin grafting.

Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

Freitas,Marcela Cristina Correa de; Fontes,Aparecida Maria; Fernandes,Andrielle de Castilho; Picanço-Castro,Virginia; Russo,Elisa Maria de Sousa; Covas,Dimas Tadeu
Fonte: Associação Brasileira de Hematologia e Hemoterapia e Terapia Celular Publicador: Associação Brasileira de Hematologia e Hemoterapia e Terapia Celular
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2014 Português
Relevância na Pesquisa
46.52%
OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K. METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones. RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293. CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

Scaffold Attachment Region-Mediated Enhancement of Retroviral Vector Expression in Primary T Cells

Agarwal, Manju; Austin, Timothy W.; Morel, Franck; Chen, Jingyi; Böhnlein, Ernst; Plavec, Ivan
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/1998 Português
Relevância na Pesquisa
46.52%
We have studied retroviral transgene expression in primary human lymphocytes. Our data demonstrate that transgene expression is high in activated primary CD4+ T cells but significantly decreased in mitotically quiescent cells. Incorporation of a DNA fragment from the scaffold attachment region (SAR) of the human beta interferon gene into the vector improved transgene expression, particularly in quiescent cells. The SAR element functioned in an orientation-dependent manner and enhanced expression of Moloney murine leukemia virus- and murine embryonic stem cell-based vectors. Clonal analysis of transduced T cells showed that the SAR sequence did not confer position-independent expression on a transgene but rather prevented the decrease of expression when cells became quiescent. The SAR sequence also enhanced transgene expression in T cells generated from retrovirally transduced CD34-enriched hematopoietic progenitor-stem cells in a SCID-hu thymus-liver mouse model. We have used the SAR-containing retroviral vector to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) Rev gene. Compared to a standard retroviral vector, the SAR-containing vector was up to 2 orders of magnitude more efficient in inhibiting replication of the HIV-1 virus in infected CD4+ peripheral blood lymphocyte populations in vitro. This is the first demonstration that SAR elements can be used to improve retroviral vector expression in human primary T cells.

Human Beta Interferon Scaffold Attachment Region Inhibits De Novo Methylation and Confers Long-Term, Copy Number-Dependent Expression to a Retroviral Vector

Dang, Qi; Auten, Jennifer; Plavec, Ivan
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2000 Português
Relevância na Pesquisa
46.58%
Moloney murine leukemia virus-based retroviral vector expression is gradually lost during prolonged in vitro culture of CEMSS T cells. However, when the human beta interferon scaffold attachment region (IFN-SAR) was inserted into the vector immediately upstream of the 3′ long terminal repeat (LTR), expression was maintained for the length of the study (4 months). Clonal analysis of the retrovirus vector-infected CEMSS cells showed that SAR-containing retroviral vector expression levels were positively correlated with the proviral copy numbers (P < 0.0001), while there was no correlation between the proviral copy numbers and expression levels in control vector-infected clones. Thirty-three percent of the CEMSS cell clones infected with the control vector showed evidence of partial or complete methylation in the 5′ LTR region. In sharp contrast, we detected no methylation in the clones infected with the SAR-containing vector. To demonstrate a direct inhibitory effect of methylation on retroviral vector expression, we have transfected 293 cells with in vitro-methylated proviral DNA. In transiently transfected cells, expression of methylated LTR was reduced but not completely inhibited, irrespective of the presence of the IFN-SAR sequence. In stably transfected cells...

DNA Methylation of Helper Virus Increases Genetic Instability of Retroviral Vector Producer Cells

Young, Won-Bin; Lindberg, Gary L.; Link, Charles J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2000 Português
Relevância na Pesquisa
46.46%
Retroviral vector producer cells (VPC) have been considered genetically stable. A clonal cell population exhibiting a uniform vector integration pattern is used for sustained vector production. Here, we observed that the vector copy number is increased and varied in a population of established LTKOSN.2 VPC. Among five subclones of LTKOSN.2 VPC, the vector copy number ranged from 1 to approximately 29 copies per cell. A vector superinfection experiment and Northern blot analysis demonstrated that suppression of helper virus gene expression decreased Env-receptor interference and allowed increased superinfection. The titer production was tightly associated with helper virus gene expression and varied between 0 and 2.2 × 105 CFU/ml in these subclones. In one analyzed subclone, the number of integrated vectors increased from one copy per cell to nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 × 105 CFU to an undetectable level. To understand the mechanism involved, helper virus and vectors were examined for DNA methylation status by methylation-sensitive restriction enzyme digestion. We demonstrated that DNA methylation of helper virus 5′ long terminal repeat occurred in approximately 2% of the VPC population per day and correlated closely with inactivation of helper virus gene expression. In contrast...

Retroviral Vector Biosafety: Lessons from Sheep

Van den Broeke, Anne; Burny, Arsène
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
Publicado em 19/03/2003 Português
Relevância na Pesquisa
46.6%
The safety of retroviral-based systems and the possible transmission of replication-competent virus to patients is a major concern associated with using retroviral vectors for gene therapy. While much effort has been put into the design of safe retroviral production methods and effective in vitro monitoring assays, there is little data evaluating the risks resulting from retroviral vector instability at post-transduction stages especially following in vivo gene delivery. Here, we briefly describe and discuss our observations in an in vivo experimental model based on the inoculation of retroviral vector-transduced tumor cells in sheep. Our data indicates that the in vivo generation of mosaic viruses is a dynamic process and that virus variants, generated by retroviral vector-mediated recombination, may be stored and persist in infected individuals prior to selection at the level of replication. Recombination may not only restore essential viral functions or provide selective advantages in a changing environment but also reestablish or enhance the pathogenic potential of the particular virus undergoing recombination. These observations in sheep break new ground in our understanding of how retroviral vectors may have an impact on the course of a preestablished disease or reactivate dormant or endogenous viruses. The in vivo aspects of vector stability raise important biosafety issues for the future development of safe retroviral vector-based gene therapy.

High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.

Akkina, R K; Walton, R M; Chen, M L; Li, Q X; Planelles, V; Chen, I S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1996 Português
Relevância na Pesquisa
46.47%
Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore...

Gene transfer into mammalian cells by a Rous sarcoma virus-based retroviral vector with the host range of the amphotropic murine leukemia virus.

Barsov, E V; Hughes, S H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1996 Português
Relevância na Pesquisa
46.49%
We have constructed and characterized a Rous sarcoma virus-based retroviral vector with the host range of the amphotropic murine leukemia virus (MLV). The chimeric retroviral genome was created by replacing the env coding region in the replication-competent retroviral vector RCASBP(A) with the env region from an amphotropic MLV. The recombinant vector RCASBP-M(4070A) forms particles containing MLV Env glycoproteins. The vector replicates efficiently in chicken embryo fibroblasts and is able to transfer genes into mammalian cells. Vector stocks with titers exceeding 10(6) CFU/ml on mammalian cells can be easily prepared by passaging transfected chicken embryo fibroblasts. Since the vector is inherently defective in mammalian cells, it appears to have the safety features required for gene therapy.

Novel retroviral vector transferring a suicide gene and a selectable marker gene with enhanced gene expression by using a tetracycline-responsive expression system.

Hwang, J J; Scuric, Z; Anderson, W F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1996 Português
Relevância na Pesquisa
46.57%
A retroviral vector for the enhanced expression of the herpes simplex virus thymidine kinase (HSV tk) gene was developed by using a tetracycline-responsive expression system (TRES). The two components of the TRES, the chimeric transactivator (tTA) and the corresponding tTA-binding cis element (tetO), were both incorporated into a retroviral vector and resulted in high levels of tk gene expression from tetO in target cells. Amphotropic virus supernatants from stable producer cells, generated by the retroviral vector containing the TRES, gave titers of 10(4) to 10(5) G418-resistant CFU/ml on murine NIH 3T3 cells. The retroviral vector (G1tTA-[tetOTkINa]R), in which tetO was used in the opposite orientation relative to viral transcription, was capable of transducing tk and neo genes into murine NIH 3T3 cells to yield a high level of tk gene expression. TK enzyme activity in NIH 3T3 cells transduced by this vector was 417-fold higher than in control cells. This increased TK activity was returned to basal levels in the presence of tetracycline. The level of tk gene expression driven by tetO from G1tTA-[tetOTkINa]R vector in NIH 3T3 cells was fourfold higher at both the mRNA level and the TK enzyme level than that produced by the long terminal repeat of G1Tk1SvNa...

Direct injection of a recombinant retroviral vector induces human immunodeficiency virus-specific immune responses in mice and nonhuman primates.

Irwin, M J; Laube, L S; Lee, V; Austin, M; Chada, S; Anderson, C G; Townsend, K; Jolly, D J; Warner, J F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1994 Português
Relevância na Pesquisa
46.57%
The cytotoxic T-lymphocyte (CTL) response plays an important role in controlling the severity and duration of viral infections. Immunization by direct in vivo administration of retroviral vector particles represents an efficient means of introducing and expressing genes and, subsequently, the proteins they encode in vivo in mammalian cells. In this manner foreign proteins can be provided to the endogenous, class I major histocompatibility complex antigen presentation pathway leading to CTL activation. A nonreplicating recombinant retroviral vector, encoding the human immunodeficiency virus type 1 (HIV-1) IIIB envelope and rev proteins, has been developed and examined for stimulation of immune responses in mouse, rhesus macaque, and baboon models. Animals were immunized by direct intramuscular injection of the retroviral vector particles. Vector-immunized mice, macaques, and baboons generated long-lived CD8+, major histocompatibility complex-restricted CTL responses that were HIV-1 protein specific. The CTL responses were found to be dependent on the ability of the retroviral vector to transduce cells. The vector also elicited HIV-1 envelope-specific antibody responses in mice and baboons. These studies demonstrate the ability of a retroviral vector encoding HIV-1 proteins to stimulate cellular and humoral immune responses and suggest that retrovector immunization may provide an effective means of inducing or augmenting CTL responses in HIV-1-infected individuals.

Development of a retroviral vector for inducible expression of transforming growth factor beta 1.

McGeady, M L; Arthur, P M; Seidman, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1990 Português
Relevância na Pesquisa
46.49%
A retroviral vector system for the expression of exogenous genes under the control of an inducible promoter was developed. By utilizing this system, the cDNA for human transforming growth factor beta 1 (TGF-beta 1) was inserted into a retroviral vector under the control of an internal mouse metallothionein promoter and introduced via infection into normal rat kidney fibroblasts (NRK-49F) and epithelial cells (NRK-52E), Chinese hamster ovary cells (CHO), and the human monocytic cell line U937. Control of TGF-beta 1 expression, achieved by Cd2+ induction of vector-encoded TGF-beta 1 mRNA, was cell line specific and resulted in a concomitant increase in neutralizable TGF-beta 1 production by the cells. Autocrine stimulation of vector-containing cells by vector-encoded TGF-beta 1 was detected by an increase in soft-agar colony formation of NRK-49F infectants compared with that of the control cells. In addition, the use of a second internal promoter in a retroviral vector of similar design allowed isolation of stable infectants from a cell line (CHO) in which the viral long terminal repeat does not function efficiently.

The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery

Anson, Donald S
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
Publicado em 13/08/2004 Português
Relevância na Pesquisa
46.5%
Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo.

Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations▿

Segura, María Mercedes; Garnier, Alain; Di Falco, Marcos Rafael; Whissell, Gavin; Meneses-Acosta, Angélica; Arcand, Normand; Kamen, Amine
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
46.48%
The Moloney murine leukemia virus (MMLV) belongs to the Retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin β1 (fibronectin receptor subunit beta) and HMFG-E8...

Justification of continuous packed-bed reactor for retroviral vector production from amphotropic ΨCRIP murine producer cell

Kang, Seung-Hyun; Kim, Byung-Gee; Lee, Gyun Min
Fonte: Kluwer Academic Publishers Publicador: Kluwer Academic Publishers
Tipo: Artigo de Revista Científica
Publicado em /10/2000 Português
Relevância na Pesquisa
46.5%
To indentify a plausible large-scale production system forretroviral vector, three culture systems, i.e., batch culturewith medium exchange, microcarrier culture, and packed-bedreactor culture were compared. In batch cultures with mediumexchange, high cell concentrations were maintained for about amonth, and the harvested retroviral titer remained constant. Inmicrocarrier cultures, although cell growth was rapid, theretroviral titer was unexpectedly low, suggesting that the lowtiter was due either to serious damage to the retroviral vectoror to a reduction in the production rate of retroviral vector,caused by mechanical shear forces. Although the retroviral titer(maximum titer, 1.56 × 106) in the packed-bedreactor was a little bit lower than that obtained in the batchculture with medium exchange (maximum titer, 1.91 ×106), continuous production made it possible to increasethe cumulative titer up to 16-fold of that from the batchculture with medium exchange. Moreover, as the packed-bedreactor system requires less labor and shows excellentvolumetric productivity in comparison to batch cultures withmedium exchanges, it will be an appropriate production systemfor retroviral vector in large quantities.

The use of retroviral vectors for gene therapy - what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery

Anson, D.
Fonte: BioMed Central Ltd. Publicador: BioMed Central Ltd.
Tipo: Artigo de Revista Científica
Publicado em //2004 Português
Relevância na Pesquisa
46.63%
Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo. The ability of retroviruses to integrate into the host cell chromosome also raises the possibility of insertional mutagenesis and oncogene activation. Both these phenomena are well known in the interactions of certain types of wild-type retroviruses with their hosts. However...

Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.

Hantzopoulos, P A; Sullenger, B A; Ungers, G; Gilboa, E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1989 Português
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This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR. The important result is that in its new position the gene is placed outside the retroviral transcriptional unit, eliminating or at least reducing the negative effects of the retroviral transcriptional unit. The utility of the DC vector design was tested by using a 2.1-kilobase-pair (kbp)-long adenosine deaminase (ADA; EC 3.5.4.4) minigene that was inserted into the 3' LTR of the N2 retroviral vector, generating a 2.7-kbp-long chimeric LTR. DNA blot analysis was used to show that the chimeric LTR was faithfully duplicated in cells infected with the corresponding virus, generating two copies of the ADA minigene, one copy in each LTR. Insertion of the ADA minigene into the 3' LTR of the N2 vector led to a 10- to 20-fold increase in ADA transcripts and human ADA isozyme synthesized in NIH 3T3 cells as compared to cells harboring the same vector in which the ADA minigene was inserted between the two LTRs. A similar increase in ADA expression was observed in two human lymphoid cell lines tested...