RESUMEN
BACKGROUND: Following entry, uncoating, and reverse transcription, a number of cellular proteins become associated with the human immunodeficiency virus type 1 (HIV-1) pre-integration complex (PIC). With the goal of obtaining reagents for the analysis of the HIV-1 PIC composition and localisation, we have constructed functional integrase (IN) and matrix (MA) proteins that can be biotinylated during virus production and captured using streptavidin-coated beads. RESULTS: Although the labelled C-terminus allows for the sensitive detection of virion-associated IN, it becomes inaccessible in the presence of cellular proteins. This masking is not dependent on the nature of the tag and does not occur with the tagged MA. It was not observed either with an IN mutant unable to interact with LEDGF/p75, or when LEDGF/p75 was depleted from cells. CONCLUSION: Our observation suggests that a structural rearrangement or oligomerization of the IN protein occurs during the early steps of infection and that this process is related to the presence of LEDGF/p75.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Integrasa de VIH/metabolismo , VIH-1/fisiología , Factores de Transcripción/metabolismo , Biotina/metabolismo , Línea Celular , Integrasa de VIH/química , Humanos , Modelos Biológicos , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Coloración y Etiquetado/métodosRESUMEN
From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.
Asunto(s)
Terapia Genética , Vectores Genéticos/biosíntesis , Vectores Genéticos/genética , Microbiología Industrial/métodos , Lentivirus/genética , Técnicas de Cultivo de Célula , Línea Celular , Contaminación de Medicamentos/legislación & jurisprudencia , Contaminación de Medicamentos/prevención & control , Regulación de la Expresión Génica , Orden Génico , Vectores Genéticos/fisiología , Células HEK293 , Células Madre Hematopoyéticas/metabolismo , Humanos , Lentivirus/fisiología , Plásmidos/genética , Provirus/genética , Control de Calidad , Transducción Genética , Transgenes/genética , Síndrome de Wiskott-Aldrich/terapiaRESUMEN
This work addresses the problem of efficient control of gene expression in the context of viral vectors, which still represents a difficult challenge. A number of lentiviral vectors incorporating the different elements of regulatable transcriptional systems have been described, but they fail to perform satisfactorily either because of a poor dynamic range of transcription levels or because they display high background activities in the uninduced state and mediocre inducer response. We report here on the systematic comparison of vector designs containing the elements of the doxycycline-inducible Tet-on system in their most advanced versions (rtTA2S-M2 transactivator and tTS(Kid) repressor). We show that a simple "all-in-one" vector can be obtained and used for efficient control of transgene expression in long-term tissue culture and in the hematopoietic system of mice following bone marrow transplantation. Using this vector, the uninduced state can be kept at background levels and induction factors of 100-fold are repeatedly obtained over months both in tissue culture and in vivo. Interestingly, the low background activity of the all-in-one vector renders the use of the tTS repressor dispensable, avoiding the problem of progressive loss of inducibility over time associated with irreversible modifications of the chromatin surrounding proviral sequences.