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Titolo:
Novel tools for production and purification of recombinant adenoassociatedvirus vectors
Autore:
Grimm, D; Kern, A; Rittner, K; Kleinschmidt, JA;
Indirizzi:
Deutsch0Krebsforschungszentrum, Forsch Schwerpunkt Angew Tumorvirol, D-6912 Deutsch Krebsforschungszentrum Heidelberg Germany D-69120 rvirol, D-6912 Transgene SA, F-67000 Strasbourg, France Transgene SA Strasbourg France F-67000 ne SA, F-67000 Strasbourg, France
Titolo Testata:
HUMAN GENE THERAPY
fascicolo: 18, volume: 9, anno: 1998,
pagine: 2745 - 2760
SICI:
1043-0342(199812)9:18<2745:NTFPAP>2.0.ZU;2-Y
Fonte:
ISI
Lingua:
ENG
Soggetto:
ADENO-ASSOCIATED VIRUSES; GENE-TRANSFER; IMMUNOCOMPETENT MICE; NONDIVIDING CELLS; TERMINAL REPEATS; MAMMALIAN-CELLS; DNA-SEQUENCES; REP PROTEINS; AAV VECTOR; HIGH-LEVEL;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
60
Recensione:
Indirizzi per estratti:
Indirizzo: Kleinschmidt, JA Deutscheimersforschungszentrum, Forsch Schwerpunkt Angew Tumorvirol, Neuenh Deutsch Krebsforschungszentrum Neuenheimer Feld 280 Heidelberg Germany D-69120
Citazione:
D. Grimm et al., "Novel tools for production and purification of recombinant adenoassociatedvirus vectors", HUM GENE TH, 9(18), 1998, pp. 2745-2760

Abstract

Standard protocols for the generation of adenoassociated virus type 2 (AAV-2)-based vectors for human gene therapy applications require cotransfection of cells with a recombinant AAV (rAAV) vector plasmid and a packaging plasmid that provides the AAV Pep and cap genes. The transfected cells must also be overinfected with a helper virus, e.g., adenovirus (Ad), which delivers multiple helper functions necessary for rAAV production. Therefore, rAAVstocks produced using these protocols are contaminated with helper adenovirus. The generation of a novel packaging/helper plasmid, pDG, containing all AAV and Ad functions required for amplification and packaging of AAV vector plasmids, is described here. Cotransfection of cells with pDG and an AAVvector plasmid was sufficient for production of infectious rAAV, resultingin helper virus-free rAAV stocks. The rAAV titers obtained using pDG as packaging plasmid were up to Ill-fold higher than those achieved using conventional protocols for rAAV production. Replacement of the AAV-2 p5 promoter by an MMTV-LTR promoter in pDG led to reduced expression of Rep78/68; however, expression of the VP proteins was significantly increased compared withVP levels from standard packaging plasmids. Immunofluorescence analyses showed that the strong accumulation of VP proteins in pDG-transfected cells resulted in enhanced AAV capsid assembly, which is limiting for efficient rAAV production. Furthermore, using a monoclonal antibody highly specific forAAV-2 capsids (A20), an rAAV affinity purification procedure protocol was established. The application of the tools described here led to a significant improvement in recombinant AAV vector production and purification.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 06/06/20 alle ore 14:17:59