Abstract: Methods are provided for purifying non-chromosomal nucleic acid molecules from cells, comprising: the general steps of (a) lysing cells to form a nucleic acid-containing lysate, and (b) applying the lysate to a depth filter in order to obtain a clarified solution containing purified non-chromosomal nucleic acid molecules.
Type:
Grant
Filed:
July 29, 1999
Date of Patent:
July 31, 2001
Assignee:
Chiron Corporation
Inventors:
Denice M. Mittelstaedt, David Chi-Tang Hsu
Abstract: Nucleic acid molecule are provided comprising a nucleic acid sequence which encodes, in order, an alphavirus capsid, a signal peptide, and an alphavirus E1 or E2 glycoprotein. Also provided are vectors encoding such nucleic acid molecules, and use of such vectors or expression cassettes to generate recombinant alphavirus particles and alphavirus packaging cell lines. In addition, modified alphavirus vector constructs are provided that permit reduced transgene expression during vector packaging, as well as methods of using such vector constructs for the production of alphavirus vector particles.
Type:
Grant
Filed:
December 30, 1999
Date of Patent:
June 5, 2001
Assignee:
Chiron Corporation
Inventors:
John Polo, Barbara Beli, Thomas W. Dubensky, Jr., Stephen F. Hardy, Silvia Perri
Abstract: The invention provides a method for producing purified replication-defective recombinant AAV virions. The method comprises introducing into a suitable host cell an AAV vector, an AAV helper construct and an adenoplasmid accessory construct into the host cell. The adenoplasmid accessory plasmid is composed adenovirus plasmid DNA unable to be packaged into adenoviral particles because it lacks packaging signal sequence(s) or it contains additional sequences making it too large to package. The host cell is cultured to produce crude rAAV virions and then lysed. The resulting cell lysate is applied to a chromatographic column containing sulfonated cellulose or subjected to cesium chloride equilibrium gradient centrifugation and the purified rAAV virions are recovered.