Adenovirus and methods of use thereof
AssigneeThe Trustees of the University of Pennsylvania
InventorsJames M. Wilson, Krishna J. Fisher, Shu-Jen Chen, Matthew Weitzman
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Abstract
A recombinant adenovirus and a method for producing the virus are provided which utilize a recombinant shuttle vector comprising adenovirus DNA sequence for the 5' and 3' cis-elements necessary for replication and virion encapsidation in the absence of sequence encoding viral genes and a selected minigene linked thereto, and a helper adenovirus comprising sufficient adenovirus gene sequences necessary for a productive viral infection. Desirably, the helper gene is crippled by modifications to its 5' packaging sequences, which facilitates purification of the viral particle from the helper virus.
Claims23
A polycation conjugate comprising:
- (a) a crippled adenovirus helper virus comprising a modified adenovirus nucleic acid sequence in place of native adenovirus nucleic acid sequence map units 0-1', which modification reduces the packaging efficiency of said virus, said virus also containing selected adenovirus nucleic acid sequences necessary to direct a productive viral infection;
- and
- (b) a recombinant shuttle vector comprising adenovirus nucleic acid sequences and a minigene, wherein said adenovirus nucleic acid sequences consist of adenovirus 5' and 3' cis-elements necessary for replication and virion encapsidation and said cis-elements flank said minigene;
- and wherein said minigene comprises a selected gene and regulatory sequences which direct expression of said selected gene in a target cell,
- wherein said helper virus is conjugated via a polycation sequence to the recombinant shuttle vector.
A method for producing a recombinant adenovirus, which comprises the steps of:(a) transfecting a selected host cell with
- (i) a recombinant shuttle vector comprising adenovirus nucleic acid sequences and a minigene, wherein said adenovirus nucleic acid sequences consist of adenovirus 5' and 3' cis-elements necessary for replication and virion encapsidation and said cis-elements flank said minigene;
- and wherein said minigene comprises a selected gene operatively linked to regulatory sequences which direct expression of said selected gene in a target cell;
- and
- (ii) a helper adenovirus comprising sufficient adenovirus nucleic acid sequences necessary for a productive viral infection,
- wherein said helper adenovirus is associated with said shuttle vector via a polycation sequence, and
- (b) isolating and purifying said recombinant virus from said cell.
A recombinant adenovirus particle comprising, in an adenovirus capsid:
- a recombinant shuttle vector comprising adenovirus nucleic acid sequences and a minigene, wherein said adenovirus nucleic acid sequences consist of adenovirus 5' and 3' cis-elements necessary for replication and virion encapsidation and said cis-elements flank said minigene;
- and wherein said minigene comprises a selected gene and regulatory sequences which direct expression of said selected gene in a target cell;
- wherein said vector is conjugated via a poly-cation sequence with a helper adenovirus.
A method of purifying a recombinant adenovirus from a host cell in which it is produced, said method comprising the steps of:
- (a) providing a suspension of host cells containing a recombinant adenovirus comprising adenovirus 5' and 3' cis-elements necessary for replication and virion encapsidation and a minigene operatively linked to regulatory sequences which direct expression of said gene in a host cell, wherein said minigene is located between the 5' and 3' cis-elements;
- (b) lysing the host cells by subjecting the suspension to three rounds of freezing and thawing;
- (c) centrifuging the suspension, thereby obtaining clarified extract containing the recombinant adenovirus;
- (d) subjecting the clarified extract to a CsCl step gradient and centrifugation;
- and
- (e) collecting fractions from the CsCl gradient containing the purified recombinant adenovirus.
Dependencies are inferred from the claim text.