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US 6,440,728 B1Granted
Chimeric vectors comprising a phage packaging site and a portion derived from the genome of a eukaryotic virus
AssigneeGenVec, Inc.
InventorsDuncan L. McVey, Douglas E. Brough, Mohammed Zuber, Imre Kovesdi
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Abstract
The present invention provides an improved method of making eukaryotic gene transfer vectors comprising homologous recombining lambdid vectors with a second DNA in a bacterium to generate novel recombinant eukaryotic viral gene transfer vectors as well as a novel lambdid vector used in the inventive method and an inventive system comprising the novel lambdid vector.
Claims18
A method of making and packaging a DNA vector comprising:
- (i) transducing a first bacterial cell with a first DNA vector and a second DNA vector, wherein the first DNA vector comprises (a) a portion of a eukaryotic viral genome comprising an ITR and a regulatable anti-selective gene flanked by a first DNA segment and a second DNA segment that have sufficient homology to the second DNA vector to mediate a double homologous recombination event with the second DNA vector and (b) a phage packaging site, wherein (a) does not comprise (b),
- (ii) maintaining the first bacterial cell under conditions to allow double homologous recombination to occur and produce a product DNA vector comprising the phage packaging site that does not contain the regulatable anti-selective gene, and
- (iii) placing the product DNA vector under in vivo conditions such that the product DNA vector is packaged in a phage capsid in the first bacterial cell.
A method of generating a eukaryotic gene transfer vector, the method comprising:
- (i) transducing a bacterial cell with (a) a first DNA, wherein the first DNA comprises a DNA vector comprising (1) a portion of eukaryotic viral genome comprising an ITR, (2) a regulatable anti-selection gene, (3) a phage packaging site, (4) an independent positive selection gene, and (5) a deficient or conditionally-deficient lambdoid origin of replication, and (b) a second DNA, which comprises at least one DNA segment having high homology to the first DNA;
- (ii) supplying (a) a source of gene products that complement the deficiency or conditional deficiency in the deficient or conditionally deficient lambdoid origin of replication and (b) a source of phage capsid components sufficient to encapsidate the DNA vector comprising the phage packaging site of the first DNA;
- (iii) obtaining a population of encapsidated DNA vectors;
- (iv) transducing the population of encapsidated DNA vectors into a population of bacteria;
- (v) growing the population of bacteria transduced by the population of encapsidated DNA vectors under selective conditions sufficient to select for bacteria harboring the independent positive selection gene and to select against bacteria harboring the negative selection gene or the stringently regulated promoter operably linked to a strong bacterial signal for the initiation of translation;
- and
- (vi) culturing a single bacterium that propagates under the selective conditions of step (v) in order to obtain a culture of a bacterium comprising a DNA vector comprising a eukaryotic gene transfer vector.
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Dependencies are inferred from the claim text.