Expression in insect cells of genes with overlapping open reading frames, methods and compositions therefor
AssigneeVirovek, Inc.
InventorHaifeng Chen
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Abstract
The present teachings disclose nucleic acid cassettes for expressing in an insect cell a plurality of polypeptides encoded by a gene comprising overlapping open reading frames (ORFs). A cassette comprises, in 5′ to 3′ order, a) a first insect cell-operable promoter, b) a 5′ portion of a gene comprising a first ORF of the gene, c) an intron comprising a second insect cell-operable promoter, and d) a 3′ portion of the gene comprising at least one additional ORF. Vectors and insect cells comprising the cassettes are also disclosed, as well as methods for production of recombinant adeno-associated virus in insect cells using the cassettes.
Claims18
An in vitro method of producing a plurality of polypeptides encoded by a parvovirus Capsid (Cap) gene, the method comprising:
- a) providing one or more insect cells infected or transfected with a nucleic acid encoding the parvovirus Cap gene, the cassette comprising in 5′ to 3′ order:
- (i) a first cell operable promoter linked operably to a 5′ portion of a first ORF of the parvovirus Cap gene, the first ORF comprising a translation initiation codon;
- and
- (ii) an intron comprising a second insect cell operable promoter, the second promoter operably linked to a 5′ portion of an at least one additional ORF of a parvovirus Cap gene, wherein the at least one additional ORF comprises at least one additional translation initiation codon and overlaps with the 3′ portion of the first ORF, and
- b) culturing the one or more insect cells in a culture medium such that it produces the plurality of polypeptides encoded by the polyomavirus Cap gene.
An in vitro method of producing a plurality of polypeptides encoded by a parvovirus Rep gene, the method comprising:
- a) providing one or more insect cells infected or transfected with a nucleic acid endoding the parvovirus Rep gene, the cassette comprising in 5′ to 3′ order:
- (i) a first cell-operable promoter linked operably to a 5′ portion of a first ORF of the parvovirus Rep gene, the first ORF comprising a translation initiation codon;
- and
- (ii) an intron comprising a second insect cell-operable promoter, the second promoter operably linked to a 5′ portion of an at least one additional ORF of a parvovirus Rep gene, wherein the at least one additional ORF comprises at least one additional translation initiation codon and overlaps with the 3′ portion of the first ORF;
- and
- b) culturing the one or more insect cells in a culture medium such that it produces the plurality of polypeptides encoded by the parvovirus Rep gene.
An in vitro method of producing in an insect cell, a plurality of polypeptides encoded by a polyomavirus Capsid (Cap) gene, the method comprising:
- a) providing one or more insect cells infected or transfected with a nucleic acid expressing in the insect cell a plurality of polypeptides encoded by a polyomavirus Cap gene, the cassette comprising in 5′ to 3′ order:
- (i) a first insect cell operable-promoter linked operably to a 5′ portion of a first ORF of a polyomavirus Cap gene, the first ORF comprising a translation initiation codon;
- and
- (ii) an intron comprising a second insect cell-operable promoter, the second promoter operably linked to a 5′ portion of an at least one additional ORF of a polyomavirus Cap gene, wherein the at least one additional ORF comprises at least one additional translation codon and overlaps with the 3′ portion of the first ORF;
- and
- b) culturing the one or more insect cells in a culture medium such that it produces the plurality of polypeptides encoded by the polyomavirus Cap gene.
Dependencies are inferred from the claim text.