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.
Claims26
A nucleic acid cassette for expressing in an insect cell a plurality of polypeptides encoded by a parvovirus capsid 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 the parvovirus capsid gene, the first ORF comprising a translation initiation codon,
- (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 capsid 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.
A nucleic acid cassette for expressing in an insect cell a plurality of polypeptides encoded by a parvovirus Rep 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 the parvovirus Rep gene, the first ORF comprising a translation initiation codon,
- (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.
A nucleic acid cassette for expressing in an insect cell a plurality of polypeptides encoded by a polyomavirus 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 capsid gene, the first ORF comprising a translation initiation codon,
- (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 capsid 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.
Dependencies are inferred from the claim text.