Expression in insect cells of genes with overlapping open reading frames, methods and compositions therefor
AssigneeVirovek, Inc.
InventorHaifeng Chen
Life of this patent
Estimating the term…
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.
Claims17
An in vitro method of expressing a plurality of polypeptides encoded by a parvovirus Rep gene, the Rep gene comprising overlapping ORFs in an insect cell, the method comprising:
- a) infecting or transfecting one or more insect cells with a nucleic acid cassette, 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 expresses the plurality polypeptides encoded by the parvovirus Rep gene.
An in vitro method of expressing a plurality of polypeptides encoded by a polyomavirus capsid Cap gene comprising overlapping ORFs in an insect cell, the method comprising:
- a) infecting or transfecting one or more insect cells with a nucleic acid cassette, 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 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 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 expresses the plurality polypeptides encoded by the Cap gene.
An in vitro method of expressing a plurality of polypeptides encoded by a parvovirus capsid (Cap) gene, the Cap gene comprising overlapping ORFs in an insect cell, the method comprising:
- a) infecting or transfecting one or more insect cells with a nucleic acid cassette, 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 expresses the plurality of polypeptides encoded by the parvovirus Cap gene.
Dependencies are inferred from the claim text.