Expression in insect cells of genes with overlapping open reading frames, methods and compositions therefor
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.
1. 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.
2. The method of claim 1 , wherein the nucleic acid cassette further comprises a polyadenylation signal situated 3′ to the at least one additional ORF of ii).
3. The method of claim 1 , wherein each of the first insect cell-operable promoter and the second insect cell-operable promoter is independently selected from the group consisting of a p10 promoter and a polh promoter.
4. The method of claim 1 , wherein the nucleic acid cassette is comprised within a plasmid, a virus, another vector, or a combination thereof.
5. 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.
6. The method of claim 5 , wherein the at least one insect cell is selected from the group consisting of a Trichoplusiani BTI-Tn-5B1-4 cell, a Spodoptera frugiperda Sf9 cell, and a Spodoptera frugiperda Sf21 cell.
7. The method of claim 5 , wherein the Cap gene comprises a VP1 ORF and a VP2/VP3 ORF.
8. The method of claim 5 , wherein the nucleic acid cassette further comprises a polyadenylation signal situated 3′ to the at least one additional ORF of ii).
9. The method of claim 5 , wherein each of the first insect cell-operable promoter and the second insect cell-operable promoter is independently selected from the group consisting of a p10 promoter and a polh promoter.
10. The method of claim 5 , wherein the nucleic acid cassette is comprised within a plasmid, a virus, another vector, or a combination thereof.
11. 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.
12. The method of claim 11 , wherein the at least one insect cell is selected from the group consisting of a Trichoplusiani BTI-Tn-5B1-4 cell, a Spodoptera frugiperda Sf9 cell and a Spodoptera frugiperda Sf21 cell.
13. The method of claim 1 , wherein the Rep gene comprises a Rep 78/68 ORF and a Rep 52/40 ORF.
14. The method of claim 11 , wherein the Cap gene comprises a VP1 ORF and a VP2/VP3 ORF.
15. The method of claim 11 , wherein the nucleic acid cassette further comprises a polyadenylation signal situated 3′ to the at least one additional ORF of ii).
16. The method of claim 11 , wherein each of the first insect cell-operable promoter and the second insect cell-operable promoter is independently selected from the group consisting of a p10 promoter and a polh promoter.
17. The method of claim 11 , wherein the nucleic acid cassette is comprised within a plasmid, a virus, another vector, or a combination thereof.