Expression in insect cells of genes with overlapping open reading frames, methods and compositions therefor

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.

1 . A nucleic acid cassette for expressing in an insect cell a plurality of polypeptides encoded by a gene comprising overlapping open reading frames (ORFs), the cassette comprising, 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 that overlaps with at least one additional ORF, wherein the first insect cell-operable promoter is operably linked to the first ORF; the first ORF comprises a first translation initiation codon; the second insect cell-operable promoter is operably linked to the at least one additional ORF, and the at least one additional ORF comprises at least one additional translation initiation codon, and wherein at least one of a), b), c) and d) is heterologous to at least one other of a), b), c), and d). 
 
     
     
         2 . A nucleic acid cassette in accordance with  claim 1 , further comprising e) a polyadenylation signal situated 3′ to d). 
     
     
         3 . A nucleic acid cassette in accordance with  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 . A nucleic acid cassette in accordance with  claim 1 , wherein the gene comprising overlapping ORFs is selected from the group consisting of a Rep gene of an adeno-associated virus (AAV) and a Cap gene of an AAV. 
     
     
         5 . A nucleic acid cassette in accordance with  claim 4 , wherein the first ORF is a Rep 78/68 ORF and the at least one additional ORF is a Rep 52/40 ORF. 
     
     
         6 . A nucleic acid cassette in accordance with  claim 5 , wherein the first promoter is a p10 promoter and the second promoter is a polh promoter. 
     
     
         7 . A nucleic acid cassette in accordance with  claim 4 , wherein the first ORF is a VP1 ORF and the at least one additional ORF is a VP2/VP3 ORF. 
     
     
         8 . A nucleic acid cassette in accordance with  claim 7 , wherein the first promoter is a first polh promoter and the second promoter is a second polh promoter. 
     
     
         9 . A vector comprising the nucleic acid cassette of  claim 1 . 
     
     
         10 . A vector in accordance with  claim 9 , wherein the vector is selected from the group consisting of a plasmid, a virus and a combination thereof 
     
     
         11 . An insect cell in vitro comprising a nucleic acid cassette of  claim 1 . 
     
     
         12 . An insect cell in vitro comprising a first nucleic acid cassette of  claim 1  and a second nucleic acid cassette of  claim 1 , wherein the first nucleic acid cassette comprises a Rep gene comprising a Rep 78/68 ORF and a Rep 52/40 ORF and the second nucleic acid cassette comprises a Cap gene comprising a VP1 ORF and a VP2/VP3 ORF. 
     
     
         13 . An insect cell in accordance with  claim 12 , further comprising an additional nucleic acid cassette comprising, in 5′ to 3′ order,
 a first inverted terminal repeat (ITR) of an adeno-associated virus; 
 a mammalian cell-operable promoter; 
 a transgene; 
 a polyadenylation signal; and 
 a second ITR of an AAV. 
 
     
     
         14 . An insect cell in accordance with  claim 13 , wherein the transgene is a reporter gene encoding a polypeptide selected from the group consisting of a chloramphenicol acetyl transferase, a β-galactosidase, a β-glucuronidase, a  renilla luciferase , a firefly luciferase, a green fluorescent protein (GFP), a red fluorescent protein (RFP) and an alkaline phosphatase. 
     
     
         15 . An insect cell in accordance with  claim 13 , wherein the transgene comprises an ORF encoding a polypeptide selected from the group consisting of a polypeptide hormone, an interferon, a blood clotting factor, a vaccine and an erythropoietin. 
     
     
         16 . An insect cell in accordance with  claim 11 , wherein the nucleic acid cassette is integrated into the genome of the insect cell. 
     
     
         17 . An insect cell in accordance with  claim 13 , wherein at least one of the nucleic acid cassette and the additional nucleic acid cassette is integrated into the genome of the insect cell. 
     
     
         18 . An insect cell in accordance with  claim 11 , wherein the cell is selected from the group consisting of a  Trichoplusia ni  BTI-Tn-5B1-4 cell, a  Spodoptera frugiperda  Sf9 cell and a  Spodoptera frugiperda  Sf21 cell. 
     
     
         19 . A cell culture comprising:
 a plurality of insect cells of  claim 11 ; and   a culture medium.   
     
     
         20 . A cell culture comprising:
 a plurality of insect cells of  claim 13 ; and   a culture medium comprising at least 10 13  AAV genomes/liter.   
     
     
         21 . A cell culture in accordance with  claim 20 , wherein the culture medium comprising at least 10 13  AAV genomes/liter is a culture medium comprising at least 10 14  AAV genomes/liter. 
     
     
         22 - 27 . (canceled) 
     
     
         28 . A nucleic acid comprising the cassette of  claim 2 , further comprising a second cassette, the second cassette comprising, in 5′ to 3′ order:
 a second polyadenylation signal; 
 a 3′ portion of a second gene comprising overlapping ORFs; 
 a second intron comprising a third insect-operable promoter; 
 a 5′ portion of the second gene; and 
 a fourth insect-operable promoter. 
 
     
     
         29 . A nucleic acid in accordance with  claim 28 , wherein the second cassette is in an anti-sense orientation relative to the first cassette. 
     
     
         30 . A nucleic acid in accordance with  claim 28 , wherein the second cassette is in a sense orientation relative to the first cassette. 
     
     
         31 . A nucleic acid in accordance with  claim 28 , wherein the first cassette comprises an AAV Rep gene comprising a Rep 78/68 ORF and a Rep 52/40 ORF and the second cassette comprises an AAV Cap gene comprising a VP1 ORF and a VP2/VP3 ORF.