Tet-r expression cassettes and cell lines

A recombinant polynucleotide comprising at least one Tetracycline operon repressor protein (TetR) expression cassette that is optionally SEQ ID NO:

• 7 or an expression cassette having at least about 90% identity therewith;
• wherein each TetR expression cassette comprises a CMV promoter sequence that is optionally SEQ ID NO:
• 8 or a sequence having at least about 90% identity therewith, a rabbit beta-globin intron sequence that is optionally SEQ ID NO:
• 9 or a sequence having at least about 90% identity therewith, and a poly(A) polynucleotide sequence operably linked to a coding sequence for the TetR expression sequence is optionally SEQ ID NO:
• 3 or a sequence having at least about 90% identity therewith.

A method for producing an adenoviral vector encoding a transgene, the method comprising:

• a) obtaining a Tetracycline operon repressor protein (TetR) expressing cell comprising an exogenous polynucleotide in its genome, the polynucleotide comprising at least one Tetracycline operon repressor protein (TetR) expression cassette, each TetR expression cassette comprising a CAG promoter/chimeric intron sequence that is optionally SEQ ID NO:
• 2, and a poly(A) polynucleotide sequence that is optionally SEQ ID NO:
• 4 or SEQ ID NO:
• 6, operably linked to a coding sequence for the TetR gene that is optionally SEQ ID NO:
• 3;
• b) transfecting the TetR expressing cells with at least one Tetracycline operon operator (TetO) polynucleotide sequence linked to the promoter in a recombinant Ad genomic plasmid to produce a replication-deficient Ad vector containing at least one TetO polynucleotide sequence and encoding at least one transgene;
• and,
• c) isolating the Ad vector particles containing at least one TetO polynucleotide sequence and encoding at least one transgene;
• wherein:
• the polynucleotides of part a) have at least about 90% identity with any of SEQ ID NOs. 2, 3, 4 or 6;
• and,
• the number of Ad vector particles containing at least one TetO polynucleotide sequence and encoding at least one transgene isolated in step c) is at least about any of two, 10, 15, 20, 50, 100, 150, 200, 250, or 300 times the number of Ad vector particles obtained from an Ad vector package cell lacking the TetR polynucleotide.

A method for producing an adenoviral vector encoding a transgene, the method comprising:

• a) obtaining a Tetracycline operon repressor protein (TetR) recombinant cell comprising an exogenous polynucleotide in its genome, the polynucleotide comprising at least one Tetracycline operon repressor protein (TetR) expression cassette, each TetR expression cassette comprising a CMV promoter sequence (SEQ ID NO:
• 8), a rabbit beta globin intron sequence (SEQ ID NO:
• 9), and a poly(A) polynucleotide sequence (SEQ ID NO:
• 4) operably linked to a coding sequence for the TetR gene (SEQ ID NO:
• 3);
• b) transfecting the TetR expressing cells with at least one Tetracycline operon operator (TetO) polynucleotide sequence linked to the promoter in a recombinant Ad genomic plasmid to a replication-deficient Ad vector containing at least one TetO polynucleotide sequence and encoding at least one transgene;
• and,
• c) isolating the Ad vector particles containing at least one TetO polynucleotide sequence and encoding at least one transgene;
• wherein:
• the polynucleotides of part a) have at least about 90% identity with any of SEQ ID NOs. 3, 4 8, or 9;
• and,
• the number of Ad vector particles containing at least one TetO polynucleotide sequence and encoding at least one transgene isolated in step c) is at least about any of two, 10, 15, 20, 50, 100, 150, 200, 250, or 300 times the number of Ad vector particles obtained from an Ad vector package cell lacking the TetR polynucleotide.