Packaging cell line for diphtheria toxin expressing non-replicating adenovirus
The invention provides a packaging cell line for production of diphtheria toxin (DT) expressing, non-replicating adenovirus for use in suicide gene therapy of cancer cells, as well as production of immunotoxins. Also provided are methods for producing diphtheria toxin (DT) expressing, non-replicating adenovirus, methods for producing immunotoxins, and adenovirus and immunotoxins produced by those methods. Further provided are methods for making a cell resistant to diphtheria toxin.
1. A method of killing a cell that is sensitive to DT-A or PEA, comprising infecting the cell with an adenovirus produced by a packaging cell line, wherein the adenovirus comprises an adenoviral vector comprising a promoter operably linked to a nucleic acid encoding the A subunit of diphtheria toxin (DT-A) or Pseudomonas Exotoxin A (PEA), and wherein the cell line is capable of producing adenovirus that expresses the A subunit of diphtheria toxin (DT-A) or Pseudomonas Exotoxin A (PEA), wherein the cell line does not produce replication-competent adenovirus when used in conjunction with non-overlapping E1-deleted adenovirus, wherein the cell line is resistant to DTA and PEA and wherein the cell line has a mutated human EF-2 gene that encodes an EF-2 protein that is mutated at codon 705.
2. The method of claim 1 , wherein the cell is a cancer cell.
3. A method of selectively killing a cell in a subject, comprising administering a therapeutically effective amount of an adenovirus to the subject wherein the adenovirus is produced by a packaging cell line, wherein the adenovirus comprises an adenoviral vector comprising a promoter operably linked to a nucleic acid encoding the A subunit of diphtheria toxin (DT-A) or Pseudomonas Exotoxin A (PEA), and wherein the cell line is capable of producing adenovirus that expresses the A subunit of diphtheria toxin (DT-A) or Pseudomonas Exotoxin A (PEA), and wherein the cell line does not produce replication-competent adenovirus when used in conjunction with non-overlapping E1-deleted adenovirus, wherein the cell line has a mutated human EF-2 gene that encodes an EF-2 protein that is mutated at codon 705, wherein the adenovirus comprises a tissue-specific promoter or enhancer that controls the expression of the DT-A or PEA wherein the tissue-specific promoter or enhancer is active only in the cell and not in other cells, thereby killing the cell but not other cells.
4. The method of claim 3 , wherein the cell is a cancer cell.
5. A method of treating a subject suffering from cancer comprising administering a therapeutically effective amount of the adenovirus to the subject, wherein the adenovirus is produced by a packaging cell line, wherein the adenovirus comprises an adenoviral vector comprising a promoter operably linked to a nucleic acid encoding the A subunit of diphtheria toxin (DT-A) or Pseudomonas Exotoxin A (PEA), and wherein the cell line is capable of producing adenovirus that expresses the A subunit of diphtheria toxin (DT-A) or Pseudomonas Exotoxin A (PEA), and wherein the cell line does not produce replication-competent adenovirus when used in conjunction with non-overlapping E1-deleted adenovirus, wherein the cell line has a mutated human EF-2 gene that encodes an EF-2 protein that is mutated at codon 705, and wherein the cell line is resistant to DTA and PEA thereby testing said cancer.
6. The method of any one of claims 1 , 3 , or 5 , wherein the glycine residue at codon 705 of the EF-2 protein is mutated to arginine.
7. The method of claim 1 , wherein the packaging cell lines are resistant to about 10 −9 M diphtheria toxin.
8. The method of claim 1 , wherein the packaging cell lines contain the adenovirus E1 region.
9. The method of claim 1 , wherein the packaging cell lines contain the adenoviruss serotype 5 (Ad5) E1-A and E1-B encoding sequences.
10. The method of claim 1 , wherein the packaging cell lines are derived from PER.C6 cells.