Method for propagating adenoviral vectors encoding inhibitory gene products
View Patent ↗The invention provides a method of propagating an adenoviral vector. The method comprises (a) providing a cell comprising a cellular genome comprising a nucleic acid sequence encoding a tetracycline operon repressor protein (tetR), and (b) contacting the cell with an adenoviral vector comprising a heterologous nucleic acid sequence encoding a toxic protein. The heterologous nucleic acid sequence is operably linked to a promoter and one or more tetracycline operon operator sequences (tetO), and expression of the heterologous nucleic acid sequence is inhibited in the presence of tetR, such that the adenoviral vector is propagated. The invention also provides a system comprising the aforementioned cell and adenoviral vector.
1. A method of propagatin a non-subgroup C adenoviral vector, which method comprises:
(a) providing a cell comprising a cellular genome comprising a nucleic acid sequence encoding a tetracycline operon repressor protein (tetR),
(b) expressing the nucleic acid sequence encoding tetR to produce tetR, and
(c) contacting the cell with a non-subgroup C adenoviral vector in the absence of tetracycline, wherein the non-subgroup C adenoviral vector has an adenoviral genome comprising a heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence (i) encodes a protein that inhibits propagation of the adenoviral vector in the cell and (ii) is operably linked to a promoter and one or more tetracycline operon operator sequences (tetO), so as to transfect the cell with the non-subgroup C adenoviral vector, wherein expression of the heterologous nucleic acid sequence is inhibited in the presence of tetR, and a non-subgroup C adenoviral vector is propagated,
wherein the non-subgroup C adenoviral vector is replication-deficient, and
wherein the non-subgroup C adenoviral vector requires, at most, complementation of the E4 region of the adenoviral genome for replication.
2. The method of 1 wherein the non-subgroup C adenoviral vector lacks all or part of the E3 region of the adenoviral genome.
3. The method of claim 1 , wherein the heterologous nucleic acid sequence encodes an env, gag, or pol protein from clades A, B, or C of a human immunodeficiency virus (HIV), or a fusion protein comprising any of the foregoing.
4. The method of claim 1 , wherein the heterologous nucleic acid sequence encodes an E protein, an M protein, or a spike protein of a severe acute respiratory syndrome (SARS) virus.
5. The method of claim 1 , wherein the heterologous nucleic acid sequence encodes a transforming growth factor β(TGFβ), an antibiotic, a malaria protein, or a nitric oxide synthase.
6. The method of claim 1 , wherein the heterologous nucleic acid sequence encodes protein 1A, 1B, 1C, 1D, 2A, 2B, 2C, 3A, 3B, 3C, or 3D of a foot-and-mouth disease virus (FMD).
7. A system comprising:
(a) a cell comprising a cellular genome comprising a nucleic acid sequence encoding a tetracycline operon repressor protein (tetR), which can be expressed to produce tetR, and
(b) a non-subgroup C adenoviral vector having an adenoviral genome comprising a heterologous nucleic acid sequence, wherein
(1) the heterologous nucleic acid sequence (i) encodes a protein that inhibits propagation of the non-subgroup C adenoviral vector in the cell and (ii) is operably linked to a promoter and one or more tetracycline operon operator sequences (tetO),
(2) the non-subgroup C adenoviral vector can transfect the cell and be propagated in the cell, and
(3) the system lacks tetracycline,
wherein the non-subgroup C adenoviral vector is replication-deficient,
wherein the non-subgroup C adenoviral vector requires, at most, complementation of the E4 region of the adenoviral genome for replication.
8. The system of claim 7 , wherein the non-subgroup C adenoviral vector lacks all or part of the E3 region of the adenoviral genome.
9. The system of claim 7 , wherein the heterologous nucleic acid sequence encodes an env, gag, or pol protein from clades A, B, or C of a human immunodeficiency virus (HIV), or a fusion protein comprising any of the foregoing.
10. The system of claim 7 , wherein the heterologous nucleic acid sequence encodes an E protein, an M protein, or a spike protein of a severe acute respiratory syndrome (SARS) virus.
11. The system of claim 7 , wherein the heterologous nucleic acid sequence encodes a transforming growth factor β(TGFβ), an antibiotic, a malaria protein, or a nitric oxide synthase.
12. The system of claim 7 , wherein the heterologous nucleic acid sequence encodes protein 1A, 1B, 1C, 1D, 2A, 2B, 2C, 3A, 3B, 3C, or 3D of a foot-and-mouth disease virus (FMD).
13. The method of claim 1 , wherein the heterologous nucleic acid sequence encodes a viral protein from family Coronaviridae.
14. The method of claim 1 , wherein the heterologous nucleic acid sequence encodes a viral protein from family Picornaviridae.
15. The method of claim 14 , wherein the heterologous nucleic acid sequence encodes a poliovirus protein.
16. The method of claim 14 , wherein the heterologous nucleic acid sequence encodes an aphthovirus protein.
17. The method of claim 16 , wherein the heterologous nucleic acid sequence encodes a foot-and-mouth disease viral protein.
18. The method of claim 7 , wherein the heterologous nucleic acid sequence encodes a viral protein from family Coronaviridae.
19. The method of claim 7 , wherein the heterologous nucleic acid sequence encodes a viral protein from family Picornaviridae.
20. The method of claim 19 , wherein the heterologous nucleic acid sequence encodes a poliovirus protein.
21. The method of claim 19 , wherein the heterologous nucleic acid sequence encodes an aphthovirus protein.
22. The method of claim 21 , wherein the heterologous nucleic acid sequence encodes a foot-and-mouth disease viral protein.