Inducible expression systems

Abstract

Provided is an rtTA and single chain rtTA variants and uses thereof for inducible expression of a nucleic acid of interest. Nucleic acid molecules comprising an improved rtTA and/or sc rtTA sequence according to the invention are also provided, as well as vectors, replicons and cells comprising such nucleic acid molecules.

Claims

1. A method for inducibly expressing a nucleic acid sequence of interest, the method comprising: providing a nucleic acid construct comprising said nucleic acid sequence of interest operably linked to an inducible gene expression system that comprises a reverse tetracycline-controlled transactivator (rtTA) encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprising a mutation in a codon at rtTA amino acid position 9, and/or 19, and/or 37, and/or 56, and/or 67, and/or 68, and/or 138, and/or 157, and/or 171, and/or 177, and/or 195; introducing said nucleic acid construct to a suitable expression system; and allowing for inducible expression of said nucleic acid sequence of interest.

2. The method according to claim 1, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence further comprise a mutation in a codon at rtTA amino acid position 12, and/or 86, and/or 209.

3. The method according to claim 1, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon, and/or a codon at rtTA position 37 that differs in at least two nucleotides from an alanine, a lysine or a serine codon, and/or a glutamine or lysine codon at rtTA amino acid position 56.

4. The method according to claim 1, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprise a glycine codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon.

5. The method according to claim 1, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprise an alanine, cysteine, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, arginine, serine, threonine, valine, tryptophan or tyrosine codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon.

6. The method according to claim 1, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprise a histidine, a leucine or an arginine codon at rtTA amino acid position 37 that differs in at least two nucleotides from an alanine, a lysine or a serine codon.

7. The method according to claim 1, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 9 encoding isoleucine, and/or a codon at rtTA amino acid position 19 encoding alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine, and/or a codon at rtTA amino acid position 37 encoding cysteine, methionine, glutamine, threonine, histidine, leucine or arginine, and/or a codon at rtTA amino acid position 56 encoding lysine or glutamine, and/or a codon at rtTA amino acid position 67 encoding serine, and/or a codon at rtTA amino acid position 68 encoding arginine, and/or a codon at rtTA amino acid position 86 encoding tyrosine, and/or a codon at rtTA amino acid position 138 encoding aspartate or serine, and/or a codon at rtTA amino acid position 157 encoding lysine, and/or a codon at rtTA amino acid position 171 encoding lysine, and/or a codon at rtTA amino acid position 177 encoding leucine, and/or a codon at rtTA amino acid position 195 encoding serine, and/or a codon at rtTA amino acid position 209 encoding threonine.

8. The method according to claim 1, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprise at least one mutation as depicted in FIG. 14B or FIG. 14C.

9. The method according to claim 1, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprise at least one codon mutation as compared to a rtTA encoding nucleic acid sequence depicted in FIG. 19.

10. The method according to claim 1, wherein said nucleic acid of interest is expressed in a higher eukaryotic expression system.

11. The method according to claim 10, wherein said nucleic acid of interest is expressed in a mammalian cell.

12. The method according to claim 1, wherein said nucleic acid of interest comprises a viral sequence essential for replication.

13. The method according to claim 1, wherein said nucleic acid of interest comprises at least part of an HIV genome essential for replication.

14. A synthetic or recombinant nucleic acid sequence comprising a rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutated codon at rtTA amino acid position 9, and/or 19, and/or 37, and/or 56, and/or 67, and/or 68, and/or 138, and/or 157, and/or 171, and/or 177, and/or 195.

15. The synthetic or recombinant nucleic acid sequence according to claim 14, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence further comprises a mutation in a codon at rtTA amino acid position 12, and/or 86, and/or 209.

16. The synthetic or recombinant nucleic acid sequence according to claim 14, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon and/or a codon at rtTA position 37 that differs in at least two nucleotides from an alanine, a lysine or a serine codon, and/or a glutamine or lysine codon at rtTA amino acid position 56.

17. The synthetic or recombinant nucleic acid sequence according to claim 14, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a glycine codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon.

18. The synthetic or recombinant nucleic acid sequence according to claim 14, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises an alanine, cysteine, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, arginine, serine, threonine, valine, tryptophan or tyrosine codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon.

19. The synthetic or recombinant nucleic acid sequence according to claim 14, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a histidine, a leucine or an arginine codon at rtTA amino acid position 37 that differs in at least two nucleotides from an alanine, a lysine or a serine codon.

20. The synthetic or recombinant nucleic acid sequence according to claim 14, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 9 encoding isoleucine, and/or a codon at rtTA amino acid position 19 encoding alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine, and/or a codon at rtTA amino acid position 37 encoding cysteine, methionine, glutamine, threonine, histidine, leucine or arginine, and/or a codon at rtTA amino acid position 56 encoding lysine or glutamine, and/or a codon at rtTA amino acid position 67 encoding serine, and/or a codon at rtTA amino acid position 68 encoding arginine, and/or a codon at rtTA amino acid position 86 encoding tyrosine, and/or a codon at rtTA amino acid position 138 encoding aspartate or serine, and/or a codon at rtTA amino acid position 157 encoding lysine, and/or a codon at rtTA amino acid position 171 encoding lysine, and/or a codon at rtTA amino acid position 177 encoding leucine, and/or a codon at rtTA amino acid position 195 encoding serine, and/or a codon at rtTA amino acid position 209 encoding threonine.

21. The synthetic or recombinant nucleic acid sequence according to claim 14, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises at least one mutation as depicted in FIG. 14B or FIG. 14C.

22. The synthetic or recombinant nucleic acid sequence according to claim 14, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises at least one mutation as compared to an rtTA encoding nucleic acid sequence depicted in FIG. 19.

23. A synthetic or recombinant amino acid sequence encoded by the nucleic acid sequence according to claim 14.

24. A synthetic or recombinant amino acid sequence comprising a rtTA sequence and/or a single chain rtTA sequence, which rtTA sequence and/or single chain rtTA sequence comprises an isoleucine at position 9, and/or an alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine at position 19, and/or a cysteine, methionine, glutamine, threonine, histidine, leucine or arginine at position 37, and/or a lysine or glutamine at position 56, and/or a serine at position 67, and/or an arginine at position 68, and/or a tyrosine at position 86, and/or an aspartate or serine at position 138, and/or a lysine at position 157, and/or a lysine at position 171, and/or a leucine at position 177, and/or a serine at position 195, and/or a threonine at position 209.

25. In a method of inducing expression of a nucleic acid sequence of interest, the improvement comprising: utilizing the synthetic or recombinant nucleic acid sequence of claim 14 for inducible expression of a nucleic acid sequence of interest.

26. In a method of inducing expression of a nucleic acid sequence of interest, the improvement comprising: utilizing the amino acid sequence encoded by any one of the nucleic acid sequences of claim 24 for inducible expression of a nucleic acid sequence of interest.

27. In a method of tetracycline-inducible and/or minocycline-inducible expression of a nucleic acid sequence of interest, the improvement comprising: utilizing the recombinant nucleic acid sequence comprising an rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutation or a combination of mutations as depicted in FIG. 15, except for the wild type rtTA and the F86Y A209T variant, for tetracycline-inducible and/or minocycline-inducible expression of a nucleic acid of interest.

28. A vector comprising the nucleic acid sequence of claim 14.

29. An inducible viral replicon, comprising: the nucleic acid sequence of claim 14, and at least one viral sequence that is essential for replication under direct or indirect control of said nucleic acid sequence.

30. The inducible viral replicon according to claim 29, comprising all viral sequences essential for replication under direct or indirect control of said nucleic acid sequence.

31. The inducible viral replicon according to claim 29, which is derived from a human immunodeficiency virus.

32. The inducible viral replicon of claim 29, wherein the nucleic acid sequence is inserted into the nef gene.

33. The inducible viral replicon of claim 29, further comprising at least one tetO motif in at least one functional LTR.

34. The inducible viral replicon of claim 33, further comprising at least 2, 4, 6, or 8 such elements in at least one functional LTR.

35. The inducible viral replicon of claim 29, wherein at least one LTR is modified to avoid reversion to wild type virus.

36. A method for producing a virus dependent upon an inducing agent for replication, the method comprising: providing a permissive cell with the inducible viral replicon of claim 29, culturing said cell in the presence of said inducing agent, and harvesting said dependent virus from said culture.

37. The method according to claim 36, in which said dependent virus is a human immunodeficiency virus.

38. The method according to claim 36, in which said virus is an attenuated virus.

39. A virus dependent on an inducing agent for replication obtainable by the method according to claim 36.

40. The virus according to claim 39, which is a human immunodeficiency virus.

41. A method for the controlled replication of a virus or a viral replicon, the method comprising: providing a permissive cell with the inducible viral replicon of claim 29; culturing said cell in the presence of said inducing agent; and manipulating the amount of inducing agent present.

42. An isolated cell comprising the nucleic acid sequence of claim 14.

.Iadd.43. A method for inducibly expressing a nucleic acid sequence of interest, the method comprising the steps of: providing a nucleic acid construct comprising said nucleic acid sequence of interest operably linked to an inducible gene expression system that comprises a reverse tetracycline-controlled transactivator (rtTA) encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprising a mutation in a codon at rtTA amino acid position 67, optionally with one or more additional mutations in a codon at rtTA amino acid position 9, 12, 19, 37, 56, 68, 86, 138, 157, 171, 177, 195 or 209; introducing said nucleic acid construct to a suitable expression system; and allowing for inducible expression of said nucleic acid sequence of interest. .Iaddend.

.Iadd.44. The method according to claim 43, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon, and/or a codon at rtTA position 37 that differs in at least two nucleotides from an alanine, a lysine or a serine codon, and/or a glutamine or lysine codon at rtTA amino acid position 56. .Iaddend.

.Iadd.45. The method according to claim 43, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a glycine codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon. .Iaddend.

.Iadd.46. The method according to claim 43, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises an alanine, cysteine, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, arginine, serine, threonine, valine, tryptophan or tyrosine codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon. .Iaddend.

.Iadd.47. The method according to claim 43, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a histidine, a leucine or an arginine codon at rtTA amino acid position 37 that differs in at least two nucleotides from an alanine, a lysine or a serine codon. .Iaddend.

.Iadd.48. The method according to claim 43, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 9 encoding isoleucine, and/or a codon at rtTA amino acid position 19 encoding alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine, and/or a codon at rtTA amino acid position 37 encoding cysteine, methionine, glutamine, threonine, histidine, leucine or arginine, and/or a codon at rtTA amino acid position 56 encoding lysine or glutamine, and/or a codon at rtTA amino acid position 67 encoding serine, and/or a codon at rtTA amino acid position 68 encoding arginine, and/or a codon at rtTA amino acid position 86 encoding tyrosine, and/or a codon at rtTA amino acid position 138 encoding aspartate or serine, and/or a codon at rtTA amino acid position 157 encoding lysine, and/or a codon at rtTA amino acid position 171 encoding lysine, and/or a codon at rtTA amino acid position 177 encoding leucine, and/or a codon at rtTA amino acid position 195 encoding serine, and/or a codon at rtTA amino acid position 209 encoding threonine. .Iaddend.

.Iadd.49. The method according to claim 43, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises at least one mutation as depicted in FIG. 14B or FIG. 14C. .Iaddend.

.Iadd.50. The method according to claim 43, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises at least one codon mutation as compared to a rtTA encoding nucleic acid sequence depicted in FIG. 19. .Iaddend.

.Iadd.51. The method according to claim 43, wherein said nucleic acid of interest is expressed in a higher eukaryotic expression system. .Iaddend.

.Iadd.52. The method according to claim 51, wherein said nucleic acid of interest is expressed in a mammalian cell. .Iaddend.

.Iadd.53. The method according to claim 43, wherein said nucleic acid of interest comprises a viral sequence essential for replication. .Iaddend.

.Iadd.54. The method according to claim 43, wherein said nucleic acid of interest comprises at least part of an HIV genome essential for replication. .Iaddend.

.Iadd.55. A synthetic or recombinant nucleic acid sequence comprising a rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutated codon at rtTA amino acid position 67, optionally with one or more additional mutations in a codon at rtTA amino acid position 9, 12, 19, 37, 56, 68, 86, 138, 157, 171, 177, 195 or 209. .Iaddend.

.Iadd.56. The synthetic or recombinant nucleic acid sequence according to claim 55, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon and/or a codon at rtTA position 37 that differs in at least two nucleotides from an alanine, a lysine or a serine codon, and/or a glutamine or lysine codon at rtTA amino acid position 56. .Iaddend.

.Iadd.57. The synthetic or recombinant nucleic acid sequence according to claim 55, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a glycine codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon. .Iaddend.

.Iadd.58. The synthetic or recombinant nucleic acid sequence according to claim 55, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises an alanine, cysteine, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, arginine, serine, threonine, valine, tryptophan or tyrosine codon at rtTA amino acid position 19 that differs in at least two nucleotides from a glutamate codon. .Iaddend.

.Iadd.59. The synthetic or recombinant nucleic acid sequence according to claim 55, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a histidine, a leucine or an arginine codon at rtTA amino acid position 37 that differs in at least two nucleotides from an alanine, a lysine or a serine codon. .Iaddend.

.Iadd.60. The synthetic or recombinant nucleic acid sequence according to claim 55, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 9 encoding isoleucine, and/or a codon at rtTA amino acid position 19 encoding alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine, and/or a codon at rtTA amino acid position 37 encoding cysteine, methionine, glutamine, threonine, histidine, leucine or arginine, and/or a codon at rtTA amino acid position 56 encoding lysine or glutamine, and/or a codon at rtTA amino acid position 67 encoding serine, and/or a codon at rtTA amino acid position 68 encoding arginine, and/or a codon at rtTA amino acid position 86 encoding tyrosine, and/or a codon at rtTA amino acid position 138 encoding aspartate or serine, and/or a codon at rtTA amino acid position 157 encoding lysine, and/or a codon at rtTA amino acid position 171 encoding lysine, and/or a codon at rtTA amino acid position 177 encoding leucine, and/or a codon at rtTA amino acid position 195 encoding serine, and/or a codon at rtTA amino acid position 209 encoding threonine. .Iaddend.

.Iadd.61. The synthetic or recombinant nucleic acid sequence according to claim 55, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises at least one mutation as depicted in FIG. 14B or FIG. 14C. .Iaddend.

.Iadd.62. The synthetic or recombinant nucleic acid sequence according to claim 55, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises at least one mutation as compared to an rtTA encoding nucleic acid sequence depicted in FIG. 19. .Iaddend.

.Iadd.63. A synthetic or recombinant amino acid sequence encoded by the nucleic acid sequence according to claim 55. .Iaddend.

.Iadd.64. In a method of inducing expression of a nucleic acid sequence of interest, the improvement comprising: utilizing the synthetic or recombinant nucleic acid sequence of claim 55 for inducible expression of a nucleic acid sequence of interest. .Iaddend.

.Iadd.65. A vector comprising the nucleic acid sequence of claim 55. .Iaddend.

.Iadd.66. An inducible viral replicon, comprising: the nucleic acid sequence of claim 55, and at least one viral sequence that is essential for replication under direct or indirect control of said nucleic acid sequence. .Iaddend.

.Iadd.67. The inducible viral replicon according to claim 66, comprising all viral sequences essential for replication under direct or indirect control of said nucleic acid sequence. .Iaddend.

.Iadd.68. The inducible viral replicon according to claim 66, which is derived from a human immunodeficiency virus. .Iaddend.

.Iadd.69. The inducible viral replicon of claim 66, wherein the nucleic acid sequence is inserted into the nef gene. .Iaddend.

.Iadd.70. The inducible viral replicon of claim 66, further comprising at least one tetO motif in at least one functional LTR. .Iaddend.

.Iadd.71. The inducible viral replicon of claim 70, further comprising at least 2, 4, 6, or 8 such elements in at least one functional LTR. .Iaddend.

.Iadd.72. The inducible viral replicon of claim 66, wherein at least one LTR is modified to avoid reversion to wild type virus. .Iaddend.

.Iadd.73. A method for producing a virus dependent upon an inducing agent for replication, the method comprising: providing a permissive cell with the inducible viral replicon of claim 66, culturing said cell in the presence of said inducing agent, and harvesting said dependent virus from said culture. .Iaddend.

.Iadd.74. The method according to claim 73, in which said dependent virus is a human immunodeficiency virus. .Iaddend.

.Iadd.75. The method according to claim 73, in which said virus is an attenuated virus. .Iaddend.

.Iadd.76. A virus dependent on an inducing agent for replication obtainable by the method according to claim 73. .Iaddend.

.Iadd.77. The virus according to claim 76, which is a human immunodeficiency virus. .Iaddend.

.Iadd.78. A method for the controlled replication of a virus or a viral replicon, the method comprising: providing a permissive cell with the inducible viral replicon of claim 66; culturing said cell in the presence of said inducing agent and manipulating the amount of inducing agent present. .Iaddend.

.Iadd.79. An isolated cell comprising the nucleic acid sequence of claim 55. .Iaddend.

.Iadd.80. A transactivator, having the DNA sequence (5′- 3′ orientation): TABLE-US-00002 (SEQ ID NO: 28) atgtctagactggacaagagcaaagtcataaactctgctctggaattact caatggagtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagc tgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggcc ctgctcgatgccctgccaatcgagatgctggacaggcatcatacccactc ctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgcca agtcataccgctgtgctctcctctcacatcgcgacggggctaaagtgcat ctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagct cgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctc tgtccgccgtgggccactttacactgggctgcgtattggaggaacaggag catcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcc cccacttctgaaacaagcaattgagctgttcgaccggcagggagccgaac ctgccttccttttcggcctggaactaatcatatgtggcctggagaaacag ctaaagtgcgaaagcggcgggccgaccgacgeccttgacgattttgactt agacatgctcccagccgatgcccttgacgactttgaccttgatatgctgc ctgctgacgctcttgacgattttgaccttgacatgctccccgggtaa. .Iaddend.

.Iadd.81. A transactivator, having the amino acid sequence: MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEML DRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFL CQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQ GAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG*(SEQ ID NO: 29). .Iaddend.

.Iadd.82. A method for inducibly expressing a nucleic acid sequence of interest, the method comprising the steps of: providing a nucleic acid construct comprising said nucleic acid sequence of interest operably linked to an inducible gene expression system that comprises a reverse tetracycline-controlled transactivator (rtTA) encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprising a mutation in a codon at rtTA amino acid position 67; introducing said nucleic acid construct to a suitable expression system; and allowing for inducible expression of said nucleic acid sequence of interest. .Iaddend.

.Iadd.83. A synthetic or recombinant nucleic acid sequence comprising a rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutated codon at rtTA amino acid position 67. .Iaddend.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1. Mutation of the rtTA gene through viral evolution. (A) In the HIV-rtTA virus, the Tat-TAR axis of transcription regulation has been inactivated by mutation of both Tat and TAR (crossed boxes). Transcription and replication of the virus were made dox-dependent by introduction of tetO elements in the LTR promoter region and replacing the nef gene by the rtTA gene. This 248-amino acid protein is a fusion of the E. coli Tet repressor (TetR) and the VP16 activation domain (AD) of the herpes simplex virus. The TetR part can be subdivided in a DNA binding domain (BD) (α-helices 1-3) and a regulatory core domain (α-helices 5-10) with a dimerization surface (α-helices 7-10). The F86Y (dark grey triangle) and A209T (black triangle) mutations were present in the starting virus and maintained in all long-term cultures. Light grey triangles indicate additional amino acid exchanges in rtTA that were observed in multiple, independent cultures of HIV-rtTA.sub.-F86Y A209T. (B) The crystal structure of the TetR homodimer (one monomer in dark grey, the other in light grey) complexed with Tc (light grey) and Mg.sup.2+ (grey ball) (Hinrichs et al. 1994; Kisker et al. 1995). Residue 86 is shown in dark grey. Additional mutated amino acids (positions 9, 67, 138, and 171) are shown in light grey. Residue 157 is not shown, because the segment 156 to 164 is flexible and not determined in the TetR crystal structure. A close up of the Tc-binding region is shown at the right. There are seven classes of TetR proteins (A-E, G, H) with a highly conserved sequence. The high resolution crystal structure that is shown is based on class D (TetR.sup.D). rtTA is based on class B (TetR.sup.B), which shares 63% sequence identity with TetR.sup.D. The crystal structure of TetR.sup.B at medium resolution revealed an identical polypeptide fold (Hinrichs et al. 1994). Therefore, we can assume that the interactions of TetR with Tc and Mg.sup.2 will be nearly identical in both classes. Figures are drawn using the 2TCT coordinates from the Protein Data Bank and the MOL-SCRIPT (Kraulis, 1991) and RASTER3D (Merritt et al. 1997) programs.

(2) FIG. 2. Novel rtTA variants show increased activity and dox-sensitivity in different Tet systems. The transcriptional activity of rtTA variants was measured in C33A cells transfected with a plasmid carrying the firefly luciferase reporter gene under the control of the viral LTR-2ΔtetO promoter (LTR-2ΔtetO; A) or under the control of a minimal CMV-derived promoter coupled to seven tetO elements (CMV7-tetO; B). Furthermore, rtTA activity was measured in HeLa X1/6 cells (Baron et al. 1997) that contain a chromosomally integrated copy of the CMV-7tetO reporter construct (CMV7tetO-integrated; C, D). Variants V1 to V10 were compared in all three Tet systems (panels A-C) and variants V11 to V18 in the cells with the integrated reporter (panel D). Cells were transfected with the indicated rtTA expression plasmid or pBluescript as a negative control, and a plasmid constitutively expressing Renilla luciferase to correct for differences in transfection efficiency. Cells were cultured in the presence of different dox concentrations (0-1000 ng/ml). The ratio of the firefly and Renilla luciferase activities measured 2 days after transfection reflects the rtTA activity. All values were related to the wild-type (wt) rtTA activity at 1000 ng/ml dox, which was arbitrarily set at 100%. In (C and D), average values of three transfections are shown with error bars indicating the standard deviation.

(3) FIG. 3. Transcriptional activity and dox-sensitivity of the naturally evolved and constructed rtTA variants. Transfection assays were performed in HeLa X1/6 cells, see FIG. 2 for details. Transcriptional activity observed at 1000 ng/ml dox is shown as average value of three transfections with error bars indicating the standard deviation. The wild-type rtTA activity was set at 100%. Dox-sensitivity is compared with the wild-type rtTA of which the sensitivity is arbitrarily set at 1. For each rtTA variant, the dox concentration (ng/ml) that results in an activity comparable to that of the wild-type rtTA activity at 1000 ng/ml dox is indicated between brackets. (nd, not determined)

(4) FIG. 4. Mutations do not affect the intracellular rtTA protein level. HeLa X1/6 cells were transfected with the indicated rtTA expression plasmid (lanes 3 to 6) or pBluescript as a negative control (lane 2). Total cellular extracts were prepared at 2 days after transfection and analyzed on Western blot that was stained with polyclonal anti-TetR rabbit serum (Krueger et al. 2003). Detection of purified TetR protein (2 ng) is shown in lane 1. The position and molecular weight (in kDa) of the PTA and TetR proteins are indicated.

(5) FIG. 5. Novel rtTA variants can be activated by dox-like compounds. The rtTA activity was measured in HeLa X1/6 cells, see FIG. 2 for details. Cells were cultured in the presence of different concentrations of Tc or Mc (0-10000 ng/ml). The wild-type (wt) rtTA activity at 1000 ng/ml dox (not shown) was set at 100%. Average values of three transfections are plotted with error bars indicating the standard deviation.

(6) FIG. 6. rtTA variants improve HIV-rtTA replication. The PTA variants V7 and V14 were cloned into the HIV-PTA proviral genome. SupT1 cells were transfected with 5 μg of the molecular clones and cultured in the presence of different dox concentrations (0-1000 ng/ml). Virus replication was monitored by CA-p24 ELISA on culture supernatant samples.

(7) FIG. 7. HIV-rtTA replication induced by dox-like compounds. SupT1 cells were transfected with 5 μg of the HIV-PTA clones and cultured in the presence of 500 ng/ml Tc or Mc. Virus replication was monitored by CA-p24 ELISA on culture supernatant samples.

(8) FIG. 8. Evolution of HIV-rtTA can result in loss of dox-control. (A) Schematic of the HIV-PTA genome. The inactivated Tat-TAR elements (crossed boxes) and the introduced rtTA-tetO elements are indicated. rtTA is a fusion protein of the E. coli Tet repressor (TetR) and the VP16 activation domain (AD) of herpes simplex virus. TetR contains a DNA-binding domain (DNA BD) (residues 1-44) and a regulatory core domain (residues 75-207) with a dimerization surface. (B) Flow-chart of the 24-well evolution experiment. Further details are provided in the text. (C) Gradual loss of dox-control in HIV-PTA, HIV-PTA 2ΔtetO (carrying the improved 2ΔtetO promoter configuration (Marzio et al. 2001; Marzio et al. 2002) and HIV-rtTA.sub.F86Y A209T (carrying the LTR-2ΔtetO promoter and the improved rtTA.sub.F86Y A209T gene (Das et al. 2004a). The HIV-rtTA.sub.G19F E37L variant developed in this study does not escape from dox-control. Plotted is the number of dox-dependent cultures as a function of the culture time. Each experiment was started with 24 independent cultures. (D) Amino acid substitutions observed in HIV-rtTA cultures that lost dox-control. In all cases, the G19E substitution resulted from a GGA to GAA codon mutation and the E37K substitution from a GAG to AAG mutation.

(9) FIG. 9. Replication of evolved HIV-rtTA variants. Replication of the original HIV-rtTA virus, the virus from culture C6 or from culture C5 (both harvested after 50 days of culturing) was compared by infecting SupT1 T cells with equal amounts of virus (5 ng/ml CA-p24) in the absence or presence of dox (1 μg/ml). Sequence analysis revealed that the C6 virus carried the G19E and E 156K mutations in the rtTA gene, and the C5 virus carried the E37K mutation (FIG. 1D).

(10) FIG. 10. Amino acid substitutions at rtTA position 19 or 37 confer the loss of dox-control. The G19E and E37K mutated rtTA sequences were cloned into the HIV-rtTA 2ΔtetO proviral genome (Marzio et al. 2001; Marzio et al. 2002). SupT1 cells were transfected with 2.5 μg of the molecular clones and cultured in the presence of 0-1000 ng/ml dox. Virus replication was monitored by CA-p24 ELISA on culture supernatant samples.

(11) FIG. 11. Replication of HIV-rtTA variants with alternative amino acids at position 37. SupT1 cells were transfected with HIV-rtTA 2ΔtetO proviral plasmids (2.5 μg) carrying the wild-type(E) or an alternative amino acid (K, D, L, N, F, Q, R, S) at rtTA position 37, and cultured with or without 1 μg/ml dox. All viruses, except for the E37K mutant, have the alternative G codon (GGU instead of GGA) at rtTA position 19, which does not affect viral replication (data not shown), and the F86Y and A209T mutations (Das et al. 2004a).

(12) FIG. 12. Transcriptional activity of rtTA variants with alternative amino acids at position 19 or 37. (A and B) rtTA activity was measured in HeLa X1/6 cells (Baronet al. 1997) that contain stably integrated copies of the CMV-7tetO firefly luciferase reporter construct (Gossen et al. 1992). Cells were transfected with the indicated rtTA expression plasmid (all rtTA variants contain the F86Y and A209T mutations that improve rtTA activity (Das et al. 2004a) or pBluescript as a negative control (−), and a plasmid constitutively expressing Renilla luciferase to correct for differences in transfection efficiency. Cells were cultured in the presence of different dox concentrations (0-1000 ng/ml). The ratio of the firefly and Renilla luciferase activities measured two days after transfection reflects rtTA activity. All values were related to the wild-type (37E in A, and 19G in B) rtTA activity at 1000 ng/ml dox, which was arbitrarily set at 100%. Average values of two transfections are plotted with the error bar indicating the standard deviation. (C and D) Codon tables of rtTA variants with all possible amino acids at position 19 or 37. The dox-dependent phenotype is marked in light grey, variants active in the absence of dox in dark grey, and inactive variants in black. See the text for details.

(13) FIG. 13. Activity of the novel rtTA variant with safety-lock mutations. (A) The activity of wild-type and safety-lock rtTA (G19F E37L) was measured in HeLa X1/6 cells, see FIG. 5 for details. Cells were cultured in the presence of different dox concentrations (0-1000 ng/ml). All values were related to the wild-type rtTA activity at 1000 ng/ml dox, which was arbitrarily set at 100%. Average values of two transfections are plotted with the error bar indicating the standard deviation. (B and C) Replication of HIV-rtTA.sub.F86Y A209T and HIV-rtTA.sub.G19F E37L (which also carries the F86Y and A209T mutations (Das et al. 2004a). SupT1 cells were transfected with 5 μg of the molecular clones and cultured with or without 1 μg/ml dox. Virus replication was monitored by CA-p24 ELISA on culture supernatant samples.

(14) FIGS. 14A and 14B. Transcriptional activity and dox-sensitivity of wild type, naturally evolved and constructed rtTA variants. Transfection assays were performed in HeLa X1/6 cells, see FIG. 2 for details. Transcriptional activity observed at 1000 ng/ml dox is shown as average value of three transfections with error bars indicating the standard deviation. The wild-type rtTA activity was set at 100%. Dox-sensitivity is compared with the wild-type rtTA of which the sensitivity is arbitrarily set at 1. For each rtTA variant, the dox concentration (ng/ml) that results in an activity comparable to that of the wild-type rtTA activity at 1000 ng/ml dox is indicated between brackets (Part of these results is also shown in FIG. 3).

(15) FIG. 14C. rtTA variants. Each column row depicts suitable rtTA variants.

(16) FIG. 15. Novel rtTA variants can be activated by dox-like compounds. The rtTA activity was measured in HeLa X1/6 cells, see FIG. 2 for details. Cells were cultured in the presence of different concentrations of Tc or Mc (0-10000 ng/ml). The wild-type (wt) rtTA activity at 1000 ng/ml dox (not shown) was set at 100%.

(17) FIG. 16. TetR-based transactivators. (A and B) In the homodimeric rtTA, each monomer contains an N-terminal E. coli-derived TetR domain and a C-terminal herpes simplex virus VP16-derived activation domain. The V91, F67S, F86Y and G138D mutations that enhance rtTA activity are all located in the TetR domain. sc rtTA is a single-chain version of rtTA. It contains two TetR domains connected head to tail by a peptide linker and a single activation domain at the C-terminal end.

(18) FIG. 17. Mutations that enhance rtTA activity do not improve tTA activity. The transcriptional activity of tTA variants was measured in HeLa X1/6 cells (Baron et al. 1997) containing chromosomally integrated copies of the CMV-7tetO luciferase reporter construct. Cells were transfected with the indicated tTA expression plasmids or pBluescript (−) as a negative control and a plasmid constitutively expressing Renilla luciferase to correct for differences in transfection efficiency. Cells were cultured in the presence of different dox concentrations (0-20 ng/ml). The ratio of the firefly and Renilla luciferase activities measured two days after transfection reflects the tTA activity. All values were related to the original (wild-type) tTA activity in the absence of dox, which was arbitrarily set at 100%. Average values of two transfections are shown with the error bar indicating the standard deviation.

(19) FIG. 18. Mutations observed in rtTA can improve sc rtTA activity. The transcriptional activity of rtTA and sc rtTA was measured in HeLa X1/6 cells, see FIG. 17 for details. Cells were cultured in the presence of different dox concentrations (0-1000 ng/ml). All values were related to the original (wild-type) sc rtTA activity at 1000 ng/ml dox, which was arbitrarily set at 100%. Average values of two transfections are plotted with the error bar indicating the standard deviation.

(20) FIG. 19. Nucleotide and amino acid sequence of rtTA .Iadd.(SEQ ID NO: 26 and SEQ ID NO: 27).Iaddend.. Shown is the nucleotide sequence (upper line) and amino acid sequence (lower line) of the rtTA2.sup.S-S2 variant (Urlinger et al. 2000).

(21) FIG. 20. Evolution of HIV-rtTA after transient dox administration. (A) Schematic of the HIV-rtTA genome. The inactivated Tat-TAR elements (crossed boxes) and the introduced rtTA-tetO elements are indicated. rtTA is a fusion protein of the E. coli Tet repressor (TetR) and the VP16 activation domain (AD) of herpes simplex virus. TetR contains a DNA-binding domain (DNA BD) (amino acids 1-44) and a regulatory core domain (amino acids 75-207) with a dimerization surface. (B-D) Loss of dox-control in cultures of HIVrtTA after transient activation. SupT1 cells were transfected with HIV-rtTA and cultured at 100 ng/ml dox (B), HIV-rtTA.sub.V9I G138D at 10 ng/ml dox (C), or HIV-rtTA.sub.G19F E37L at 1000 ng/ml dox (D). Each experiment was started with 12 independent cultures (different symbols represent different cultures). At day 3, dox was washed out and the cultures were continued with dox-free medium. The cultures in which the virus did not lose dox-control were split in two parts at day 64 (C) or day 66 (D) and dox was added to one of the samples. Virus production was monitored by CA-p24 ELISA on culture supernatant samples.

(22) FIG. 21. The P56S mutation causes a tTA-like phenotype. The activity of wild-type and P56S-mutated rtTA was measured in C33A cells transfected with a reporter plasmid carrying the firefly luciferase gene under the control of the viral LTR-2ΔtetO promoter (LTR-2ΔtetO; A) or under the control of a minimal CMV promoter coupled to an array of seven tetO elements (CMV7tetO; B). Furthermore, rtTA activity was measured in HeLa X1/6 cells (Baron et al. 1997) that contain chromosomally integrated copies of the CMV-7tetO luciferase construct (CMV-7tetO-integrated; C). Cells were transfected with the indicated rtTA expression plasmid (both rtTA variants carry the F86Y and A209T mutations (Das et al. 2004a) or pBluescript as a negative control (−), and a plasmid constitutively expressing Renilla luciferase to correct for differences in transfection efficiency. Cells were cultured with different dox concentrations (0-1000 ng/ml). The ratio of the firefly and Renilla luciferase activities measured two days after transfection reflects the rtTA activity. All values were related to the wild-type rtTA activity at 1000 ng/ml dox, which was arbitrarily set at 100%.

(23) FIG. 22. Activity of rtTA.sub.G19F E37L variants with all possible amino acids at position 56. (A) The activity of rtTA was measured in HeLa X1/6 cells, see FIG. 21 for details. All variants carry the G19F, E37L, F86Y and A209T mutations in combination with different amino acids at position 56. The wild-type rtTA (wt) carrying only the F86Y and A209T mutations was included as a control, of which the activity at 1000 ng/ml dox was arbitrarily set at 100%. Average values of two transfections are shown with the error bar indicating the standard deviation. (B) Codon table of rtTA.sub.G19F E37L variants with all possible amino acids at position 56. The corresponding codons of inactive variants are marked in black, of dox-dependent variants in light grey, and of variants that are active without dox in dark grey. See the text for details.

(24) FIG. 23. Replication of HIV-rtTA.sub.G19F E37L variants with different amino acids at position 56. SupT1 cells were transfected with 5 μg of HIV-rtTA molecular clones encoding different rtTA alleles, and cultured with or without 1 μg/ml dox. All rtTA variants contain the F86Y and A209T mutations. Virus replication was monitored by CA-p24 ELISA on culture supernatant samples.

(25) FIG. 24. Blocking the loss of dox-control by triple safety-lock mutations. SupT1 cells were transfected with HIV-rtTA containing triple safety-lock mutations (HIV-rtTA.sub.G19F E37L P56K) at 1000 ng/ml dox and split into 24 independent cultures (different symbols represent different cultures). At day 3, dox was washed out and the cultures were continued with dox-free medium. At day 60, all cultures were split in two parts and dox (1000 ng/ml) was added to one of the samples. Virus production was monitored by CA-p24 ELISA on culture supernatant samples.

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