Claims
- 1. A retroviral LTR comprising a lentiviral R region which lacks all or a portion of the TAR sequence.
- 2. The retroviral LTR of claim 1, wherein the TAR sequence or portion thereof is replaced by a comparable portion of the R region from a non-lentiviral retrovirus, thereby forming a hybrid R region.
- 3. The retroviral LTR of claim 1, wherein the lentivirus is selected from the group consisting of HIV, EIV, SIV and FIV.
- 4. The retroviral LTR of claim 1, wherein the lentivirus is HIV-1 or HIV-2.
- 5. The retroviral LTR of claim 2, wherein the non-lentiviral retrovirus is selected from the group consisting of MoMSV, MoMLV, MLV, Friend, MSCV, RSV and spumavirus.
- 6. The retroviral LTR of claim 2, wherein the lentivirus is HIV and the non-lentiviral retrovirus is MoMSV.
- 7. The retroviral LTR of claim 6, wherein the hybrid R region comprises a portion of the HIV R region comprising the nucleotide sequence shown in SEQ ID NO: 10 and a portion of the MoMSV R region comprising the nucleotide sequence shown in SEQ ID NO: 9.
- 8. The retroviral LTR of claim 6, wherein the hybrid R region comprises the nucleotide sequence shown in SEQ ID NO: 11.
- 9. A retroviral LTR comprising a hybrid R region, wherein the hybrid R region comprises: (1) a portion of a lentiviral R region lacking the TAR sequence; and (b) a comparable portion of an R region from a non-lentiviral retrovirus.
- 10. The retroviral LTR of claim 9, wherein the portion of the lentiviral R region is from a lentivirus selected from the group consisting of HIV, EIV, SIV and FIV.
- 11. The retroviral LTR of claim 9, wherein the portion of the R region from the non-lentiviral retrovirus is from a retrovirus selected from the group consisting of MoMSV, MoMLV, MLV, Friend, MSCV, RSV and spumavirus.
- 12. The retroviral LTR of claim 9, wherein the portion of the lentiviral R region is from HIV and the portion of the R region from the non-lentiviral retrovirus is from MoMSV.
- 13. The retroviral LTR of claim 12, wherein the hybrid R region comprises a portion of the HIV R region comprising the nucleotide sequence shown in SEQ ID NO: 10 and a portion of the MoMSV R region comprising the nucleotide sequence shown in SEQ ID NO: 9.
- 14. The retroviral LTR of claim 12, wherein the hybrid R region comprises the nucleotide sequence shown in SEQ ID NO: 11.
- 15. The retroviral LTR of claim 9, wherein the LTR is a left (5′) LTR and further comprises a promoter sequence upstream from the hybrid R region.
- 16. The retroviral LTR of claim 15, wherein the promoter sequence comprises the U3 region of a non-lentiviral retrovirus.
- 17. The retroviral LTR of claim 15, wherein the promoter sequence comprises the MoMSV U3 region.
- 18. The retroviral LTR of claim 15 further comprising a lentiviral U5 region downstream from the hybrid R region.
- 19. The retroviral LTR of claim 18, wherein the U5 region is the HIV U5 region including the HIV att sequence.
- 20. The retroviral LTR of claim 9, wherein the LTR is a right (3′) LTR and further comprises a portion of a lentiviral U3 region upstream from the hybrid R region, wherein the portion includes the lentiviral U3 att sequence but lacks any promoter activity.
- 21. The retroviral LTR of claim 20, wherein the portion comprises the 3′ end of the lentiviral U3 region up to and including the lentiviral U3 att sequence.
- 22. The retroviral LTR of claim 20, wherein the portion of the U3 region is from HIV.
- 23. The retroviral LTR of claim 20, wherein the portion of the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 15.
- 24. The retroviral LTR of claim 20 further comprising a polyadenylation sequence downstream from the hybrid R region.
- 25. The retroviral LTR of claim 24, wherein the polyadenylation sequence comprises the rabbit β-globin gene polyadenylation sequence.
- 26. The retroviral LTR of claim 24, wherein the polyadenylation sequence comprises the nucleotide sequence shown in SEQ ID NO: 16.
- 27. A retroviral LTR comprising a hybrid R region, wherein the hybrid R region comprises: (1) a portion of the HIV R region lacking the TAR sequence; and (b) a portion of the MoMSV R region.
- 28. The retroviral LTR of claim 20, wherein the portion of the HIV R region comprises the nucleotide sequence shown in SEQ ID NO: 10.
- 29. The retroviral LTR of claim 20, wherein the portion of the MoMSV R region comprises the nucleotide sequence shown in SEQ ID NO: 9.
- 30. The retroviral LTR of claim 20, wherein the hybrid R region comprises the nucleotide sequence shown in SEQ ID NO: 11.
- 31. A left (5′) retroviral LTR comprising three regions, U3, R and U5, wherein:
the U3 region comprises a promoter sequence; the R region comprises a combination of (a) ) a portion of an R region from a non-lentiviral retrovirus, and (b) a portion of an R region from a lentivirus which does not include the TAR sequence; and the U5 region comprises a lentiviral U5 region.
- 32. The left (5′) retroviral LTR of claim 31, wherein the U3 region comprises the U3 region from a non-lentiviral retrovirus.
- 33. The left (5′) retroviral LTR of claim 31, wherein the U3 region comprises the MoMSV U3 region.
- 34. The left (5′) retroviral LTR of claim 31, wherein the R region comprises a portion of the MoMSV R region, and a portion of the HIV R region lacking the TAR sequence..
- 35. The left (5′) retroviral LTR of claim 31, wherein the U5 region comprises the HIV U5 region.
- 36. A left (5′) retroviral LTR comprising three regions referred to, from left to right, as U3-R-U5, wherein:
the U3 region comprises the MoMSV U3 region; the R region comprises the left half of the MoMSV R region and the right half of the HIV R region lacking the HIV TAR sequence but including the HIV polyadenylation sequence; and the U5 region comprises the HIV U5 region including the HIV att sequence.
- 37. The left (5′) retroviral LTR of claim 36, wherein the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 12.
- 38. The left (5′) retroviral LTR of claim 36, wherein the R region comprises the nucleotide sequence shown in SEQ ID NO: 11.
- 39. The left (5′) retroviral LTR of claim 36, wherein the U5 region comprises the a nucleotide sequence shown in SEQ ID NO: 13.
- 40. A left (5′) retroviral LTR comprising the nucleotide sequence shown in SEQ ID NO: 14.
- 41. A right (3′) retroviral LTR comprising three regions, U3, R and P, wherein:
the U3 region comprises the att sequence from a lentiviral U3 region but lacks any sequences from the U3 region having promoter activity; the R region comprises a combination of (a) a portion of an R region from a non-lentiviral retrovirus, and (b) a portion of an R region from a lentivirus which does not include the TAR sequence; and the P region comprises a polyadenylation sequence.
- 42. The right (3′) retroviral LTR of claim 41, wherein the U3 region comprises the HIV att sequence.
- 43. The right (3′) retroviral LTR of claim 41, wherein the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 15.
- 44. The right (3′) retroviral LTR of claim 41, wherein the R region comprises a portion of the MoMSV R region, and a portion of the HIV R region lacking the TAR sequence.
- 45. The right (3′) retroviral LTR of claim 41, wherein the R region comprises the nucleotide sequence shown in SEQ ID NO: 11.
- 46. The right (3′) retroviral LTR of claim 41, wherein the P region comprises the rabbit β-globin gene polyadenylation sequence.
- 47. The right (3′) retroviral LTR of claim 41, wherein the P region comprises the nucleotide sequence shown in SEQ ID NO: 16.
- 48. A right (3′) retroviral LTR comprising three regions referred to, from left to right, as U3-R-P, wherein:
the U3 region comprises a portion of the HIV U3 region containing the HIV att sequence but not containing any sequences from the HIV U3 region having promoter activity; the R region comprises the left half of the MoMSV R region and the right half of the HIV R region lacking the HIV TAR sequence but including the HIV polyadenylation sequence; and the P region comprises a polyadenylation sequence.
- 49. The right (3′) retroviral LTR of claim 48, wherein the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 15.
- 50. The right (3′) retroviral LTR of claim 48, wherein the R region comprises the nucleotide sequence shown in SEQ ID NO: 11.
- 51. The right (3′) retroviral LTR of claim 48, wherein the P region comprises the a nucleotide sequence shown in SEQ ID NO: 16.
- 52. The right (3′) retroviral LTR of claim 48 further comprising the HIV polypurine track (ppt) sequence upstream from the U3 region.
- 53. A right (3′) retroviral LTR comprising the nucleotide sequence shown in SEQ ID NO: 17.
- 54. A hybrid retroviral LTR comprising all or a portion of the R region from a non-lentiviral retrovirus and all or a portion of the U3 region or the U5 region from a lentivirus.
- 55. The hybrid retroviral LTR of claim 54, wherein the LTR is a left (5′) LTR and comprises all or a portion of the U3 region of a non-lentiviral retrovirus, all or a portion of the R region of a non-lentiviral retrovirus, and all or a portion of the U5 region from a lentivirus.
- 56. The hybrid retroviral LTR of claim 54, wherein the LTR is a right (5′) LTR and comprises all or a portion of the U3 region of a lentivirus and all or a portion of the R region of a non-lentiviral retrovirus.
- 57. The hybrid retroviral LTR of any one of claims 54, 55 or 56, wherein the lentivirus is HIV and the non-lentiviral retrovirus is MoMSV.
- 58. An expression vector comprising the retroviral LTR of any one of claims 1, 9, 20, 31, 36, 41, 48 or 54.
- 59. The expression vector of claim 58, wherein the expression vector is a SIN vector.
- 60. An expression vector comprising
(1) a left (5′) retroviral LTR comprising three regions, U3, R and U5, wherein:
the U3 region comprises a promoter sequence; the R region comprises a combination of (a) ) a portion of an R region from a non-lentiviral retrovirus, and (b) a portion of an R region from a lentivirus which does not include the TAR sequence; and the U5 region comprises a lentiviral U5 region; and further comprising (2) a right (3′) retroviral LTR comprising three regions, U3, R and P, wherein:
the U3 region comprises the att sequence from a lentiviral U3 region but lacks any sequences from the U3 region having promoter activity; the R region comprises a combination of (a) a portion of an R region from a non-lentiviral retrovirus, and (b) a portion of an R region from a lentivirus which does not include the TAR sequence; and the P region comprises a polyadenylation sequence.
- 61. The expression vector of claim 60, wherein the expression vector is a SIN vector.
- 62. The expression vector of claim 60 further comprising a polypurine tract upstream from the U3 region of the right (3′) LTR.
- 63. The expression vector of claim 60 further comprising an RNA export element.
- 64. The expression vector of claim 60, wherein the RNA export element comprises the HIV Rev responsive element (RRE).
- 65. The expression vector of claim 60 further comprising an exogenous gene.
- 66. The expression vector of claim 60, wherein the gene is a marker gene.
- 67. The expression vector of claim 66, wherein the marker gene comprises the green fluorescence protein (GFP) gene.
- 68. The expression vector of claim 60, wherein the gene is a therapeutic gene.
- 69. The expression vector of claim 68, wherein the gene encodes a protein which promotes angiogenesis.
- 70. An expression vector comprising
(1) a left (5′) retroviral LTR comprising three regions referred to, from left to right, as U3-R-U5, wherein:
the U3 region comprises the MoMSV U3 region; the R region comprises the left half of the MoMSV R region and the right half of the HIV R region lacking the HIV TAR sequence but including the HIV polyadenylation sequence; and the U5 region comprises the HIV U5 region including the HIV att sequence; and further comprising (2) a right (3′) retroviral LTR comprising three regions referred to, from left to right, as U3-R-P, wherein:
the U3 region comprises a portion of the HIV U3 region containing the HIV att sequence but not containing any sequences from the HIV U3 region having promoter activity; the R region comprises the left half of the MoMSV R region and the right half of the HIV R region lacking the HIV TAR sequence but including the HIV polyadenylation sequence; and the P region comprises a polyadenylation sequence.
- 71. The expression vector of claims 70, wherein the expression vector is a SIN vector.
- 72. The expression vector of claim 70 further comprising a polypurine tract upstream from the U3 region of the right (3′) LTR.
- 73. The expression vector of claim 70 further comprising an RNA export element.
- 74. The expression vector of claim 70, wherein the RNA export element comprises the HIV Rev responsive element (RRE).
- 75. The expression vector of claim 70 further comprising an exogenous gene.
- 76. The expression vector of claim 70, wherein the gene is a marker gene.
- 77. The expression vector of claim 76, wherein the marker gene comprises the green fluorescence protein (GFP) gene.
- 78. The expression vector of claim 70, wherein the gene is a therapeutic gene.
- 79. The expression vector of claim 78, wherein the gene encodes a protein which promotes angiogenesis.
- 80. The expression vector of any one of claims 65-69 or 75-79 further comprising a promoter sequence upstream from the gene.
- 81. The expression vector of claim 80, wherein the promoter is selected from the group consisting of the PGK, EF1 alpha and CMV promoter.
- 82. The expression vector of claim 80, wherein the promoter is the natural promoter associated with the gene.
- 83. The expression vector of claim 80 further comprising an internal ribosome entry site (IRES).
- 84. The expression vector of claim 80 further comprising all or a portion of a lentiviral GAG sequence.
- 85. The expression vector of claim 80, wherein the gene is selected from the group consisting of genes encoding soluble Interleukin-1α Receptor Type I, Soluble Interleukin-1α Receptor Type II, Interleukin-1α Receptor Antagonist Protein (IRAP), Insulin-Like Growth Factor (IGF), Tissue Inhibitors of Matrix Metallo-Proteinases (TIMP)-1,-2,-3,-4, Bone Morphogenic Protein (BMP)-2 and -7, Indian Hedgehog, Sox-9, Interleukin-4, Transforming Growth Factor (TGF)-β, Superficial Zone Protein, Cartilage Growth and Differentiation Factors (CGDF), Bcl-2, Soluble Tumor Necrosis Factor (TNF)-α Receptor, Fibronectin and/or Fibronectin Fragments, Leukemia Inhibitory Factor (LIF), LIF binding protein (LBP), Interleukin-4, Interleukin-10, Interleukin-11, Interleukin-13, Hyaluronan Synthase, soluble TNF-α receptors 55 and 75, Insulin Growth Factor (IGF)-1, activators of plasminogen, urokinase plasminogen activator (uPA), parathyroid hormone-related protein (PTHrP), and platelet derived growth factor (PDGF)-AA -AB or -BB.
- 86. Use of the expression vector of any one of claims 60-79 in gene therapy.
- 87. A virion for use in gene therapy comprising the expression vector of any one of claims 60-79.
- 88. A method of delivering a gene to a cell comprising contacting the cell with the virion of claim 87.
- 89. The method of claim 88, wherein the cell is selected from the group comprising an autologous cell, a bone marrow cell, a mesenchymal stem cell obtained from adipose tissue, a synovial fibroblast or a chondrocyte, a non-autologous cell (i.e., is allogeneic or xenogenic) and a cell from a cell line or from primary cells derived from a human or animal source.
- 90. The method of claim 88, wherein the virion is contacted with the cell in vivo.
- 91. The method of claim 88, wherein the virion is contacted with the cell ex vivo.
- 92. A method of producing a lentivirus suitable for use in gene therapy comprising transfecting the expression vector of any one of claims 60-79 into a packaging cell line containing the necessary envelop (env) and polymerase (pol) gene sequences to produce the lentivirus.
- 93. The method of claim 92, wherein the envelope (env) gene is from a non-lentiviral virus so that the lentivirus is pseudotyped.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Serial No. 06/288,042, filed May 1, 2001, the entire contents of which are incorporated herein by this reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60288042 |
May 2001 |
US |