Claims
- 1. A retroviral vector genome comprising two or more nucleotides of interest (NOIs) operably linked by one or more Internal Ribosome Entry Site(s) (IRES).
- 2. The genome according to claim 1, wherein each NOI encodes a protein associated with a neurodegenerative disorder.
- 3. The genome according to claim 1, which is a lentiviral vector genome.
- 4. The genome according to claim 3, wherein the lentiviral vector genome is a non-primate lentiviral vector genome.
- 5. The genome according to claim 3, wherein the lentiviral vector genome is derived from EIAV.
- 6. The genome according to claim 3, wherein the lentiviral vector genome is derived from HIV.
- 7. The genome according to claim 1, wherein each NOI encodes a protein selected from the group consisting of Tyrosine Hydroxylase (TH), GTP-cyclohydrolase I (GTP-CH1), Aromatic Amino Acid Dopa Decarboxylase (AADD) and Vesicular Monoamine Transporter 2 (VMAT2).
- 8. The genome according to claim 1, wherein at least one of the NOIs is operably linked to a promoter or promoter element(s).
- 9. The genome according to claim 1, which lacks the rev responsive element (RRE).
- 10. The genome according to claim 1, further comprising a cPPT sequence.
- 11. The genome according to claim 1, further comprising a post-transcriptional regulatory element or a translational enhancer.
- 12. The genome according to claim 1, wherein the NOIs encode:
a) TH and GTP-CH1; or b) TH, GTP-CH1 and AADD; c) AADD and VMAT1; or d) TH and AADD.
- 13. The genome according to claim 12, wherein the NOIs encode TH, GTP-CH1 and AADD.
- 14. The genome according to claim 12, which is a lentiviral vector genome.
- 15. The genome according to claim 14, wherein the lentiviral vector genome is derived from EIAV.
- 16. A vector system comprising the genome according to claim 1.
- 17. A vector system comprising the genome according to claim 3, further comprising:
(i) a nucleotide sequence coding for lentiviral gag and pol proteins; and (ii) nucleotide sequence(s) encoding an env protein.
- 18. The vector system of claim 17, wherein the genome is longer than the lentivirus wild type genome.
- 19. The vector system according to claim 18, which is an EIAV vector system.
- 20. The vector system according to claim 16 or 17, which is devoid of any additional viral functional genes.
- 21. The vector system according to claim 17, which is pseudotyped with at least part of a heterologous env protein.
- 22. A vector system according to claim 21, in which the heterologous env protein is derivable from Rabies-G or VSV G.
- 23. The vector system according to claim 17, wherein the genome comprises a packaging signal.
- 24. A method for producing a lentiviral particle comprising introducing into a producer cell:
i) the genome of claim 3, ii) a nucleotide sequence coding for lentiviral gag and pol proteins; and iii) nucleotide sequence(s) encoding an env protein, thereby producing a lentiviral particle.
- 25. The method according to claim 24, wherein the nucleotide sequence coding for gag and pol is codon optimized for expression in the producer cell.
- 26. The method according to claim 24, wherein the genome comprises a packaging signal.
- 27. A lentiviral particle produced by the method of claim 24, which comprises the two or more NOIs, operably linked by one or more IRES(s).
- 28. A pharmaceutical composition comprising the genome of claim 1, the system of claim 16 or 17, or the lentiviral particle of claim 27, together with a pharmaceutically acceptable carrier or diluent.
- 29. A method of treating a disease in a subject in need thereof, comprising administering the composition of claim 28 to the subject, whereby the two or more NOIs are expressed, such that the subject is treated.
- 30. The method according to claim 29, wherein the disease is a neurodegenerative disease.
- 31. The method according to claim 30, wherein the neurodegenerative disease is Parkinson's disease.
- 32. A cell which has been transduced with the system according to claim 16 or 17.
- 33. A bicistronic cassette comprising a nucleotide sequence which encodes TH and a nucleotide sequence which encodes GTP-CH1, operably linked by one or more IRES(s).
- 34. A bicistronic cassette comprising a nucleotide sequence which encodes AADD and a nucleotide sequence which encodes VMAT2, operably linked by one or more IRES(s).
- 35. A tricistronic cassette comprising a nucleotide sequence which encodes TH, a nucleotide sequence which encodes GTP-CH 1, and a nucleotide sequence which encodes AADD, operably linked by two or more IRES(s).
- 36. The cassette of claim 33, 34 or 35, wherein the IRES is a viral IRES.
- 37. The cassette of claim 36, wherein the viral IRES is from a picornavirus.
- 38. The cassette of claim 37, wherein the picornavirus is encephalomyocarditis virus (EMCV) or poliovirus (PV).
- 39. The cassette of claim 33, 34 or 35, wherein the IRES is a cellular IRES.
- 40. The cassette of claim 39, wherein the cellular IRES is FGF2 IRES or NRF IRES.
- 41. A retroviral vector genome comprising three or more NOIs operably linked by two or more Internal Ribosome Entry Sites (IRESs).
- 42. The genome according to claim 41, wherein each NOI encodes a protein associated with a neurodegenerative disorder.
- 43. The genome according to claim 41, which is a lentiviral vector genome.
- 44. The genome according to claim 43, wherein the lentiviral vector genome is a non-primate lentiviral vector genome.
- 45. The genome according to claim 43, wherein the lentiviral vector genome is derived from EIAV.
- 46. The genome according to claim 43, wherein the lentiviral vector genome is derived from HIV.
- 47. The genome according to claim 41, wherein each NOI encodes a protein selected from the group consisting of Tyrosine Hydroxylase (TH), GTP-cyclohydrolase I (GTP-CH1), Aromatic Amino Acid Dopa Decarboxylase (AADD) and Vesicular Monoamine Transporter 2 (VMAT2).
- 48. The genome according to claim 41, wherein at least one of the NOIs is operably linked to a promoter or promoter element(s).
- 49. The genome according to claim 41, which lacks the rev responsive element (RRE).
- 50. The genome according to claim 41, further comprising a cPPT sequence.
- 51. The genome according to claim 41, further comprising a post-transcriptional regulatory element or a translational enhancer.
- 52. The genome according to claim 41, wherein the NOIs encode TH, GTP-CH1 and AADD.
- 53. The genome according to claim 41, which is a lentiviral vector genome.
- 54. The genome according to claim 53, wherein the lentiviral vector genome is derived from EIAV.
- 55. A vector system comprising the genome according to claim 41.
- 56. A vector system comprising the genome according to claim 43, further comprising:
i) a nucleotide sequence coding for lentiviral gag and pol proteins; and ii) nucleotide sequence(s) encoding an env protein.
- 57. The vector system of claim 56, wherein the genome is longer than the lentivirus wild type genome.
- 58. The vector system according to claim 57, which is an EIAV vector system.
- 59. The vector system according to claim 55 or 56, which is devoid of any additional viral functional genes.
- 60. The vector system according to claim 56, which is pseudotyped with at least part of a heterologous env protein.
- 61. The vector system according to claim 60, in which the heterologous env protein is derivable from Rabies-G or VSV G.
- 62. The vector system according to claim 56, wherein the genome comprises a packaging signal.
- 63. A method for producing a lentiviral particle comprising introducing into a producer cell:
i) the genome of claim 43, ii) a nucleotide sequence coding for lentiviral gag and pol proteins; and iii) nucleotide sequence(s) encoding an env protein, thereby producing a lentiviral particle.
- 64. The method according to claim 63, wherein the nucleotide sequence coding for gag and pol is codon optimized for expression in the producer cell.
- 65. The method according to claim 63, wherein the genome comprises a packaging signal.
- 66. A lentiviral particle produced by the method of claim 63, which comprises the three or more NOIs, operably linked by two or more IRESs.
- 67. A pharmaceutical composition comprising the genome of claim 41, the system of claim 55 or 56, or the lentiviral particle of claim 66, together with a pharmaceutically acceptable carrier or diluent.
- 68. A method of treating a disease in a subject in need thereof, comprising administering the composition of claim 67 to the subject, whereby the three or more NOIs are expressed, such that the subject is treated.
- 69. The method according to claim 68, wherein the disease is a neurodegenerative disease.
- 70. The method according to claim 69, wherein the neurodegenerative disease is Parkinson's disease.
- 71. A cell which has been transduced with the system according to claim 55 or 56.
Priority Claims (1)
Number |
Date |
Country |
Kind |
0024550.6 |
Oct 2000 |
GB |
|
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of PCT/GB01/04433, filed on Oct. 5, 2001, designating the U.S., published on Apr. 11, 2002 as WO 02/29062 A2, and claiming priority from GB 0024550.6, filed on Oct. 6, 2000. All of the above-mentioned applications, as well as all documents cited herein, and documents referenced or cited in documents cited herein, are incorporated by reference.
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
PCT/GB01/04433 |
Oct 2001 |
US |
Child |
10408456 |
Apr 2003 |
US |