Vector system
The present invention provides a vector system comprising a mutated post-transcriptional regulatory element. In particular, the present invention relates to a mutated WPRE sequence that can efficiently express nucleotides of interest in a retroviral vector system. The present invention also relates to methods of delivering and expressing nucleotides of interest to a target cell.
1. An isolated nucleic acid molecule comprising a woodchuck post-transcriptional regulatory element (WPRE) containing an X region, wherein the WPRE has a mutation in the X region whereby expression of a functional X protein is prevented, and wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 1.
2. An isolated nucleic acid molecule comprising a woodchuck post-transcriptional regulatory element (WPRE) containing an X region, wherein the WPRE has a mutation in the X region whereby expression of a functional X protein is prevented, wherein the X region comprises a promoter sequence, and wherein the mutation is in the promoter sequence.
3. An isolated nucleic acid molecule comprising a woodchuck post-transcriptional regulatory element (WPRE) containing an X region, wherein the WPRE has a mutation in the X region whereby expression of a functional X protein is prevented, and wherein the X region comprises an initiation codon and wherein the mutation is in the initiation codon.
4. A retroviral vector genome comprising at least one nucleotide of interest (NOI) and the nucleic acid molecule of claim 1 , 2 , or 3 .
5. The retroviral vector genome of claim 4 , which is a lentiviral vector genome.
6. The retroviral vector genome of claim 4 , wherein the retroviral vector genome comprises a self-inactivating (SIN) LTR.
7. The retroviral vector genome of claim 4 , wherein the retroviral vector genome is multicistronic.
8. A retroviral vector system for producing a retroviral vector particle, comprising:
(i) the retroviral vector genome of claim 4 ;
(ii) a nucleotide sequence encoding retroviral gag and pol proteins; and
(iii) nucleotide sequences encoding other essential viral packaging components not encoded by the nucleotide sequence of ii).
9. A composition comprising the retroviral vector genome of claim 4 , together with a carrier or diluent.
10. A method of delivering at least one nucleotide sequence of interest (NOI) to a target cell, comprising introducing the retroviral vector genome of claim 8 into the target cell, whereby the NOL is delivered to the target cell.
11. The lentiviral vector genome of claim 5 , which is a minimal lentiviral vector genome.
12. The lentiviral vector genome according to claim 5 , wherein a nucleic acid sequence encoding Rev is disrupted such that the nucleic acid sequence is incapable of encoding functional Rev or the nucleic acid sequence encoding Rev is removed from the vector genome.
13. The lentiviral vector genome according to claim 5 , wherein a nucleic acid sequence encoding Tat is disrupted such that the nucleic acid sequence is incapable of encoding functional Tat or the nucleic acid sequence encoding Tat is removed from the lentiviral vector genome.
14. The lentiviral vector genome of claim 5 , wherein the lentiviral vector genome is from a viral species selected from the group consisting of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), visna/maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
15. The lentiviral vector genome of claim 5 , wherein the lentiviral vector genome is from a non-primate lentivirus.
16. The lentiviral vector genome of claim 5 , wherein the lentiviral vector genome comprises a central polypurine tract (cPPT) sequence.
17. The lentiviral vector genome of claim 5 , wherein the lentiviral vector genome comprises a gag packaging signal having ATG motifs, and wherein the ATG motifs are ATTG motifs.
18. The retroviral vector genome of claim 7 , wherein the retroviral vector genome comprises at least one internal regulatory element.
19. The retroviral vector genome of claim 18 , wherein the internal regulatory element is a promoter or an internal ribosomal entry site (IRES).
20. The retroviral vector system of claim 8 , wherein the retroviral vector system is a lentiviral vector system, and wherein nucleic acid sequence(s) encoding at least one of Vpr, Vif, Tat, Nef, or analogous lentiviral auxiliary proteins are disrupted such that the nucleic acid sequence(s) are incapable of encoding functional Vpr, Vif, Tat, Nef, or the nucleic acid sequence(s) encoding Vpr, Vif, Tat, Nef, or analogous auxiliary proteins are removed from the lentiviral vector system.
21. The retroviral vector system of claim 8 , wherein the vector system is pseudotyped with at least part of a heterologous env protein.
22. A retroviral particle produced from the retroviral vector system of claim 8 .
23. An isolated cell that has been transduced with the retroviral vector system of claim 8 .
24. The retroviral vector system of claim 21 , wherein the heterologous env protein is from Rabies-G or VSV-G.
25. A composition comprising the viral particle of claim 22 , together with a carrier or diluent.
26. A retroviral vector comprising at least one nucleotide sequence of interest (NOI) and a nucleic acid molecule comprising a woodchuck post-transcriptional regulatory element (WPRE) containing an X region, wherein the WPRE has a mutation in the X region whereby expression of a functional X protein is prevented, and wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 1.
27. A retroviral vector comprising at least one nucleotide sequence of interest (NOI) and a nucleic acid molecule comprising a woodchuck post-transcriptional regulatory element (WPRE) containing an X region, wherein the WPRE has a mutation in the X region whereby expression of a functional X protein is prevented, wherein the X region comprises a promoter sequence, and wherein the mutation is in the promoter sequence.
28. A retroviral vector comprising at least one nucleotide sequence of interest (NOI) and a nucleic acid molecule comprising a woodchuck post-transcriptional regulatory element (WPRE) containing an X region, wherein the WPRE has a mutation in the X region whereby expression of a functional X protein is prevented, wherein the X region comprises an initiation codon and wherein the mutation is in the initiation codon.
29. A method of making a retroviral particle comprising the steps of:
(i) introducing the retroviral vector of claim 26 , 27 , or 28 into a packaging cell, or introducing the retroviral vector of claim 26 , 27 , or 28 together with nucleic acid sequence(s) encoding gag/pol and envelope proteins into a producer cell; and
(ii) obtaining the retroviral vector particle therefrom, wherein said retroviral vector particle comprises the at least one NOI and the nucleic acid molecule comprising the mutated WPRE.
30. A method of delivering at least one NOI to a target cell, comprising introducing the retroviral vector of claim 26 , 27 , or 28 into the target cell, whereby the NOI is delivered to the target cell.
31. The method of claim 29 , wherein the envelope is a heterologous envelope protein selected from the group consisting of Rabies G and VSV-G.