Vector System

Abstract

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. 
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the mutation in the X region further prevents reversion to wild type WPRE. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , comprising the sequence of SEQ ID NO:1. 
     
     
         4 . The isolated nucleic acid molecule of  claim 1 , wherein the X region comprises a promoter sequence and wherein the mutation is in the promoter sequence. 
     
     
         5 . The isolated nucleic acid molecule of  claim 1 , wherein the X region comprises an initiation codon and wherein the mutation is in the initiation codon. 
     
     
         6 . The isolated nucleic acid molecule of  claim 1 , wherein the X protein is not expressed. 
     
     
         7 . The isolated nucleic acid molecule of  claim 1 , wherein the X protein is non-functional. 
     
     
         8 . A retroviral vector genome comprising at least one NOI and the isolated nucleic acid molecule of  claim 1 . 
     
     
         9 . The retroviral vector genome of  claim 8 , which is a lentiviral vector genome. 
     
     
         10 . The retroviral vector genome of  claim 8 , wherein the retroviral vector genome comprises a self-inactivating (SIN) LTR. 
     
     
         11 . The retroviral vector genome of  claim 9 , wherein the lentiviral vector genome is a minimal lentiviral vector genome. 
     
     
         12 . The retroviral vector genome according to  claim 9 , wherein a nucleic acid sequence encoding Rev, or a functional equivalent thereof, is disrupted such that the nucleic acid sequence is incapable of encoding the functional Rev or is removed from the vector genome. 
     
     
         13 . The retroviral vector genome according to  claim 9 , wherein a nucleic acid sequence encoding Tat is disrupted such that the nucleic acid sequence is incapable of encoding functional Tat or is removed from the vector genome. 
     
     
         14 . The retroviral vector genome of  claim 9 , wherein the lentiviral vector genome is derived 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 retroviral vector genome of  claim 9 , wherein the lentiviral vector genome is derived from a non-primate lentivirus. 
     
     
         16 . The retroviral vector genome of  claim 9 , wherein the retroviral vector genome comprises a central polypurine tract (cPPT) sequence. 
     
     
         17 . The retroviral vector genome of  claim 9 , wherein the retroviral 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 8 , wherein the retroviral vector genome is multicistronic. 
     
     
         19 . The retroviral vector genome of  claim 18 , wherein the retroviral vector genome comprises at least one internal regulatory element. 
     
     
         20 . The retroviral vector genome of  claim 19 , wherein the internal regulatory element is a promoter or an internal ribosomal entry site (IRES). 
     
     
         21 . A retroviral vector system for producing a retrovirus-derived vector particle, comprising:
 (i) the retroviral vector genome of  claim 8 ;   (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).   
     
     
         22 . The retroviral vector system of  claim 21 , wherein nucleic acid sequence(s) encoding at least one of Vpr, Vif, Tat, Nef, or analogous auxiliary genes, from the retrovirus from which the particles are derived, are disrupted such as said nucleic acid sequence(s) are incapable of encoding functional Vpr, Vif, Tat, Nef, or analogous auxiliary proteins, or are removed from the system. 
     
     
         23 . The retroviral vector system of  claim 21 , wherein the vector system is pseudotyped with at least part of a heterologous env protein. 
     
     
         24 . The retroviral vector system of  claim 23 , wherein the heterologous env protein is derivable from Rabies-G or VSV-G. 
     
     
         25 . A retroviral particle produced from the retroviral vector system of  claim 21 . 
     
     
         26 . A cell that has been transduced with the retroviral vector system of  claim 21 . 
     
     
         27 . A composition comprising the retroviral vector genome of  claim 8 , together with a carrier or diluent. 
     
     
         28 . A composition comprising the viral particle of  claim 25 , together with a carrier or diluent. 
     
     
         29 . A method of delivering at least one NOI to a target cell, comprising introducing the retroviral vector genome of  claim 8  into the target cell, whereby the NOI is delivered to the target cell. 
     
     
         30 . A retroviral vector comprising at least one 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. 
     
     
         31 . The retroviral vector of  claim 30 , wherein the mutation in the X region further prevents reversion to wildtype WPRE. 
     
     
         32 . A method of making a retroviral particle comprising the steps of:
 (i) introducing the retroviral vector of  claim 30  into a packaging cell, or introducing the retroviral vector of  claim 30  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.   
     
     
         33 . The method of  claim 32 , wherein the envelope is a heterologous envelope protein selected from the group consisting of Rabies G and VSV-G. 
     
     
         34 . A method of delivering at least one NOI to a target cell, comprising introducing the retroviral vector of  claim 30  into the target cell, whereby the NOI is delivered to the target cell. 
     
     
         35 . A method of identifying a gene involved in tumorigenesis, said method comprising the steps of:
 (i) introducing the isolated nucleic acid of  claim 1  into a cell of interest, whereby the nucleic acid is recombined into chromosomal DNA of the cell of interest;   (ii) determining whether the cell of interest forms a tumor; and, if the cell of interest forms a tumor;   (iii) locating a site of recombination in the chromosomal DNA; and   (iv) identifying a gene near or adjacent to the site of recombination;   thereby identifying the gene involved in tumorigenesis.