Single lentiviral vector system for induced pluripotent (ips) stem cells derivation

Abstract

The present invention is based on the discovery that a single lentiviral vector expressing multiple individual transcription factor proteins from a single multi-cistronic mRNA can reprogram a fibroblast cell to a stem cell-like cell. These reprogrammed induced pluripotent stem (iPS) cells are pluripotent. Additions of the Cre-LoxP sequences into the single lentiviral vector facilitate excision of the vector after reprogramming in achieved. Addition of a maker gene into the single lentiviral vector facilitates detection of the presence of the vector in an iPS. The invention provides compositions and methods of producing iPS cells using a single multi-cistronic lentiviral vector.

What is claimed: 
     
         1 . A lentiviral vector comprising a nucleic acid sequence operatively linked to a promoter, wherein the nucleic acid sequences comprises:
 a. a ‘self-cleaving’ 2A peptide;   b. an internal ribosome entry site (IRES); and   c. a first gene, a second gene, and third gene selected from the group of genes consisting of Oct4, Klf4 and Sox2;   wherein the first gene, second gene and third gene can be in any order and wherein the ‘self-cleaving’ 2A peptide is positioned between the first gene and second gene, and wherein the IRES is positioned between the second and third genes, and wherein the nucleic acid sequence encoding the first gene, second gene and third gene, the ‘self-cleaving’ 2A peptide and the IRES are transcribed from the promoter as a multi-cistronic RNA.   
     
     
         2 . The lentiviral vector particle of  claim 1  further comprising a marker gene, wherein the marker gene encodes an optically visible protein or an enzyme. 
     
     
         3 . The lentiviral vector particle of  claim 1 , wherein the ‘self-cleaving” 2A peptide is selected from the group consisting of F2A, E2A, T2A and P2A. 
     
     
         4 . The lentiviral vector particle of  claim 1 , wherein the order of the first, second and third genes is Oct4, Klf4 and Sox2. 
     
     
         5 . The lentiviral vector particle of  claim 1  further comprises a Cre-LoxP excision sequence. 
     
     
         6 . A method of producing an induced pluripotent stem (iPS) cell comprising transducing a somatic cell with the lentiviral vector of  claim 1 . 
     
     
         7 . The method of  claim 6 , wherein the somatic cell is a mammalian cell. 
     
     
         8 . The method of  claim 7 , wherein the mammalian cell is a human cell. 
     
     
         9 . A cell comprising the lentiviral vector of  claim 1 . 
     
     
         10 . The cell of  claim 9 , wherein the cell is a mammalian cell. 
     
     
         11 . The cell of  claim 11 , wherein the mammalian cell is a human cell.