Vectors and methods for the efficient generation of integration/transgene-free induced pluripotent stem cells from peripheral blood cells

Abstract

A vector for generating induced pluripotent stem cells from human target cells comprising a) a vector backbone, b) exactly two, three or four transcription and reprogramming factor genes, each gene separated by a 2a self-cleavage peptide sequence, c) a spleen focus-forming virus promoter, and d) a post-transcriptional regulatory element Wpre, with or without an anti-apoptotic factor gene. A method for generating integration-free induced pluripotent stem cells, the method comprising: a) providing target cells, b) providing one or more than one vector according to the present invention, c) transducing or transfecting the target cells with the one or more than one vector, and d) culturing the transduced or transfected cells in a cell culture, thereby generating integration-free induced pluripotent stem cells.

What is claimed is: 
     
         1 . An episomal vector for generating induced pluripotent stem cells from human target cells, the vector comprising:
 a) an oriP/EBNA1-based plasmid backbone;   b) exactly two transcription and reprogramming factor genes, oct4 and sox2, separated by a 2a self-cleavage peptide sequence;   c) a spleen focus-forming virus promoter;   d) a post-transcriptional regulatory element Wpre; and   e) anti-apoptotic factor gene selected from the group consisting of bcl-xl and bcl2.   
     
     
         2 . An episomal vector for generating induced pluripotent stem cells from human target cells, the vector comprising:
 a) an oriP/EBNA1-based plasmid backbone;   b) exactly three transcription and reprogramming factor genes, oct4, sox2 and klf4, each separated by a 2a self-cleavage peptide sequence;   c) a spleen focus-forming virus promoter;   d) a post-transcriptional regulatory element Wpre; and   e) anti-apoptotic factor gene selected from the group consisting of bcl-xl and bcl2.   
     
     
         3 . An episomal vector for generating induced pluripotent stem cells from human target cells, the vector comprising:
 a) an oriP/EBNA1-based plasmid backbone;   b) exactly four transcription and reprogramming factor genes, oct4, sox2, klf4 and myc, each separated by a 2a self-cleavage peptide sequence;   c) a spleen focus-forming virus promoter;   d) a post-transcriptional regulatory element Wpre; and   e) anti-apoptotic factor gene selected from the group consisting of bcl-xl and bcl2.   
     
     
         4 . A vector for generating induced pluripotent stem cells from human target cells, the vector comprising:
 a) a vector backbone;   b) exactly two, three or four transcription and reprogramming factor genes, each gene separated by a 2a self-cleavage peptide sequence;   c) a spleen focus-forming virus promoter; and   d) a post-transcriptional regulatory element Wpre.   
     
     
         5 . The vector of  claim 4 , where the vector backbone is an oriP/EBNA1-based episomal vector. 
     
     
         6 . The vector of  claim 4 , where the vector backbone is an oriP/EBNA1-based plasmid backbone. 
     
     
         7 . The vector of  claim 4 , where the vector is an episomal vector. 
     
     
         8 . The vector of  claim 4 , where the vector is selected from the group consisting of a plasmid, a non-plasmid, a non-integrating plasmid, a non-integrating vector, a viral vector, a non-integrating viral vector, a self-inactivating vector and a lentivirus vector. 
     
     
         9 . The vector of  claim 4 , where the transcription and reprogramming factor genes are selected from the group consisting of one or more than one Yamanaka factor gene and one or more than one Thomson/Yu factor gene, and a combination of the preceding. 
     
     
         10 . The vector of  claim 4 , where one or more than one of the transcription and reprogramming factor genes are selected from the group consisting of klf4, lin28, myc, nanog, oct4, sox1, sox2, sox3, sox15 and sox18. 
     
     
         11 . The vector of  claim 4 , where a plurality of the transcription and reprogramming factor genes are selected from the group consisting of klf4, lin28, myc, nanog, oct4, sox1, sox2, sox3, sox15 and sox18. 
     
     
         12 . The vector of  claim 4 , where all of the transcription and reprogramming factor genes are selected from the group consisting of klf4, lin28, myc, nanog, oct4, sox1, sox2, sox3, sox15 and sox18. 
     
     
         13 . The vector of  claim 4 , where all of the transcription and reprogramming factor genes are selected from the group consisting of oct4, sox2, klf4 and myc. 
     
     
         14 . The vector of  claim 4 , where the transcription and reprogramming factor genes are exactly two transcription and reprogramming factor genes, oct4 and sox2. 
     
     
         15 . The vector of  claim 4 , where the transcription and reprogramming factor genes are exactly three transcription and reprogramming factor genes, oct4, sox2 and klf4. 
     
     
         16 . The vector of  claim 4 , where the transcription and reprogramming factor genes are exactly four transcription and reprogramming factor genes, oct4, sox2, klf4 and myc. 
     
     
         17 . The vector of  claim 4 , where the 2a self-cleavage peptide sequence is selected from the group consisting of equine rhinitis A virus, foot-and-mouth disease virus, porcine teschovirus-1 and  Thosea asigna  virus. 
     
     
         18 . The vector of  claim 4 , further comprising one or more than one gene coding for an inhibitor, siRNA, or shRNA construct of a pro-apoptotic factor. 
     
     
         19 . The vector of  claim 4 , further comprising one or more than one gene coding for an inhibitor, siRNA, or shRNA construct of a pro-apoptotic factor, where the pro-apoptotic factor is a BAX subfamily pro-apoptotic factor selected from the group consisting of BAK, BAX and BOK. 
     
     
         20 . The vector of  claim 4 , further comprising one or more than one gene coding for an inhibitor, siRNA, or shRNA construct of a pro-apoptotic factor, where the pro-apoptotic factor is a BH3 subfamily pro-apoptotic factor selected from the group consisting of BAD, BID, BIK, BIML, BLK, BNIP3 and HRK. 
     
     
         21 . The vector of  claim 4 , further comprising one or more than one anti-apoptotic factor gene encoding one or more than one anti-apoptotic factor. 
     
     
         22 . The vector of  claim 4 , further comprising one or more than one anti-apoptotic factor gene encoding one or more than one anti-apoptotic factor, where the anti-apoptotic factor is a BCL-2 family anti-apoptotic factor. 
     
     
         23 . The vector of  claim 4 , further comprising one or more than one anti-apoptotic factor gene encoding one or more than one anti-apoptotic factor, where the anti-apoptotic factor is a BCL-2 family anti-apoptotic factor selected from the group consisting of A1, BCL2, BCL-W, BCL-XL and MCL1. 
     
     
         24 . The vector of  claim 4 , further comprising one or more than one anti-apoptotic factor gene encoding one or more than one anti-apoptotic factor, where the anti-apoptotic factor is BCL2 or BCL-XL. 
     
     
         25 . A method for generating integration-free induced pluripotent stem cells, the method comprising:
 a) providing target cells;   b) providing one or more than one vector of  claim 4 ;   c) transducing or transfecting the target cells with the one or more than one vector; and   d) culturing the transduced or transfected cells in a cell culture, thereby generating integration-free induced pluripotent stem cells.   
     
     
         26 . The method of  claim 25 , where the one or more than one vector provided is one vector. 
     
     
         27 . The method of  claim 25 , where the one or more than one vector provided is a plurality of vectors. 
     
     
         28 . The method of  claim 25 , where the one or more than one vector provided is two vectors. 
     
     
         29 . The method of  claim 25 , where the one or more than one vector provided is three vectors. 
     
     
         30 . The method of  claim 25 , where the one or more than one vector is a first vector and a second vector, and transducing or transfecting the target cells comprises transducing or transfecting the target cells with a first amount of the first vector and a second amount of a second vector, where the first amount is equal to the second amount. 
     
     
         31 . The method of  claim 25 , where the one or more than one vector is a first vector and a second vector, and transducing or transfecting the target cells comprises transducing or transfecting the target cells with a first amount of the first vector and a second amount of a second vector, where the first amount is half of the second amount. 
     
     
         32 . The method of  claim 25 , where the one or more than one vector is three vectors. 
     
     
         33 . The method of  claim 25 , where the one or more than one vector is four vectors. 
     
     
         34 . The method of  claim 25 , where the one or more than one vector is five vectors. 
     
     
         35 . The method of  claim 25 , where the one or more than one vector is an episomal vector comprising a strong spleen focus-forming virus promoter, a post-transcriptional regulatory element Wpre, and exactly two transcription and reprogramming factor genes, oct4 and sox2, and the method further comprises transducing or transfecting the target cells with an additional episomal vector comprising a strong spleen focus-forming virus promoter, a post-transcriptional regulatory element Wpre, an anti-apoptotic factor gene bcl-xl, and exactly one transcription and reprogramming factor gene, klf4. 
     
     
         36 . The method of  claim 25 , where the one or more than one vector is an episomal vector comprising a strong spleen focus-forming virus promoter, a post-transcriptional regulatory element Wpre, and exactly two transcription and reprogramming factor genes, oct4 and sox2, and the method further comprises transducing or transfecting the target cells with a first additional episomal vector comprising a strong spleen focus-forming virus promoter, a post-transcriptional regulatory element Wpre, and exactly one transcription and reprogramming factor gene, klf4, and with a second additional episomal vector comprising a strong spleen focus-forming virus promoter, a post-transcriptional regulatory element Wpre, and an anti-apoptotic factor gene bcl-xl, but without any transcription and reprogramming factor gene. 
     
     
         37 . The method of  claim 25 , where the target cells are hematopoietic stem cells. 
     
     
         38 . The method of  claim 25 , where the target cells are peripheral blood mononuclear cells. 
     
     
         39 . The method of  claim 25 , where the target cells are peripheral blood myeloid cells. 
     
     
         40 . The method of  claim 25 , where the target cells are peripheral blood cells that have been enriched for one or more than one cell type selected from the group consisting of CD33+ cells, CD34+ cells and CD133+ cells. 
     
     
         41 . The method of  claim 25 , where the target cells are peripheral blood mononuclear cells that have been enriched for CD33+ cells. 
     
     
         42 . The method of  claim 25 , where the target cells are peripheral blood cells that have been depleted of cells that express T cell marker CD3 or B cell maker CD19. 
     
     
         43 . The method of  claim 25 , further comprising harvesting the target cells from a body fluid or tissue. 
     
     
         44 . The method of  claim 43 , where the body fluid or tissue is selected from the group consisting of bone marrow and cord blood. 
     
     
         45 . The method of  claim 43 , where the body fluid or tissue is peripheral blood. 
     
     
         46 . The method of  claim 25 , further comprising providing cord blood, and purifying the cord blood to obtain the target cells. 
     
     
         47 . The method of  claim 46 , where the cord blood is obtained from a cord blood bank. 
     
     
         48 . The method of  claim 25 , further comprising enhancing or purifying the target cells for cells that express a CD33 marker. 
     
     
         49 . The method of  claim 25 , further comprising enhancing or purifying the target cells for cells that express a CD34 marker or a CD133 marker. 
     
     
         50 . The method of  claim 25 , further comprising depleting the target cells of cells that express a T cell marker CD3 or a B cell maker CD19. 
     
     
         51 . The method of  claim 25 , further comprising enhancing or purifying the target cells for cells that express a CD33 marker, and depleting the target cells of cells that express a T cell marker CD3 or a B cell maker CD19. 
     
     
         52 . The method of  claim 25 , further comprising purifying integration-free induced pluripotent stem cells from the cell culture after generating the integration-free induced pluripotent stem cells. 
     
     
         53 . The method of  claim 25 , further comprising culturing the target cells in a cell culture for a duration of between three days and six days before transducing or transfecting the target cells. 
     
     
         54 . The method of  claim 25 , further comprising culturing the target cells in a cell culture for a duration of four days before transducing or transfecting the target cells. 
     
     
         55 . Integration-free induced pluripotent stem cells generated by the method of  claim 25 . 
     
     
         56 . Integration-free induced pluripotent stem cells of  claim 55  that express one or more than one marker for a mature cell type selected from the group consisting of cardiomyocytes, hepatocytes and mesenchymal stem cells. 
     
     
         57 . Integration-free induced pluripotent stem cell colonies formed by the integration-free induced pluripotent stem cells generated by the method of  claim 25 . 
     
     
         58 . Integration-free induced pluripotent stem cell colonies of  claim 57  that express one or more than one marker for a mature cell type selected from the group consisting of cardiomyocytes, hepatocytes and mesenchymal stem cells. 
     
     
         59 . A method of treating a patient having a condition or disease, the method comprising:
 a) identifying a patient with a condition or disease suitable for treatment by the present method; and   b) administering integration-free induced pluripotent stem cells according to the present invention or generated by a method according to  claim 25 .   
     
     
         60 . The method of  claim 59 , where the patient is a human. 
     
     
         61 . The method of  claim 59 , where the condition or disease is selected from the group consisting of an autoimmune disease, cancer, cardiovascular disease, a connective tissue disease, an injury, and a neurodegenerative disease. 
     
     
         62 . The method of  claim 59 , where identifying the patient comprises diagnosing the patient with one or more than one condition or disease suitable for treatment by the method. 
     
     
         63 . The method of  claim 62 , where diagnosing the patient comprises performing one or more than one of action selected from the group consisting of performing a physical examination, performing a non-invasive imaging examination, and identifying one or more than one marker for a condition or disease in the blood or other body fluid of the patient. 
     
     
         64 . The method of  claim 59 , where identifying the patient comprises consulting patient records to determine if the patient has a condition or disease suitable for treatment by the method. 
     
     
         65 . A method for generating integration-free induced pluripotent stem cells, the method comprising:
 a) providing target cells;   b) providing one or more than one vector of  claim 1 ;   c) transducing or transfecting the target cells with the one or more than one vector; and   d) culturing the transduced or transfected cells in a cell culture, thereby generating integration-free induced pluripotent stem cells.   
     
     
         66 . A method for generating integration-free induced pluripotent stem cells, the method comprising:
 a) providing target cells;   b) providing one or more than one vector of  claim 2 ;   c) transducing or transfecting the target cells with the one or more than one vector; and   d) culturing the transduced or transfected cells in a cell culture, thereby generating integration-free induced pluripotent stem cells.   
     
     
         67 . A method for generating integration-free induced pluripotent stem cells, the method comprising:
 a) providing target cells;   b) providing one or more than one vector of  claim 3 ;   c) transducing or transfecting the target cells with the one or more than one vector; and   d) culturing the transduced or transfected cells in a cell culture, thereby generating integration-free induced pluripotent stem cells.