Polycistronic Vector For Human Induced Pluripotent Stem Cell Production

Abstract

Methods of producing induced pluripotent stem (iPS) cells are provided. For example, a method of producing an iPS cell from a differentiated cell, which includes transforming the differentiated cell with a first vector comprising a nucleic acid sequence comprising a nucleic acid sequence encoding an Oct4, a nucleic acid sequence encoding a Sox2, and a nucleic acid sequence encoding a Klf4. Each of the nucleic acid sequences are separated from each other by a first and second viral 2A sequence. The method described can further comprise culturing the transformed cell under conditions that allow for the production of an iPS cell and isolating the cultured iPS cell.

1 . A method of producing an induced pluripotent stem (iPS) cell from a differentiated cell comprising transforming the differentiated cell with a first vector, wherein the first vector comprises a nucleic acid sequence comprising (i) a nucleic acid sequence encoding an Oct4, (ii) a nucleic acid sequence encoding a Sox2, and (iii) a nucleic acid sequence encoding a Klf4, wherein each of the nucleic acid sequences, (i)-(iii), are separated by a first and second nucleic acid encoding a viral 2A sequence. 
     
     
         2 . The method of  claim 1 , wherein the vector comprises SEQ ID NO:7. 
     
     
         3 . The method of  claim 1 , wherein the vector comprises a nucleic acid sequence encoding SEQ ID NO:9. 
     
     
         4 . The method of  claim 1 , further comprising culturing the transformed cell under conditions that allow for the production of a population of iPS cells. 
     
     
         5 . The method of  claim 1 , further comprising isolating the population of iPS cells. 
     
     
         6 . The method of  claim 1 , wherein the first vector comprises in order from the 5′ end the nucleic acid sequence encoding the Oct4, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Sox2, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Klf4. 
     
     
         7 . The method of  claim 1 , wherein the first vector comprises in order from the 5′ end the nucleic acid sequence encoding the Oct4, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Klf4, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Sox2. 
     
     
         8 . The method of  claim 1 , wherein the first vector comprises in order from the 5′ end the nucleic acid sequence encoding the Sox2, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Oct4, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Klf4. 
     
     
         9 . The method of  claim 1 , wherein the first vector comprises in order from the 5′ end the nucleic acid sequence encoding the Sox2, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Klf4, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Oct4. 
     
     
         10 . The method of  claim 1 , wherein the first vector comprises in order from the 5′ end the nucleic acid sequence encoding the Klf4, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Oct4, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Sox2. 
     
     
         11 . The method of  claim 1 , wherein the first vector comprises in order from the 5′ end the nucleic acid sequence encoding the Klf4, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Sox2, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Oct4. 
     
     
         12 . The method of  claim 1 , wherein the differentiated cell is a mammalian cell. 
     
     
         13 . The method of  claim 12 , wherein the mammalian cell is a human cell. 
     
     
         14 . The method of  claim 13 , wherein the mammalian cell is selected from the group consisting of a(n) epithelial cell, keratinocyte, fibroblast, hepatocyte, neuron, osteoblast, myocyte, kidney cell, lung cell, thyroid cell, and pancreatic cell. 
     
     
         15 . The method of  claim 14 , wherein the mammalian cell is a keratinocyte. 
     
     
         16 . The method of  claim 1 , wherein the first and second nucleic acid sequences encoding a viral 2A sequence are selected from picornaviral 2A sequences, tetraviral 2A sequences, or a combination thereof. 
     
     
         17 . The method of  claim 16 , wherein the picornaviral 2A sequences are selected from the group consisting of the Enteroviral 2A sequences, Rhinoviral 2A sequences, Cardioviral 2A sequences, Aphthoviral 2A sequences, Hepatoviral 2A sequences, Erboviral 2A sequences, Kobuviral 2A sequences, Teschoviral 2A sequences, and the Parechoviral 2A sequences. 
     
     
         18 . The method of  claim 16 , wherein the tetraviral 2A sequences are Betatetraviral 2A sequences or Omegatetraviral 2A sequences. 
     
     
         19 . The method of  claim 1 , wherein the first and second nucleic acid sequences encoding a viral 2A sequence comprises a nucleic acid sequence encoding the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO:2) or EGRGSLLTCGDVEENPGP (SEQ ID NO:3). 
     
     
         20 . The method of  claim 1 , wherein the first nucleic acid sequence encoding a viral 2A sequence comprises a nucleic acid sequence encoding the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO:2) and the second nucleic acid sequence encoding a viral 2A sequence comprises a nucleic acid sequence encoding the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO:3). 
     
     
         21 . The method of  claim 1 , wherein the first vector is a plasmid, an adenoviral vector or a retroviral vector. 
     
     
         22 . The method of  claim 21 , wherein the retroviral vector is a lentiviral vector. 
     
     
         23 . The method of  claim 22 , wherein the lentiviral vector is a lentiviral SIN vector. 
     
     
         24 . The method of  claim 21 , wherein the retroviral vector comprises a 3′ long terminal repeat. 
     
     
         25 . The method of  claim 24 , wherein the retroviral vector further comprises a loxP sequence. 
     
     
         26 . The method of  claim 25 , wherein the loxP sequence is in a 3′ long terminal repeat of the lentiviral vector. 
     
     
         27 . The method of  claim 25 , further comprising transforming the iPS cell with a second vector, wherein the second vector comprises a nucleic acid encoding a Cre recombinase, wherein expression of the Cre recombinase results in the deletion of the first vector from the genome of the iPS cells. 
     
     
         28 . The method of  claim 27 , further comprising isolating a population of iPS cells lacking the first vector. 
     
     
         29 . An isolated iPS cell produced by the method described in  claim 28 . 
     
     
         30 . The method of  claim 1 , further comprising correcting a genetic mutation in the differentiated cell, wherein the first vector further comprises a nucleic acid sequence comprising an unmutated nucleic acid sequence of interest and homologous nucleic acid sequences flanking the genetic mutation to be corrected. 
     
     
         31 . The method of  claim 30 , wherein the genetic mutation is a mutation in the nucleic acid sequence encoding β-globin, the nucleic acid sequence encoding cystic fibrosis transmembrane conductance regulator, the nucleic acid sequence encoding phenylalanine hydroxylase, and the nucleic acid sequence encoding dystrophin. 
     
     
         32 . The method of  claim 31 , wherein the genetic mutation is a mutation in the nucleic acid sequence encoding β-globin. 
     
     
         33 . The method of  claim 32 , wherein the mutation in the nucleic acid sequence encoding β-globin results in a glutamic acid to valine substitution at the sixth amino acid of the β-globin protein. 
     
     
         34 . The method of  claim 33 , wherein the glutamic acid to valine substitution is caused by an A to T transversion at base pair +20 relative to the A(+1) of the ATG start codon of the nucleic acid sequence encoding β-globin. 
     
     
         35 . The method of  claim 30 , wherein the first vector further comprises a first and second loxP sequence. 
     
     
         36 . The method of  claim 35 , wherein the first vector further comprises a nucleic acid sequence encoding a Cre recombinase operably linked to an inducible promoter. 
     
     
         37 . The method of  claim 36 , wherein the inducible promoter comprises a Nanog-responsive thymidine kinase promoter. 
     
     
         38 . The method of  claim 30 , wherein the first vector comprises SEQ ID NO:44. 
     
     
         39 . A vector comprising (i) a nucleic acid sequence encoding an Oct4, (ii) a nucleic acid sequence encoding a Sox2, and (iii) a nucleic acid sequence encoding a Klf4, wherein each of the nucleic acid sequences, (i)-(iii), are separated by a first and second nucleic acid sequence encoding a viral 2A sequence. 
     
     
         40 . The vector of  claim 39 , wherein the vector comprises SEQ ID NO:7. 
     
     
         41 . The vector of  claim 39 , wherein the vector comprises a nucleic acid sequence encoding SEQ ID NO:9. 
     
     
         42 . The vector of  claim 39 , wherein the vector comprises in order from the 5′ end the nucleic acid sequence encoding the Oct4, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Sox 2, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Klf4. 
     
     
         43 . The vector of  claim 39 , wherein the vector comprises in order from the 5′ end the nucleic acid sequence encoding the Oct4, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Klf4, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Sox2. 
     
     
         44 . The vector of  claim 39 , wherein the vector comprises in order from the 5′ end the nucleic acid sequence encoding the Sox2, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Oct 4, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Klf4. 
     
     
         45 . The vector of  claim 39 , wherein the vector comprises in order from the 5′ end the nucleic acid sequence encoding the Sox2, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Klf4, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Oct4. 
     
     
         46 . The vector of  claim 39 , wherein the vector comprises in order from the 5′ end the nucleic acid sequence encoding the Klf4, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Oct 4, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Sox2. 
     
     
         47 . The vector of  claim 39 , wherein the vector comprises in order from the 5′ end the nucleic acid sequence encoding the Klf4, the first nucleic acid sequence encoding a viral 2A sequence, the nucleic acid sequence encoding the Sox2, the second nucleic acid sequence encoding a viral 2A sequence, and the nucleic acid sequence encoding the Oct4. 
     
     
         48 . The vector of  claim 39 , wherein the first and second nucleic acid sequences encoding a viral 2A sequence are selected from picornaviral 2A sequences, tetraviral 2A sequences, or a combination thereof. 
     
     
         49 . The vector of  claim 48 , wherein the picornaviral 2A sequences are selected from the group consisting of the Enteroviral 2A sequences, Rhinoviral 2A sequences, Cardioviral 2A sequences, Aphthoviral 2A sequences, Hepatoviral 2A sequences, Erboviral 2A sequences, Kobuviral 2A sequences, Teschoviral 2A sequences, and the Parechoviral 2A sequences. 
     
     
         50 . The vector of  claim 48 , wherein the tetraviral 2A sequences are Betatetraviral 2A sequences or Omegatetraviral 2A sequences. 
     
     
         51 . The vector of  claim 39 , wherein the first and second nucleic acid sequences encoding a viral 2A sequence comprise a nucleic acid sequence encoding the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO:2) or EGRGSLLTCGDVEENPGP (SEQ ID NO:3). 
     
     
         52 . The vector of  claim 39 , wherein the first nucleic acid sequence encoding a viral 2A sequence comprises a nucleic acid sequence encoding the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO:2) and the second nucleic acid sequence encoding a viral 2A sequence comprises a nucleic acid sequence encoding the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO:3). 
     
     
         53 . The vector of  claim 39 , wherein the vector is designed to correct a genetic mutation, the vector further comprising an unmutated nucleic acid sequence of interest and homologous nucleic acid sequences flanking the genetic mutation. 
     
     
         54 . The vector of  claim 53 , wherein the unmutated nucleic acid sequence of interest comprises the nucleic acid sequence encoding β-globin. 
     
     
         55 . The vector of  claim 54 , wherein the vector further comprises a first and second loxP sequence. 
     
     
         56 . The vector of  claim 55 , wherein the vector further comprises a nucleic acid sequence encoding a Cre recombinase operably linked to an inducible promoter. 
     
     
         57 . The vector of  claim 56 , wherein the inducible promoter comprises a Nanog-responsive thymidine kinase promoter. 
     
     
         58 . The vector of  claim 57 , wherein the vector comprises SEQ ID NO:44. 
     
     
         59 . The vector of  claim 39 , wherein the vector is a plasmid, an adenoviral vector or a retroviral vector. 
     
     
         60 . The vector of  claim 59 , wherein the retroviral vector is a lentiviral vector. 
     
     
         61 . The vector of  claim 60 , wherein the lentiviral vector is a lentiviral SIN vector. 
     
     
         62 . The vector of  claim 59 , wherein the retroviral vector comprises a 3′ long terminal repeat. 
     
     
         63 . The vector of  claim 62 , wherein the retroviral vector further comprises a loxP sequence. 
     
     
         64 . The vector of  claim 63 , wherein the loxP sequence is in the 3′ long terminal repeat of the lentiviral vector. 
     
     
         65 . A differentiated cell comprising the vector of  claim 39 . 
     
     
         66 . The differentiated cell of  claim 65 , wherein the differentiated cell is a mammalian cell. 
     
     
         67 . The differentiated cell of  claim 65 , wherein the mammalian cell is a human cell. 
     
     
         68 . The differentiated cell of  claim 65 , wherein the mammalian cell is selected from the group consisting of a(n) epithelial cell, keratinocyte, fibroblast, hepatocyte, neuron, osteoblast, myocyte, kidney cell, lung cell, thyroid cell, and pancreatic cell. 
     
     
         69 . The differentiated cell of  claim 65 , wherein the mammalian cell is a keratinocyte. 
     
     
         70 . A kit comprising (i) the first vector of  claim 39  and (ii) a second vector comprising a nucleic acid sequence encoding a Cre recombinase. 
     
     
         71 . A method of treating or preventing a disease associated with a genetic mutation in a subject, the method comprising:
 (a) selecting a subject with a disease associated with a genetic mutation;   (b) isolating differentiated cells from the subject;   (c) transforming the differentiated cells with a vector comprising an unmutated nucleic acid sequence of interest;   (d) culturing the transformed cells under conditions that allow for the production of a population of iPS cells;   (e) screening the iPS cells for correction of the genetic mutation; and   (f) administering the iPS cells to the subject, wherein administration of the iPS cells treats or prevents the disease associated with the genetic mutation in the subject.   
     
     
         72 . The method of  claim 71 , wherein the vector comprises a nucleic acid sequence comprising (i) an unmutated nucleic acid sequence of interest and homologous nucleic acid sequences flanking the genetic mutation, (ii) a nucleic acid sequence encoding a Cre recombinase operably linked to an inducible promoter, (iii) a first and second loxP sequence, (iv) a nucleic acid sequence encoding an Oct4, (v) a nucleic acid sequence encoding a Sox2, and (vi) a nucleic acid sequence encoding a Klf4, wherein each of the nucleic acid sequences, (iv)-(vi), are separated by a first and second nucleic acid sequence encoding a viral 2A sequence. 
     
     
         73 . The method of  claim 72 , wherein the inducible promoter comprises a Nanog-responsive thymidine kinase promoter. 
     
     
         74 . The method of  claim 71 , wherein the disease caused by the mutation in the genome is selected from the group consisting of sickle cell disease, thalassemia, cystic fibrosis, phenylketonuria, and Duchenne muscular dystrophy. 
     
     
         75 . The method of  claim 74 , wherein the disease is sickle cell disease. 
     
     
         76 . The method of  claim 75 , wherein the vector comprises SEQ ID NO:44. 
     
     
         77 . The method of  claim 71 , wherein the differentiated cell is selected from the group consisting of a(n) epithelial cell, keratinocyte, fibroblast, hepatocyte, neuron, osteoblast, myocyte, kidney cell, lung cell, thyroid cell, and pancreatic cell. 
     
     
         78 . The method of  claim 77 , wherein the differentiated cell is a keratinocyte.