Methods and Vectors to Produce Vector Free Induced Pluripotent Stem Cells

A method of producing induced human pluripotent stem cells (iPSCs) that are essentially free of episomal reprogramming vector(s), comprising:

• (a) introducing an episomal reprogramming vector(s) into a somatic cell to produce a first cell population, wherein the episomal reprogramming vector(s) comprise
• (i) an OriP replication origin,
• (ii) an expression cassette encoding an iPSC reprogramming factor, a synthetic transcription factor, or both,
• (iii) a polynucleotide encoding EBNA-1 of EBV, a derivative of EBNA-1 that has a deletion of residues 65 to 89 of EBNA-1, a derivative of EBNA-1 that has a deletion of residues 90 to 328 of EBNA-1, or a derivative of EBNA-1 that has a deletion of residues 65-328, and
• (iv) a thymidine kinase or cytosine deaminase suicide gene;
• (b) culturing the first cell population to effect expression of the reprogramming factor, the synthetic transcription factor, or both, to produce a second cell population having traits consistent with embryonic stem cells;
• (c) contacting the second cell population with a suicide gene substrate to produce iPSCs that are essentially free of an episomal reprogramming vector.

A method of producing induced human pluripotent stem cells (iPSCs) that are essentially free of an episomal reprogramming vector, comprising:

• (a) introducing an episomal reprogramming vector into a somatic cell to produce a first cell population, wherein the episomal reprogramming vector comprises
• (i) an OriP replication origin,
• (ii) an expression cassette encoding an iPSC reprogramming factor, a synthetic transcription factor, or both,
• (iii) a polynucleotide encoding EBNA-1 of EBV, a derivative of EBNA-1 that has a deletion of residues 65 to 89 of EBNA-1, a derivative of EBNA-1 that has a deletion of residues 90 to 328 of EBNA-1, or a derivative of EBNA-1 that has a deletion of residues 65 to 328 of EBNA-1, and
• (iv) a tetracycline or tetracycline derivative regulated promoter system (TetOn or TetOff);
• (b) culturing the first cell population to produce a second cell population having traits consistent with embryonic stem cells, wherein during culturing the episomal reprogramming vector is replicated;
• (c) culturing the second cell population to produce a third cell population, wherein during culturing the episomal reprogramming vector is not replicated;
• (d) selecting colonies from the third cell population to produce a fourth cell population;
• and
• (e) culturing the fourth cell population to produce iPSCs that are essentially free of the episomal reprogramming vector.

A method of producing induced human pluripotent stem cells (iPSCs) that are essentially free of a reprogramming vector, comprising:

• (a) introducing a reprogramming vector into a somatic cell to produce a first cell population, wherein the reprogramming vector comprises (i) a viral origin of replication, (ii) an expression cassette encoding an iPSC reprogramming factor, a synthetic transcription factor, or both, (iii) a gene regulating extrachromosomal replication and partitioning of the reprogramming vector, (iv) a regulated promoter system and (v) a suicide gene;
• (b) culturing the first cell population to effect expression of the reprogramming factor, the synthetic transcription factor, or both, to produce a second cell population having traits consistent with embryonic stem cells, wherein during culture the reprogramming vector is replicated;
• (c) culturing the second cell population wherein the gene regulating extrachromosomal replication and partitioning is regulated such that the reprogramming vector is lost during cell division, to produce a third cell population comprising iPSCs that are substantially free of the reprogramming vector;
• (d) contacting the third cell population with a suicide gene substrate to produce iPSCs that are essentially free of the reprogramming vector.

A method of producing induced human pluripotent stem cells (iPSCs) that are essentially free of an episomal reprogramming vector, comprising:

• (a) introducing an episomal reprogramming vector into a somatic cell to produce a first cell population, wherein the episomal reprogramming vector comprises:
• (i) an OriP replication origin,
• (ii) an expression cassette encoding an iPSC reprogramming factor, a synthetic transcription factor, or both,
• (iii) a polynucleotide encoding EBNA-1 of EBV, a derivative of EBNA-1 that has a deletion of residues 65 to 89 of EBNA-1, a derivative of EBNA-1 that has a deletion of residues 90 to 328 of EBNA-1, or or a derivative of EBNA-1 that has a deletion of residues 65-328 of EBNA-1,
• (iv) a tetracycline or derivative regulated promoter system (TetOn or TetOff), and
• (v) a thymidine kinase or cytosine deaminase suicide gene;
• (b) culturing the first cell population to effect expression of the reprogramming factors to produce a second cell population having traits consistent with embryonic stem cells, wherein during culturing the episomal reprogramming vector is replicated;
• (c) culturing the second cell population to produce a third cell population comprising iPSCs that are substantially free of the episomal reprogramming vector, wherein during culturing of the second cell population, the episomal reprogramming vector is not replicated;
• (d) contacting the third cell population with a suicide gene substrate to produce iPSCs that are essentially free of the episomal reprogramming vector.

An episomal reprogramming vector comprising:

• (a) an OriP origin of replication;
• (b) an expression cassette encoding an iPSC reprogramming factor, a synthetic transcription factor, or both, and
• (c) a polynucleotide molecule encoding EBNA-1 of EBV, a derivative of EBNA-1 that has a deletion of residues 65 to 89 of EBNA-1, a derivative of EBNA-1 that has a deletion of residues 90 to 328 of EBNA-1, or a derivative of EBNA-1 that has a deletion of residues 65-328 of EBNA-1;
• and element selected from
• (d1) a suicide gene;
• (d2) a thymidine kinase or cytosine deaminase suicide gene
• (d3) a regulated promoter system;
• (d4) a TetOn or TetOff system;
• (d5) a TetOn or TetOff system;
• and a suicide gene;
• and/or
• (d6) a TetOn or TetOff system, and a thymidine kinase or cytosine deaminase suicide gene.