Methods for the production of IPS cells using Epstein-Barr (EBV)-based reprogramming vectors

• US 9,644,184 B2
• Filed: 03/11/2013
• Issued: 05/09/2017
• Est. Priority Date: 06/04/2008
• Status: Active Grant

• Current Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Original Assignee: Cellular Dynamics International, Inc. (Fujifilm Holdings Corporation)
• Inventors: Mack, Amanda
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US13/794,297
• Publication Number:
  US 20130189778A1
• Time in Patent Office: 1,520 Days
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/605 Nanog
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2501/608 Lin28
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2710/16222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2800/107 for mammalian
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2800/24 Vectors characterised by the absence of particular element, e.g. selectable marker, viral origin of replication
• CPC Class Codes: C12N 2820/60 from viruses
• CPC Class Codes: C12N 2840/105 enhancing translation
• CPC Class Codes: C12N 2840/206 having multiple IRES
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Methods and composition of induction of pluripotent stem cells and other desired cell types are disclosed. For example, in certain aspects methods for generating essentially vector-free induced pluripotent stem cells are described. Furthermore, the invention provides induced pluripotent stem cells and desired cell types essentially free of exogenous vector elements with the episomal expression vectors to express differentiation programming factors.

Claims

1. An isolated population of human induced pluripotent stem (iPS) cells, said population comprising human pluripotent cells that comprise Epstein-Barr virus (EBV)-based reprogramming vectors comprising a replication origin and one or more expression cassettes encoding iPS reprogramming factors comprising at least Sox-2, Oct-4, wherein said cells, or one or more of said expression cassettes, comprise a nucleotide sequence encoding a trans-acting factor that binds to the replication origin to replicate an extra-chromosomal template, the cell population comprising the genome of a selected adult human individual and essentially free of chromosomally integrated retroviral elemants.

2. The isolated human iPS cell population of claim 1, wherein the genome is that of a terminally differentiated human cell.

3. The isolated population of claim 1, wherein the Epstein-Barr virus (EBV)-based vectors encode at least the reprogramming factors Oct4 and Sox2, and additionally one or more of the reprogramming factors selected from the group consisting of Lin28, Nanog, Klf4 and c-Myc.

4. The isolated population of claim 1, wherein the population further comprises human pluripotent cells that are essentially free of Epstein-Barr (EBV)-based vectors.

7. The isolated population of claim 4 or 6, wherein the human pluripotent cells that comprise Epstein-Barr (EBV)-based vectors constitute less than 10% of said population.

8. The isolated population of claim 7, wherein the human pluripotent cells that comprise Epstein-Barr (EBV)-based vectors constitute less than 1% of said population.

9. The isolated population of claim 8, wherein the human pluripotent cells that comprise Epstein-Barr (EBV)-based vectors constitute less than 0.5% of said population.

10. The isolated population of claim 9, wherein the human pluripotent cells that comprise Epstein-Barr (EBV)-based vectors constitute less than 0.1% of said population.

5. An isolated population of human induced pluripotent stem (iPS) cells, said population comprising human pluripotent cells that comprise Epstein-Barr virus (EBV)-based reprogramming vectors comprising a replication origin and one or more expression cassettes encoding at least the reprogramming factors Oct4 and Sox2, and additionally one or more of the reprogramming factors selected from the group consisting of Lin28, Nanog, Klf4 and c-Myc, wherein said cells, or one or more of said expression cassettes, comprise a nucleotide sequence encoding a trans-acting factor that binds to the replication origin to replicate an extra-chromosomal template, wherein the iPS cells of the population have been produced from somatic cells of a selected human individual and comprise the genome of that individual and are essentially free of chromosomally integrated retroviral elements.

6. The isolated population of claim 5, wherein the population further comprises human pluripotent cells that are essentially free of Epstein-Barr (EBV)-based vectors.