Methods for the production of ips cells using non-viral approach

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.

1 .- 25 . (canceled) 
     
     
         26 . A differentiation programming vector comprising a replication origin, and one or more expression cassettes encoding a trans-acting factor which binds to the replication origin to replicate the vector extra-chromosomally; and one or more differentiation programming factors. 
     
     
         27 . The differentiation programming vector of  claim 26 , wherein the differentiation programming factors are selected from the group consisting of Sox-2, Sox-7, Sox-17, Oct-4, Nanog, Lin-28, c-Myc, Klf4, Esrrb, EBF1, C/EBPα, C/EBPβ, Ngn3, Pdx and Mafa. 
     
     
         28 . The differentiation programming vector of  claim 26 , further defined as a reprogramming vector comprising a Sox family member and an Oct family member. 
     
     
         29 . The differentiation programming vector of  claim 28 , wherein the differentiation programming factors further comprise one or more selected from the group consisting of Nanog, Lin-28, Klf4, and c-Myc. 
     
     
         30 . The differentiation programming vector of  claim 26 , wherein the differentiation programming vector lacks the ability to be integrated into a host cell genome. 
     
     
         31 . The differentiation programming vector of  claim 26 , wherein the replication origin is a replication origin of a lymphotrophic herpes virus, an adenovirus, SV40, a bovine papillomavirus, or a yeast. 
     
     
         32 . The differentiation programming vector of  claim 31 , wherein the replication origin is a replication origin of a lymphotrophic herpes virus and corresponds to oriP of EBV. 
     
     
         33 . The differentiation programming vector of  claim 32 , wherein the lymphotrophic herpes virus is Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek's disease virus (MDV). 
     
     
         34 . The differentiation programming vector of  claim 26 , wherein the trans-acting factor corresponds to EBNA-1 of EBV. 
     
     
         35 . The differentiation programming vector of  claim 26 , wherein the trans-acting factor is a derivative of a wild-type protein corresponding to EBNA-1 of EBV, which derivative activates transcription at least 5% that of the corresponding wild-type protein from an extra-chromosomal template after binding to the replication origin and has a reduced ability to activate transcription from an integrated template as compared to wild-type EBNA-1. 
     
     
         36 . The differentiation programming vector of  claim 35 , wherein the derivative lacks sequences present in the wild-type EBNA-1 protein that activate transcription from an integrated template. 
     
     
         37 . The differentiation programming vector of  claim 35 , wherein the derivative has a deletion of residues corresponding to residues about 65 to about 89 of EBNA-1 and/or a deletion of residues corresponding to residues about 90 to about 328 of EBNA-1. 
     
     
         38 . The differentiation programming vector of 35, wherein the derivative encodes a derivative with at least 80% amino acid sequence identity to residues 1 to about 40 and residues about 328 to 641 of EBNA-1. 
     
     
         39 . The differentiation programming vector of  claim 35 , wherein the derivative comprises a first nucleotide sequence encoding residues 1 to about 40 of the corresponding wild-type EBNA-1 and a second nucleotide sequence encoding residues about 328 to 641 of the corresponding wild-type EBNA-1.