Non-integrating dna vectors for the genetic modification of cells

Abstract

The present invention relates to a polynucleotide comprising at least one promoter and an S/MAR element, wherein said S/MAR element is located downstream of said promoter and wherein the nucleic acid sequence of said S/MAR element (S/MAR sequence) comprises at least 3sequence motifs ATTA (SEQ ID NO:1) per 100 nucleotides over a stretch of at most 200 nucleotides; the present invention further relates to a composition and to a host cell comprising said polynucleotide, and to the polynucleotide for use in medicine and for use in treating genetic disease. The present invention also relates to a kit and to a device comprising said polynucleotide, and to methods and uses related to the polynucleotide.

1 . A polynucleotide comprising at least one promoter and an S/MAR element, wherein said S/MAR element is located downstream of said promoter and wherein the nucleic acid sequence of said S/MAR element (S/MAR sequence) comprises at least 3 sequence motifs ATTA (SEQ ID NO:1) per 100 nucleotides over a stretch of at most 200 nucleotides. 
     
     
         2 . The polynucleotide of  claim 1 , wherein said S/MAR element is flanked by a splice donor and a splice acceptor. 
     
     
         3 . The polynucleotide of  claim 1 , wherein said polynucleotide further comprises a coding sequence encoding a polypeptide, preferably a selectable marker, wherein said coding sequence encoding a polypeptide intervenes said promoter and said S/MAR element. 
     
     
         4 . The polynucleotide of  claim 1 , wherein said polynucleotide further comprises a coding sequence encoding a selectable marker (selectable marker sequence), said selectable marker sequence intervening said promoter and said S/MAR element, wherein said promoter and said selectable marker sequence together constitute a selectable marker gene, and wherein said selectable marker is a selectable marker of a eukaryotic cell. 
     
     
         5 . The polynucleotide of  claim 4 , wherein said selectable marker gene is a puromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, or a zeocin resistance gene, preferably is a puromycin resistance gene. 
     
     
         6 . The polynucleotide of  claim 2 , wherein a transcript is transcribed from said promoter, from which transcript the sequence of the S/MAR element is spliced out. 
     
     
         7 . The polynucleotide of  claim 1 , wherein said polynucleotide further comprises a bacterial origin of replication and/or a bacterial selectable marker gene, wherein said bacterial origin of replication and/or bacterial selectable marker gene is/are insulated from the residual sequences comprised in the polynucleotide by the presence of at least one insulation element. 
     
     
         8 . The polynucleotide of  claim 1 , wherein said polynucleotide is devoid of a simian virus 40 (SV40) origin of replication, a bovine papillomavirus (BPV) origin of replication, and an Epstein-Barr virus (EBV) origin of replication. 
     
     
         9 . The polynucleotide of  claim 1 , wherein said polynucleotide replicates episomally in a host cell, preferably wherein episomal replication is stable episomal replication, preferably in a mammalian cell. 
     
     
         10 . A composition comprising a polynucleotide according to  claim 1 , preferably wherein said composition is a pharmaceutical composition. 
     
     
         11 . A host cell comprising the polynucleotide according to  claim 1 . 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . A method for stably transfecting a host cell, comprising
 a) contacting said host cell with a polynucleotide according to  claim 1 , and,   b) thereby, stably transfecting a host cell.   
     
     
         16 . The method of  claim 15 , wherein said stably transfecting comprises stable episomal replication of the polynucleotide, preferably episomal replication to such an extent that the polynucleotide still is detectable in a host cell population after on average 50 cell divisions. 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . A method for treating genetic disease in a subject, comprising
 a) contacting said subject with a polynucleotide according to  claim 1 , and,   b) thereby, treating genetic disease in said subject.   
     
     
         20 . The host cell of  claim 11 , wherein said host cell is a CD34+ Progenitor Cell; a CD61+ Thrombocyte; a CD19+ B-Lymphocyte; a CD14+ Monocyte; a CD15+ Granulocyte; a CD3+ Cytotoxic T-Lymphocyte; a CD3+ Helper T-Lymphocyte; a CD3+ activated T-Lymphocyte, a Tumor infiltrating Lymphocyte, or a Natural Killer (NK) cell.