Replicative minicircle vectors with improved expression

Abstract

The present invention relates to the production and use of covalently closed circular (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly to vector modifications that improve expression of said DNA molecules.

1 - 21 . (canceled) 
     
     
         22 . A method of constructing a eukaryotic replicative pUC-free minicircle expression vector comprising:
 a. combining, under conditions so as to create a eukaryotic replicative pUC-free minicircle expression vector, i) a eukaryotic region encoding a gene of interest and comprising an intron and 5′ and 3′ ends, with ii) a spacer region linking the 5′ and 3′ ends of the eukaryotic region, said spacer region less than 500 basepairs in length, and with iii) a bacterial replication origin that is not the pUC origin and a RNA selectable marker positioned within said intron; and   b. expressing said gene of interest in said pUC-free minicircle expression vector, wherein said gene of interest in said pUC-free minicircle expression vector is expressed at a higher level than a vector comprising a pUC origin encoding spacer region greater than 500 basepairs.   
     
     
         23 . The method of  claim 22 , wherein said RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO:20, and SEQ ID NO:22. 
     
     
         24 . The method of  claim 22 , wherein said RNA selectable marker is selected from the group consisting of: an RNA-OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 35. 
     
     
         25 . The method of  claim 22 , wherein said bacterial replication origin is an R6K replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12. 
     
     
         26 . The method of  claim 22 , wherein said bacterial replication origin is an ColE2-P9 replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16. 
     
     
         27 . The method of  claim 22 , wherein said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31. 
     
     
         28 . The method of  claim 22 , wherein said bacterial replication origin that is not the pUC origin and a RNA selectable marker comprises an R6K replication origin-RNA-OUT RNA selection marker with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28. 
     
     
         29 . The method of  claim 22 , wherein said bacterial replication origin that is not the pUC origin and a RNA selectable marker comprises an ColE2-P9 replication origin-RNA-OUT RNA selection marker with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25. 
     
     
         30 . The method of  claim 22 , wherein said intron is a functional variant with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10. 
     
     
         31 . A method of constructing a eukaryotic replicative pUC-free minicircle expression vector comprising:
 a. combining, under conditions so as to create a eukaryotic replicative pUC-free minicircle expression vector, i) a eukaryotic region encoding a gene of interest and comprising an intron and 5′ and 3′ ends, with ii) a spacer region linking the 5′ and 3′ ends of the eukaryotic region sequences and a bacterial replication origin that is not the pUC origin and a RNA selectable marker, said spacer region less than 500 basepairs in length and said bacterial replication origin and said RNA selectable marker positioned within said intron;   b. transforming said replicative minicircle expression vector into cells of an RNA selectable marker regulated bacterial cell line;   c. isolating the resultant transformed bacterial cells by selection; and   d. propagating the resultant transformed bacterial cells in culture under conditions such as to manufacture said vector in yields of greater than 100 mg vector per liter culture.   
     
     
         32 . The method of  claim 31 , wherein said RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO:20, and SEQ ID NO:22. 
     
     
         33 . The method of  claim 31 , wherein said RNA selectable marker is selected from the group consisting of: an RNA-OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 35. 
     
     
         34 . The method of  claim 31 , wherein said bacterial replication origin is an R6K replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12. 
     
     
         35 . The method of  claim 31 , wherein said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31. 
     
     
         36 . A eukaryotic replicative pUC-free minicircle expression vector comprising i) a eukaryotic region sequence comprising an intron and 5′ and 3′ ends and ii) a spacer region of less than 500 basepairs in length linking the 5′ and 3′ ends of the eukaryotic region sequences and iii) a bacterial replication origin that is not the pUC origin and a RNA selectable marker positioned within said intron. 
     
     
         37 . The vector of  claim 36 , wherein said RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO:20, and SEQ ID NO:22. 
     
     
         38 . The vector of  claim 36 , wherein said RNA selectable marker is selected from the group consisting of: an RNA-OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 35. 
     
     
         39 . The vector of  claim 36 , wherein said bacterial replication origin is an R6K replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12. 
     
     
         40 . The vector of  claim 36 , wherein said bacterial replication origin is an ColE2-P9 replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16. 
     
     
         41 . The vector of  claim 36 , wherein said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.