Avoidance of undesirable replication intermediates in plasmid propagation

Abstract

Disclosed herein are improved plasmid shuttle vectors, vaccines based on them, and methods related to their construction and use. Particular arrangements of functional elements of such plasmids, namely origins of replication and eukaryotic transcription/translation control elements, which give rise to generally undesirable side-products upon propagation of the plasmids in bacterial culture are disclosed. These side-products apparently arise as terminated replication intermediates. Strategies both to eliminate accumulation of these side-products, and to make them useful as a vaccine adjuvant, are described.

1 . A plasmid adapted for high copy number replication, the plasmid comprising a high copy number prokaryotic origin of replication and at least one eukaryotic control element, wherein the origin of replication is in antiparallel orientation relative to the control element, and wherein replication of the plasmid results in a substantial decrease in replication side products as compared to replication of a second plasmid having the same origin of replication and the same at least one eukaryotic control element, wherein in the second plasmid, the origin of replication and the control element are in a parallel orientation.  
   
   
       2 . A plasmid adapted for high copy number replication, the plasmid comprising a high copy number prokaryotic origin of replication at a first distance from at least one eukaryotic control element, wherein the origin of replication is in a parallel orientation relative to the control element, and wherein replication of the plasmid results in a substantial decrease in linear replication side products as compared to replication of a second plasmid having the same origin of replication at a second distance from the same at least one eukaryotic control element, wherein in the second plasmid, the origin of replication and the control element are in a parallel orientation, and wherein the first distance is at least about 1.5 kb, and the second distance is substantially less than the first distance.  
   
   
       3 . In a plasmid having a high copy number prokaryotic origin of replication and at least one eukaryotic control element located at a first distance in a direction of replication, wherein the first distance is less than about 1200 bp from the origin of replication, and wherein the plasmid produces a first population of linear replication side products, 
 the improvement comprising: an increased distance in a direction of replication between the origin of replication and the control element, wherein the increased distance is at least about 1.5 kb, and wherein the improved plasmid produces a second population of linear replication side products, wherein the second population is substantially smaller than the first population.    
   
   
       4 . The improved plasmid of  claim 3 , wherein the origin of replication and the control element are in a parallel orientation.  
   
   
       5 . The improved plasmid of  claim 3 , wherein the origin of replication and the control element are in an antiparallel orientation.  
   
   
       6 . The improved plasmid of  claim 3 , wherein said first distance is less than about 1 kb.  
   
   
       7 . The improved plasmid of  claim 3 , wherein the prokaryotic origin of replication is pUC, pMB1 or ColE1.  
   
   
       8 . The improved plasmid of  claim 3 , wherein the control element is selected from the group consisting of an enhancer, a promoter, and an IRES.  
   
   
       9 . The improved plasmid of  claim 8 , wherein the control element comprises a CMV promoter and/or enhancer.  
   
   
       10 . The improved plasmid of  claim 8 , wherein the control element comprises an RSV LTR promoter.  
   
   
       11 . The improved plasmid of  claim 8 , wherein the control element comprises a picornaviral IRES.  
   
   
       12 . The improved plasmid of  claim 11 , wherein the IRES comprises a cardiovirus IRES.  
   
   
       13 . The improved plasmid of  claim 12 , wherein the IRES is an EMC virus IRES.  
   
   
       14 . The improved plasmid of  claim 8 , wherein the control element comprises a CMV enhancer 19 base pair repeat.  
   
   
       15 . The improved plasmid of  claim 8 , wherein the control element comprises a CMV enhancer 16 base pair repeat or 18 base pair repeat.  
   
   
       16 . The improved plasmid of  claim 3  which is derived from a plasmid selected from the group consisting of: a pVAX1 vector, a pEBFP-N1 vector, a pECFP-N1 vector, a pEGFP-N1 vector, a pEGFP-N2 vector, a pEGFP-N3 vector, a pEYFP-N1 vector, a pEBFP-C1 vector, a pECFP-C1 vector, a pEGFP-C1 vector, a pEGFP-C2 vector, a pEGFP-C3 vector, a pEYFP-C1 vector, a pEGFP-F vector, a pCMS-EGFP vector, a pIRES2-EGFP vector, a pd2ECFP-N1 vector, a pd2EGFP-N1 vector, a pd2EYFP-N1 vector, a pd1EGFP-N1 vector, a pCMV-Script™ vector, a pCMV-Tag™ vector, a pDual™ vector, a pBK-CMV vector, a pBK-RSV vector, a PTARGET™ vector, a pCI vector, and a pCI-Neo vector, wherein said derivation comprises substantially increasing the distance in the direction of replication between said origin of replication and said eukaryotic control element.  
   
   
       17 . A DNA vaccine or gene therapeutic, comprising the improved plasmid of  claim 3 .  
   
   
       18 . A DNA vaccine or gene therapeutic, comprising an improved plasmid having a high copy number prokaryotic origin of replication and at least one eukaryotic control element located at a first distance in a direction of replication, wherein the first distance is less than about 1200 bp from the origin of replication, and wherein the plasmid produces a first population of linear replication side products, wherein said plasmid has been improved; 
 the improvement comprising: an increased distance in a direction of replication between the origin of replication and the control element, wherein the increased distance is at least about 1.5 kb, and wherein the improved plasmid produces a second population of linear replication side products, wherein the second population is substantially smaller than the first population.