Expression of products from nucleic acid concatemers

Abstract

Provided are techniques for generating expression products using one or more nucleic acid concatemers that include tandem repeats of a nucleic acid sequence encoding the expression product or products. In one embodiment, different expression products may be co-expressed using a concatemer mixture of a first nucleic acid concatemer and a second nucleic acid concatemer having a predefined ratio to one another.

1 . A method comprising:
 formulating a concatemer mixture comprising at least a first nucleic acid concatemer and a second nucleic acid concatemer having a predefined ratio to one another, wherein the first nucleic acid concatemer comprises tandem repeats of a first nucleic acid sequence and wherein the second nucleic acid concatemer comprises tandem repeats of a second nucleic acid sequence; and   co-expressing the concatemer mixture to generate a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence.   
     
     
         2 . The method of  claim 1 , comprising generating the first nucleic acid concatemer and the second nucleic acid concatemer using rolling circle amplification. 
     
     
         3 . The method of  claim 2 , comprising using the rolling circle amplification to amplify circular or plasmid DNA to generate the first nucleic acid concatemer and/or the second nucleic acid concatemer. 
     
     
         4 . The method of  claim 1 , wherein the first tandem repeats and/or the second tandem repeats are tandem repeats of at least a portion of the circular or plasmid DNA. 
     
     
         5 . The method of  claim 1 , comprising transfecting cultured cells with the concatemer mixture to cause the co-expressing or wherein the co-expressing is in a cell-free expression system. 
     
     
         6 . The method of  claim 1 , wherein the first nucleic acid concatemer, the second nucleic acid concatemer, or both are over 10 kb in length. 
     
     
         7 . The method of  claim 1 , comprising collecting the first expression product and the second expression product. 
     
     
         8 . The method of  claim 1 , wherein one or both of the first nucleic acid concatemer or the second nucleic acid concatemer is unprocessed before the co-expressing. 
     
     
         9 . The method of  claim 1 , wherein the first expression product is an envelope or packing protein of a virus and the second expression product comprises a transgene of the virus, wherein the first expression product and the second expression product form a viral vector for delivering the transgene. 
     
     
         10 . The method of  claim 1 , wherein the first expression product is a nucleic acid product and the second expression product is a protein product. 
     
     
         11 . The method of  claim 10 , wherein the first expression product forms a complex with the second expression product. 
     
     
         12 . The method of  claim 10 , wherein the first expression product acts on a third nucleic acid concatemer in the concatemer mixture. 
     
     
         13 . A method comprising:
 formulating a mixture comprising at least two nucleic acid concatemers in a predefined ratio, wherein each of the nucleic acid concatemers comprises tandem repeats of two or more nucleic acid sequences; and   co-expressing the concatemer mixture to generate two or more expression products from each nucleic acid concatemer in the mixture.   
     
     
         14 . The method of  claim 13 , wherein the nucleic acid sequence of an individual concatemer in the concatemer mixture comprises a plurality of expression sequences that, when expressed to generate the two or more expression products, generate a plurality of proteins. 
     
     
         15 . The method of  claim 13 , wherein the nucleic acid sequence of an individual concatemer in the concatemer mixture comprises a plurality of expression sequences that, when expressed to generate the two or more expression products, generate a mix comprising at least one protein expression product and at least one nucleic acid expression product. 
     
     
         16 . A method, comprising:
 amplifying at least one template comprising a first nucleic acid sequence using strand-displacement rolling circle amplification to a generate a first concatemer comprising tandem repeats of the first nucleic acid sequence;   contacting the first concatemer with a second concatemer comprising tandem repeats of a second nucleic acid sequence to form a concatemer mixture having a predefined ratio of the first nucleic acid concatemer to the second nucleic acid concatemer; and   co-expressing the concatemer mixture to generate a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence, wherein a ratio of the first expression product to the second expression product is proportional to the predefined ratio of the first nucleic acid concatemer to the second nucleic acid concatemer in the concatemer mixture.   
     
     
         17 . The method of  claim 16 , comprising allowing the first expression product to form a complex with the second expression product. 
     
     
         18 . The method of  claim 16 , wherein the first expression product and the second expression product comprise different viral products from a same virus, such as a an adenovirus or a lentivirus. 
     
     
         19 . The method of  claim 16 , wherein the first expression product comprises a viral mRNA and the second expression product comprises a plurality of viral packaging proteins.