Adenoviral assembly method

Abstract

Methods of assembling modified adenoviruses, libraries of adenoviral gene modules and compositions thereof are provided herein.

1 . A method of making a recombinant adenovirus, comprising assembling an adenovirus genome by combining a hybridization competent adenoviral destination vector with one or more hybridization competent adenoviral gene modules by sequence and ligation independent cloning (SLIC), wherein the one or more hybridization competent adenoviral gene modules comprise an E2-L2 module, an L3-L4 module, an E2-L4 module, an E1 module, an E3 module, an E4 module, or any combination thereof. 
     
     
         2 . The method of  claim 1 , wherein the hybridization competent adenoviral destination vector is an adenovirus core module destination vector comprising an E2-L2 module, an L3-L4 module, both an E2-L2 module and an L3-L4 module, or an E2-L4 module; and the one or more hybridization competent adenoviral gene modules comprise an E1 module, an E3 module, an E4 module, or any combination thereof. 
     
     
         3 . The method of  claim 1 , wherein the hybridization competent destination vector backbone comprises a p15A origin of replication. 
     
     
         4 . The method of  claim 1 , wherein the hybridization competent destination vector backbone comprises a mammalian I-SceI expression cassette. 
     
     
         5 . The method of  claim 2 , wherein the core module is at least 12 kb in length. 
     
     
         6 . The method of  claim 2 , wherein the core module is at least 14 kb in length. 
     
     
         7 . The method of  claim 2 , wherein the core module comprises an E2-L2 module and an L3-L4 module. 
     
     
         8 . The method of  claim 2 , wherein the core module comprises an E2-L4 module. 
     
     
         9 . The method of  claim 1 , wherein the hybridization competent destination vector backbone and the one or more hybridization competent adenoviral gene modules comprise a single-stranded nucleic acid overhang of about 20 to about 25 base pairs in length. 
     
     
         10 . The method of  claim 9 , wherein the hybridization competent destination vector backbone and the one or more hybridization competent adenoviral gene modules comprise a single-stranded nucleic acid overhang on each terminus. 
     
     
         11 . The method of  claim 1 , wherein the one or more hybridization competent adenoviral gene modules are formed by:
 contacting adenoviral gene modules that are circular or contained within a circular plasmid with an endonuclease to form linear adenoviral gene modules; and   contacting the linear adenoviral gene modules with an exonuclease to form the one or more hybridization competent adenoviral gene modules.   
     
     
         12 . The method of  claim 1 , wherein the hybridization competent destination vector backbone is formed by:
 contacting a circular destination vector backbone with an endonuclease to form a linear destination vector backbone; and   contacting the linear destination vector backbone with an exonuclease to form the hybridization competent destination vector backbone.   
     
     
         13 . The method of  claim 1 , further comprising transfecting the adenovirus genome into a cell. 
     
     
         14 . The method of  claim 1 , wherein the adenovirus genome is capable of forming a recombinant adenovirus when expressed in a cell. 
     
     
         15 . The method of  claim 1 , wherein the adenovirus genome is a partial adenovirus genome construct that is capable of forming a recombinant adenovirus when expressed in a complementing cell line or when expressed in a cell with a helper virus. 
     
     
         16 . The method of  claim 1 , wherein at least one of the one or more hybridization competent adenoviral gene modules comprises one or more modifications relative to the wild type adenovirus from which the gene module is derived. 
     
     
         17 . A library of adenoviral gene modules, comprising:
 a plurality of different E1 modules;   a plurality of different E2-L2 modules;   a plurality of different L3-L4 modules;   a plurality of different E3 modules; and/or   a plurality of different E4 modules.   
     
     
         18 . A recombinant adenovirus, comprising a hybridization competent adenoviral destination vector, and one or more hybridization competent adenoviral gene modules comprising an E2-L2 module, an L3-L4 module, an E2-L4 module, an E1 module, an E3 module, an E4 module, or any combination thereof.