Adenoviral assembly method
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 adenoviral core module destination vector by combining a hybridization competent vector backbone 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 form a core module that comprises an E2-L2 module, an L3-L4 module, both an E2-L2 module and an L3-L4 module, or an E2-L4 module, wherein the core module is at least 12 kb in length;
inserting into the adenoviral core module destination vector recombination site nucleic acid sequences that flank the core module, thereby forming a recombination competent core module destination vector; and
assembling an adenovirus genome by combining the recombination competent core module destination vector with one or more recombination competent adenoviral gene modules by site-specific recombination, wherein the one or more recombination competent adenoviral gene modules comprise an E1 module, an E3 module, an E4 module, or any combination thereof.
2. The method of claim 1 , wherein the hybridization competent vector backbone comprises a p15A origin of replication.
3. The method of claim 1 , wherein the hybridization competent vector backbone comprises a mammalian I-SceI expression cassette.
4. The method of claim 1 , wherein the core module is at least 14 kb in length.
5. The method of claim 4 , wherein the core module comprises an E2-L2 module and an L3-L4 module.
6. The method of claim 4 , wherein the core module comprises an E2-L4 module.
7. The method of claim 1 , wherein the hybridization competent 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.
8. The method of claim 7 , wherein the hybridization competent vector backbone and the one or more hybridization competent adenoviral gene modules comprise a single-stranded nucleic acid overhang on each terminus.
9. 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.
10. The method of claim 1 , wherein the hybridization competent vector backbone is formed by:
contacting a circular vector backbone with an endonuclease to form a linear vector backbone; and
contacting the linear vector backbone with an exonuclease to form the hybridization competent vector backbone.
11. The method of claim 1 , wherein the recombination site nucleic acid sequences are attB, attP, attR or attL sites.
12. The method of claim 1 , wherein the recombination site nucleic acid sequences are inserted into the adenoviral core module destination vector by SLIC.
13. The method of claim 1 , wherein combining the recombination competent core module destination vector with one or more recombination competent adenoviral gene modules by site-specific recombination comprises contacting the recombination competent core module destination vector and the one or more recombination competent adenoviral gene modules with an integrase.
14. The method of claim 1 , further comprising transfecting the adenovirus genome into a cell.
15. The method of claim 1 , wherein the adenovirus genome is capable of forming a recombinant adenovirus when expressed in a cell.
16. 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.
17. The method of claim 1 , wherein at least one of the one or more recombination competent or hybridization competent adenoviral gene modules comprises one or more modifications relative to the wild type adenovirus from which the gene module is derived.
18. A method of making a recombinant adenovirus, comprising:
assembling an adenoviral core module destination vector by combining a hybridization competent vector backbone with one or more hybridization competent adenoviral gene modules by sequence and ligation independent cloning (SLIC), wherein the vector backbone comprises a mammalian I-SceI expression cassette, a p15A origin of replication, or both, and wherein the one or more hybridization competent adenoviral gene modules form a core module that comprises an E2-L2 module, an L3-L4 module, both an E2-L2 module and an L3-L4 module, or an E2-L4 module;
inserting into the adenoviral core module destination vector recombination site nucleic acid sequences that flank the core module, thereby forming a recombination competent core module destination vector; and
assembling an adenovirus genome by combining the recombination competent core module destination vector with one or more recombination competent adenoviral gene modules by site-specific recombination, wherein the one or more recombination competent adenoviral gene modules comprise an E1 module, an E3 module, an E4 module, or any combination thereof.
19. The method of claim 18 , wherein the core module is at least 12 kb in length.
20. The method of claim 18 , wherein the hybridization competent 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.