High throughput assay for measuring adenovirus replication kinetics

1. A method for measuring replication kinetics of a recombinant adenovirus, comprising:

(i) transfecting cells with the genome of the recombinant adenovirus, or infecting cells with particles of the recombinant adenovirus,

wherein the recombinant adenovirus comprises a recombinant adenovirus genome comprising a heterologous opening reading frame (ORF) encoding a fluorescent protein, and a self-cleaving peptide coding sequence, both operably linked to and in the same reading frame as an endogenous adenovirus ORF, wherein the self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous ORF, and wherein:

(a) the endogenous ORF is E1B-55k and the heterologous ORF is 3′ of E1B-55k;

(b) the endogenous ORF is DNA polymerase and the heterologous ORF is 5′ of DNA polymerase;

(c) the endogenous ORF is DNA-binding protein (DBP) and the heterologous ORF is 3′ of DBP;

(d) the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5′ of ADP;

(e) the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k; or

(f) the endogenous ORF is E4-ORF2 and the heterologous ORF is 5′ of E4-ORF2;

(ii) culturing the transfected cells or infected cells for at least two days;

(ii) measuring fluorescence at regular intervals throughout the culture period; and

(iv) calculating log-slope from the fluorescence measurements, thereby measuring replication kinetics of the recombinant adenovirus.

2. The method of claim 1 , wherein the recombinant adenovirus further comprises a second heterologous ORF.

3. The method of claim 1 , wherein replication kinetics of the recombinant adenovirus is measured in a first cell type and a second cell type.

4. The method of claim 3 , wherein the first cell type is a tumor cell and the second cell type is a non-tumor cell.

5. The method of claim 1 , comprising transfecting cells with the genome of the recombinant adenovirus.

6. The method of claim 5 , wherein transfection results in approximately 5-10% of cells transfected.

7. The method of claim 1 , comprising infecting cells with particles of the recombinant adenovirus.

8. The method of claim 7 , wherein the cells are infected with serial dilutions of the recombinant adenovirus particles.

9. The method of claim 1 , wherein the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k.

10. The method of claim 1 , wherein the endogenous ORF is ADP and the heterologous ORF is 5′ of ADP.

11. The method of claim 1 , wherein the endogenous ORF is E1B-55k and the heterologous ORF is 3′ of E1B-55k.

12. The method of claim 1 , wherein the self-cleaving peptide is a 2A peptide or variant thereof.

13. The method of claim 12 , wherein the 2A peptide comprises a porcine teschovirus-1 (PTV1) 2A (P2A) peptide, a foot and mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide or a Thosea asigna virus (TaV) 2A (T2A) peptide, or a variant thereof.

14. The method of claim 13 , wherein the amino acid sequence of the self-cleaving peptide is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.

15. The method of claim 13 , wherein the self-cleaving peptide comprises the amino acid sequence of any one of SEQ ID NOs: 12-19.

16. The method of claim 1 , wherein the fluorescent protein is a green fluorescent protein (GFP) a yellow fluorescent protein (YFP), a red fluorescent protein (RFP) or a blue fluorescent protein (BFP).

17. The method of claim 16 , wherein the YFP is YPet or the RFP is mCherry.

18. The method of claim 1 , wherein the recombinant adenovirus genome comprises in the 5′ to 3′ direction:

E1B-55K-P2A-YPet;

E1B-55K-P2A-mCherry;

YPet-P2A-(DNA polymerase);

DBP-P2A-YPet;

YPet-P2A-ADP;

E3-14.7k-P2A-YPet;

YPet-P2A-E4-ORF2; or

mCherry-P2A-E4-ORF2.

19. The method of claim 1 , wherein the recombinant adenovirus genome comprises the nucleotide sequence of any one of SEQ ID NOs: 3-7, 9-11, 20 and 21.

20. The method of claim 1 , wherein the recombinant adenovirus genome comprises in the 5′ to 3′ direction YPet-P2A-ADP.

21. The method of claim 20 , wherein the recombinant adenovirus genome comprises the YPet-P2A-ADP sequence of adenovirus CMBT-403, wherein adenovirus CMBT-403 has a genome of SEQ ID NO: 7.