Exogenous gene expression in therapeutic adenovirus for minimal impact on viral kinetics

1. A recombinant adenovirus genome, comprising a heterologous open reading frame (ORF) 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:

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

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

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

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

the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k;

the endogenous ORF is E4-ORF2 and the heterologous ORF is 5′ of E4-ORF2; or

the endogenous ORF is fiber and the heterologous ORF is 3′ of fiber,

wherein the heterologous ORF encodes a therapeutic protein.

2. The recombinant adenovirus genome of claim 1 , wherein the therapeutic protein comprises an immunomodulator.

3. The recombinant adenovirus genome of claim 1 , wherein the self-cleaving peptide is a 2A peptide or variant thereof.

4. The recombinant adenovirus genome of claim 3 , 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.

5. The recombinant adenovirus genome of claim 4 , 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: 14-21.

6. The recombinant adenovirus genome of claim 4 , wherein the self-cleaving peptide comprises the amino acid sequence of any one of SEQ ID NOs: 14-21.

7. A composition comprising the recombinant adenovirus genome of claim 1 and a pharmaceutically acceptable carrier.

8. A recombinant adenovirus comprising the recombinant adenovirus genome of claim 1 .

9. A composition comprising the recombinant adenovirus of claim 8 and a pharmaceutically acceptable carrier.

10. A method of delivering a therapeutic protein to a subject, comprising administering to the subject the recombinant adenovirus of claim 8 .

11. A method of inhibiting tumor cell viability and/or tumor cell growth, comprising contacting the tumor cell with the recombinant adenovirus of claim 8 .

12. The method of claim 11 , wherein the method is an in vitro method.

13. The method of claim 11 , wherein the method is an in vivo method and contacting the tumor cell comprises administering the recombinant adenovirus genome, recombinant adenovirus, or composition to a subject with a tumor.

14. A method of inhibiting tumor progression or reducing tumor volume in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant adenovirus of claim 8 , thereby inhibiting tumor progression or reducing tumor volume in the subject.

15. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant adenovirus of claim 8 , thereby treating cancer in the subject.

16. The method of claim 15 , further comprising administering an additional therapeutic agent to the subject.

17. A kit comprising:

(i) the recombinant adenovirus genome of claim 1 ; and

(ii) one or more additional therapeutic agents and/or one or more diagnostic agents.

18. The kit of claim 17 , wherein the one or more additional therapeutic agents comprise a chemotherapeutic, a biologic, or a combination thereof.

19. The kit of claim 17 , wherein the one or more diagnostic agents comprise one or more antibodies that bind a tumor marker.

20. The recombinant adenovirus genome of claim 4 , wherein the amino acid sequence of the self-cleaving peptide is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 14-21.