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

Patent metadata

Field	Value
Publication number	US-11401529-B2
Application number	US-202016916764-A
Country	US
Kind code	B2
Filing date	Jun 30, 2020
Priority date	Feb 23, 2016
Publication date	Aug 2, 2022
Grant date	Aug 2, 2022

Abstract

Recombinant adenovirus genomes that include an exogenous open reading frame (ORF) and a self-cleaving peptide coding sequence are described. Optimal placement of the exogenous genes for minimal impact on viral kinetics is further disclosed. Therapeutic applications of the recombinant adenoviruses are also described.

First claim

The invention claimed is:

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.

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

3. The recombinant adenovirus genome of claim 2 , 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.

4. The recombinant adenovirus genome of claim 3 , 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.

5. The recombinant adenovirus genome of claim 3 , wherein the self-cleaving peptide comprises the amino acid sequence of any one of SEQ ID NOs: 14-21.

6. The recombinant adenovirus genome of claim 1 , wherein the heterologous ORF encodes a fluorescent protein.

7. The recombinant adenovirus genome of claim 6 , 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).

8. The recombinant adenovirus genome of claim 7 , wherein the YFP is YPet.

9. The recombinant adenovirus genome of claim 7 , wherein the RFP is mCherry.

10. The recombinant adenovirus genome of claim 6 , comprising 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;
mCherry-P2A-E4-ORF2; or
Fiber-P2A-YPet.

11. The recombinant adenovirus genome of claim 10 , comprising the nucleotide sequence of any one of SEQ ID NOs: 3-7, 9-11 and 13.

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

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

14. A kit comprising:
(i) the recombinant adenovirus genome of claim 1 ; and
(ii) one or more diagnostic agents.

15. The kit of claim 14 , wherein the one or more diagnostic agents comprises one or more antibodies that bind a tumor marker.

16. The kit of claim 14 , wherein the one or more diagnostic agents comprises an imaging probe.

17. The recombinant adenovirus genome of claim 1 , wherein the endogenous ORF is E1B-55k and the heterologous ORF is 3′ of E1B-55k.

18. The recombinant adenovirus genome of claim 1 , wherein the endogenous ORF is DNA polymerase and the heterologous ORF is 5′ of DNA polymerase.

19. The recombinant adenovirus genome of claim 1 , wherein the endogenous ORF is DBP and the heterologous ORF is 3′ of DBP.

20. The recombinant adenovirus genome of claim 1 , wherein the endogenous ORF is ADP and the heterologous ORF is 5′ of ADP.

21. The recombinant adenovirus genome of claim 1 , wherein the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k.

22. The recombinant adenovirus genome of claim 1 , wherein the endogenous ORF is E4-ORF2 and the heterologous ORF is 5′ of E4-ORF2.

23. The recombinant adenovirus genome of claim 1 , wherein the endogenous ORF is fiber and the heterologous ORF is 3′ of fiber.

24. The recombinant adenovirus genome of claim 17 , wherein the heterologous ORF encodes a fluorescent protein.

25. The recombinant adenovirus genome of claim 18 , wherein the heterologous ORF encodes a fluorescent protein.

26. The recombinant adenovirus genome of claim 19 , wherein the heterologous ORF encodes a fluorescent protein.

27. The recombinant adenovirus genome of claim 20 , wherein the heterologous ORF encodes a fluorescent protein.

28. The recombinant adenovirus genome of claim 21 , wherein the heterologous ORF encodes a fluorescent protein.

29. The recombinant adenovirus genome of claim 22 , wherein the heterologous ORF encodes a fluorescent protein.

30. The recombinant adenovirus genome of claim 23 , wherein the heterologous ORF encodes a fluorescent protein.

31. The recombinant adenovirus genome of claim 24 , 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.

32. The recombinant adenovirus genome of claim 25 , 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.

33. The recombinant adenovirus genome of claim 26 , 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.

34. The recombinant adenovirus genome of claim 27 , 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.

35. The recombinant adenovirus genome of claim 28 , 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.

36. The recombinant adenovirus genome of claim 29 , 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.

37. The recombinant adenovirus genome of claim 30 , 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.

38. The recombinant adenovirus genome of claim 27 , wherein the self-cleaving peptide is a 2A peptide or variant thereof.

39. The recombinant adenovirus genome of claim 38 , 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.

40. The recombinant adenovirus genome of claim 27 , wherein the amino acid sequence of the self-cleaving peptide comprises the amino acid sequence of any one of SEQ ID NOs: 14-21.

41. The recombinant adenovirus genome of claim 27 , wherein the amino acid sequence of the self-cleaving peptide consists of the amino acid sequence of any one of SEQ ID NOs: 14-21.

42. The recombinant adenovirus genome of claim 27 , wherein the self-cleaving peptide i

Assignees

• Salk Inst For Biological Studi

Inventors

• O'Shea Clodagh
• Partlo William
• Powers Colin

Classifications

• C12N15/86 Primary

  Viral vectors · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C12N2710/10043

  viral genome or elements thereof as genetic vector · CPC title

• C12N2710/10011

  Adenoviridae · CPC title

• C12N2710/10051

  Methods of production or purification of viral material · CPC title