Ww-domain-activated extracellular vesicles

Patent metadata

Field	Value
Publication number	US-2023398202-A1
Application number	US-202118032105-A
Country	US
Kind code	A1
Filing date	Oct 15, 2021
Priority date	Oct 16, 2020
Publication date	Dec 14, 2023
Grant date	—

Abstract

Disclosed herein are methods, systems, compositions and strategies for the creation and use WW-domain-Activated Extracellular Vesicles, or WAEVs. These WAEVs can be harnessed to deliver and present viral or bacterial antigens useful for vaccine development; to display homing molecules for targeted delivery of therapeutic molecules to specific cells or tissues; and for packaging and delivery of therapeutic molecules via interactions with the WW domains.

First claim

What is claimed is:

1 . A fusion protein comprising:
(a) a WW-containing domain; (b) a transmembrane domain; and (c) an extracellular domain.

2 . The fusion protein of claim 1 , wherein the fusion protein does not comprise an arrestin domain containing protein 1 (ARRDC1).

3 . The fusion protein of any one of claims 1 - 2 , wherein the WW-containing domain comprises at least one WW domain.

4 . The fusion protein of any one of claims 1 - 3 , wherein the WW-containing domain comprises at least two WW domain.

5 . The fusion protein of any one of claims 1 - 4 , wherein the WW-containing domain comprises at least three WW domain.

6 . The fusion protein of any one of claims 1 - 5 , wherein the WW-containing domain comprises at least four WW domain.

7 . The fusion protein of any of claims 3 - 6 , wherein the WW-containing domain is a NEDD4 E3 ligase domain.

8 . The fusion protein of claim 7 , wherein the NEDD4 E3 ligase is selected from the group consisting of ITCH, NEDD4, NEDD4 L, WWP1, WWP2, Smurf1, Smurf2, BUL1, and NEDL2.

9 . The fusion protein of claim 7 , wherein the NEDD4 E3 ligase is the ITCH protein.

10 . The fusion protein of any one of claims 1 - 9 , wherein the WW-containing domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 1.

11 . The fusion protein of any one of claims 1 - 10 , wherein the WW-containing domain comprises the sequence of SEQ ID NO: 1.

12 . The fusion protein of any one of claims 1 - 11 , wherein the transmembrane domain is the M2 transmembrane domain.

13 . The fusion protein of any one of claims 1 - 11 , wherein the transmembrane domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 9.

14 . The fusion protein of any one of claims 1 - 11 , wherein the transmembrane domain comprises the sequence of SEQ ID NO: 9.

15 . The fusion protein of any one of claims 1 - 11 , wherein the transmembrane domain is the hemagglutinin (H2) transmembrane domain.

16 . The fusion protein of any one of claims 1 - 11 , wherein the transmembrane domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 70.

17 . The fusion protein of any one of claims 1 - 11 , wherein the transmembrane domain comprises the sequence of SEQ ID NO: 70.

18 . The fusion protein of any one of claims 1 - 17 , wherein the extracellular domain is a influenza antigen domain.

19 . The fusion protein of claim 18 , wherein the influenza antigen domain is an M2 extracellular domain.

20 . The fusion protein of claim 18 , wherein the viral antigen domain is hemagglutinin (H1) extracellular domain.

21 . The fusion protein of claim 18 , wherein the viral antigen domain is hemagglutinin (H2) extracellular domain.

22 . The fusion protein of any one of claims 1 - 17 , wherein the extracellular domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 8.

23 . The fusion protein of any one of claims 1 - 17 , wherein the extracellular domain comprises the sequence of SEQ ID NO: 8.

24 . The fusion protein of any one of claims 1 - 23 , further comprising a signal peptide.

25 . The fusion protein of claim 1 , wherein the fusion protein comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 61.

26 . The fusion protein of claim 1 , wherein the fusion protein consists of a sequence having at least 95% identity to the sequence of SEQ ID NO: 61.

27 . The fusion protein of claim 1 , wherein the fusion protein comprises the sequence of SEQ ID NO: 61.

28 . The fusion protein of claim 1 , wherein the fusion protein consists of the sequence of SEQ ID NO: 61.

29 . The fusion protein of claim 1 , wherein the fusion protein comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 62.

30 . The fusion protein of claim 1 , wherein the fusion protein consists of a sequence having at least 95% identity to the sequence of SEQ ID NO: 62.

31 . The fusion protein of claim 1 , wherein the fusion protein comprises the sequence of SEQ ID NO: 62.

32 . The fusion protein of claim 1 , wherein the fusion protein consists of the sequence of SEQ ID NO: 62.

33 . An isolated nucleic acid encoding a fusion protein of any one of claims 1 - 32 .

34 . The isolated nucleic acid of claim 33 , operably linked to a promoter.

35 . The isolated nucleic acid of claim 34 , wherein the promoter is a constitutive promoter, an inducible promoter, or a tissue specific promoter.

36 . The isolated nucleic acid of any of claims 33 - 35 , comprising at least one additional regulatory sequence.

37 . A WW protein domain activated extracellular vesicle (WAEV), comprising:
(a) a lipid bilayer; and (b) the fusion protein of any one of claims 1 - 32 .

38 . The WAEV of claim 37 , further comprising SCAMP3.

39 . The WAEV of claims 37 or 38 , wherein the fusion protein does not comprise at least one of the following exosomal markers: CD63; CD81, CD9, and PTGFRN.

40 . The WAEV of any one of claims 37 - 39 , wherein the fusion protein does not comprise any of the following exosomal markers: CD63; CD81, CD9, and PTGFRN.

41 . A WAEV-producing cell comprising a recombinant expression construct encoding the fusion protein of any one of claims 1 - 32 under the control of a heterologous promoter.

42 . A WAEV-producing cell comprising the isolated nucleic acid of any one of claims 33 - 35 .

43 . A method of delivering WAEVs displaying an antigenic peptide, comprising: delivering the fusion protein of any one of claims 1 - 34 , the isolated nucleic acid of any one of claims 33 - 36 , the WAEV of any one of claims 37 - 40 , or the WAEV-producing cell of any one of claims 41 or 42 to a subject, wherein the extracellular protein of the fusion protein comprises an antigenic peptide.

44 . The method of claim 43 , wherein the subject is mammalian.

45 . The method of claim 43 or 44 , wherein the subject is human.

46 . A kit comprising one or more of the fusion protein of any one of claims 1 - 34 , the isolated nucleic acid of any one of claims 33 - 36 , the WAEV of any one of claims 37 - 40 , or the WAEV-producing cell of any one of claims 41 or 42 .

Assignees

• Harvard College

Inventors

• Lu Quan
• Choi Sengjin

Classifications

• A61K39/145 Primary

  Orthomyxoviridae, e.g. influenza virus · CPC title

• C12N9/104 Primary

  Aminoacyltransferases (2.3.2) · CPC title

• C12Y203/02

  Aminoacyltransferases (2.3.2) · CPC title

• C07K14/005

  from viruses · CPC title

• A61P31/16

  for influenza or rhinoviruses · CPC title