Rotavirus vectors for heterologous gene delivery

Patent metadata

Field	Value
Publication number	US-2024200099-A1
Application number	US-202218555035-A
Country	US
Kind code	A1
Filing date	Apr 11, 2022
Priority date	Apr 15, 2021
Publication date	Jun 20, 2024
Grant date	—

Abstract

Rotavirus vectors encoding in their genome a heterologous gene, and nucleic acid constructs encoding such rotavirus vectors. The rotavirus vector genome may include a rotavirus non-structural protein, a 2A peptide downstream of the rotavirus non-structural protein, and a heterologous protein downstream of the 2A peptide. The heterologous gene may be, for example, a SARS-CoV-2 spike protein or a fragment thereof, or an RSV F protein or a fragment thereof.

First claim

What is claimed is:

1 . An isolated nucleic acid molecule comprising:
a promoter sequence; and a cDNA molecule encoding: a rotavirus non-structural protein; a 2A peptide downstream of the rotavirus non-structural protein; and a heterologous protein downstream of the 2A peptide.

2 . The isolated nucleic acid molecule of claim 1 , wherein the rotavirus non-structural protein is NSP1, NSP3, or NSP5.

3 . The isolated nucleic acid molecule of claim 2 , wherein the rotavirus non-structural protein is NSP1.

4 . The isolated nucleic acid molecule of claim 2 , wherein the rotavirus non-structural protein is NSP3.

5 . The isolated nucleic acid molecule of claim 2 , wherein the rotavirus non-structural protein is NSP5.

6 . The isolated nucleic acid molecule of any one of claims 1-5 , comprising a nucleic acid encoding an antigenomic hepatitis delta ribozyme, and wherein the promoter is a T7 promoter.

7 . The isolated nucleic acid molecule of any one of claims 1-6 , wherein the heterologous protein is a viral protein or fragment thereof.

8 . The isolated nucleic acid molecule of claim 7 , wherein the viral protein or fragment thereof is a SARS-COV-2 spike protein or a fragment thereof.

9 . The isolated nucleic acid molecule of claim 8 , wherein the viral protein or fragment thereof is the S1 domain of SARS-COV-2 spike protein (SEQ ID NO: 36) or the receptor binding domain of SARS-COV-2 spike protein (SEQ ID NO: 37).

10 . The isolated nucleic acid molecule of claim 7 , wherein the viral protein or fragment thereof is an RSV F protein or fragment thereof.

11 . The isolated nucleic acid molecule of claim 10 , wherein the viral protein or fragment thereof is RSV-T4PreF (SEQ ID NO: 44), RSV-T4scPreF (SEQ ID NO: 46), RSV-A2PreF (SEQ ID NO: 48), or RSV-A2scPreF (SEQ ID NO: 50).

12 . The isolated nucleic acid molecule of any one of claims 1-6 , wherein the heterologous protein is a fluorescent protein.

13 . The isolated nucleic acid molecule of claim 12 , wherein the fluorescent protein is: green fluorescent protein (GFP); enhanced GFP (eGFP); superfolder GFP; AcGFPl; ZsGreenl; enhanced blue fluorescent protein (EBFP), EBFP2, Azurite, mKalama; cyan fluorescent protein (CFP); enhanced CFP (ECFP); Cerulean; mHoneydew; CyPet; yellow fluorescent protein (YFP); Citrine; Venus; mBanana; ZsYellow1; Ypet; mOrange; tdTomato; LSSmOrange, PSmOrange PSmOrange2; DsRed; DsRed-monomer; DsRed-Express2; mRFPi; mCherry; mStrawberry; mRaspberry; niPluni; E2-Crimson; iRFP670; iRFP682; iRFP702; or iRFP720.

14 . The isolated nucleic acid molecule of claim 13 , wherein the fluorescent protein is GFP.

15 . A recombinant rotavirus comprising in its genome a cDNA sequence encoding a 2A peptide downstream of NSP1, NSP3, or NSP5, and a heterologous gene downstream of the 2A peptide.

16 . The recombinant rotavirus of claim 15 , wherein the heterologous gene is downstream of NSP1.

17 . The recombinant rotavirus of claim 15 , wherein the heterologous gene is downstream of NSP3.

18 . The recombinant rotavirus of claim 15 , wherein the heterologous gene is downstream of NSP5.

19 . The recombinant rotavirus of any one of claims 15-18 , wherein the heterologous gene encodes a viral protein or fragment thereof.

20 . The recombinant rotavirus of claim 19 , wherein the viral protein or fragment thereof is a SARS-COV-2 spike protein or a variant or fragment thereof.

21 . The recombinant rotavirus of claim 20 , wherein the viral protein or fragment thereof is the S1 domain of SARS-COV-2 spike protein (SEQ ID NO: 36) or the receptor binding domain of SARS-COV-2 spike protein (SEQ ID NO: 37).

22 . The recombinant rotavirus of claim 21 , wherein the viral protein or fragment thereof is an RSV F protein or a variant or fragment thereof.

23 . The recombinant rotavirus of claim 22 , wherein the viral protein or fragment thereof is RSV-T4PreF (SEQ ID NO: 44), RSV-T4scPreF (SEQ ID NO: 46), RSV-A2PreF (SEQ ID NO: 48), or RSV-A2scPreF (SEQ ID NO: 50).

24 . The isolated nucleic acid molecule of any one of claims 15-18 , wherein the heterologous gene encodes a fluorescent protein.

25 . The isolated nucleic acid molecule of claim 24 , wherein the fluorescent protein is: green fluorescent protein (GFP); enhanced GFP (eGFP); superfolder GFP; AcGFPl; ZsGreenl; enhanced blue fluorescent protein (EBFP), EBFP2, Azurite, mKalama; cyan fluorescent protein (CFP); enhanced CFP (ECFP); Cerulean; mHoneydew; CyPet; yellow fluorescent protein (YFP); Citrine; Venus; mBanana; ZsYellow1; Ypet; mOrange; tdTomato; LSSmOrange, PSmOrange PSmOrange2; DsRed; DsRed-monomer; DsRed-Express2; mRFPi; mCherry; mStrawberry; mRaspberry; niPluni; E2-Crimson; iRFP670; iRFP682; iRFP702; or iRFP720.

26 . The isolated nucleic acid molecule of claim 25 , wherein the fluorescent protein is GFP.

27 . An immunogenic composition comprising (i) an effective amount of the recombinant rotavirus of any one of claims 15-23 , and (ii) a pharmaceutically acceptable carrier.

28 . A method for treating or preventing an infection in a subject, comprising administering an effective amount of the immunogenic composition according to claim 27 to the subject.

29 . A method for inducing a protective immune response in a subject, comprising administering an effective amount of the immunogenic composition of claim 27 to the subject.

30 . The method of claim 29 , wherein the immunogenic composition is administered to a mucous membrane of the subject.

31 . The method of claim 30 , wherein administration of the immunogenic composition is oral.

32 . The method of any one of claims 28-31 , comprising a first administration of the immunogenic composition and a second administration of the immunogenic composition.

33 . The method of any one of claims 28-32 , wherein the protective immune response is a humoral immune response and/or a cellular immune response.

34 . The method of claim 33 , wherein the second administration is performed from one month to two months after the first administration.

35 . The method of any one of claims 28-34 , wherein the subject is a human.

36 . Use of the recombinant rotavirus of any one of claims 15-23 or the immunogenic composition of claim 27 for preventing or treating an infection.

37 . The recombinant rotavirus of any one of claims 15-23 or the immunogenic composition of claim 27 , for use in preventing or treating an infection in a subject.

38 . In vitro use of the recombinant rotavirus of any one of claims 15-23 or the immunogenic composition of claim 27 expressing the heterologous protein in eukaryotic cells.

39 . A method for rescuing recombinant rotavirus, the method comprising:
a) transfecting cells with
i) eleven individual rotavirus genomic segment plasmids (RGSP), each RGSP having a promoter and encoding one of a single rotavirus protein VP1, VP2, VP3, VP4, NSP1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5, wherein one or more of the plasmids encoding NSP1, NSP3, and NSP5 protein includes a sequence encoding a 2A protein that is downstream of the NSP protein and a sequence encoding a heterologous protein that is downstream of the sequence encoding the 2A prote

Assignees

• Merck Sharp & Dohme Llc

Inventors

• Wang Dai
• Wei Jiajie

Classifications

• C12N2720/12352

  relating to complementing cells and packaging systems for producing virus or viral particles · CPC title

• C12N2720/12343

  viral genome or elements thereof as genetic vector · CPC title

• C12N2720/12322

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• A61K2039/70

  Multivalent vaccine · CPC title

• A61P31/14

  for RNA viruses · CPC title