Kits comprising plus-sense single stranded RNA viral vectors and methods for producing polypeptides using the kits

Patent metadata

Field	Value
Publication number	US-10190128-B2
Application number	US-201414764112-A
Country	US
Kind code	B2
Filing date	Mar 21, 2014
Priority date	Mar 22, 2013
Publication date	Jan 29, 2019
Grant date	Jan 29, 2019

Abstract

The present invention relates to kits comprising plus-sense single stranded RNA viral vectors, as well as mixtures of these vectors and uses thereof, and methods for producing in a plant, or plant tissue, or plant cell simultaneously two or more polypeptides using the kits and vectors.

First claim

The invention claimed is:

1. A kit comprising
a) a first plus-sense single stranded ribonucleic acid (RNA) viral vector, and
b) a second plus-sense single stranded RNA viral vector,
wherein
(i) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector are derived from different plant viruses, and
(ii) the coat protein open reading frame (ORF) of the virus from which the first vector is derived is completely deleted in the first plus-sense single stranded RNA viral vector, and
(iii) the coat protein ORF of the virus from which the second vector is derived is completely deleted in the second plus-sense single stranded RNA viral vector, and
(iv) the first plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the second plus-sense single-stranded viral vector is derived, and
(v) the second plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the first plus- sense single-stranded viral vector is derived, and
(vi) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector comprise an RNA replicon which is able to replicate in plant cells,

and wherein
the virus from which the first plus-sense single-stranded viral vector is derived is a Potexvirus, and
the virus from which the second plus-sense single-stranded viral vector is derived is a Tobamovirus.

2. The kit according to claim 1 , wherein
a) the functional coat protein ORF of the virus from which the second plus-sense single- stranded viral vector is derived is inserted at the location from which the coat protein ORF of the first plus-sense single-stranded viral vector is deleted, and/or
b) the functional coat protein ORF of the virus from which the first plus-sense single- stranded viral vector is derived is inserted at the location from which the coat protein ORF of the second plus-sense single-stranded viral vector is deleted.

3. The kit according to claim 1 , wherein
the first plus-sense single-stranded viral vector comprises a functional heterologous ORF in addition to the functional coat protein ORF or the second plus-sense single-stranded viral vector comprises a functional heterologous ORF in addition to the functional coat protein ORF.

4. The kit according to claim 1 , wherein the kit further comprises
at least one additional plus-sense single stranded RNA viral vector, wherein
(i) the at least one additional plus-sense single-stranded viral vector comprises at least one functional heterologous ORF, and
(ii) the at least one additional plus-sense single-stranded viral vector is derived from a Potexvirus.

5. The kit according to claim 4 , wherein the at least one additional plus-sense single stranded RNA viral vector is devoid of a functional movement protein ORF(s) of the Potexvirus.

6. A mixture comprising:
a) at least one first plus-sense single stranded RNA viral vector,
b) at least one second plus-sense single stranded RNA viral vector, and, optionally,
c) at least one third plus-sense single stranded RNA viral vector,
wherein
(i) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector are derived from different plant viruses, and
(ii) the coat protein ORF of the virus from which the first vector is derived is completely deleted in the first plus-sense single stranded RNA viral vector, and
(iii) the coat protein ORF of the virus from which the second vector is derived is completely deleted in the second plus-sense single stranded RNA viral vector, and
(iv) the first plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the second plus-sense single-stranded viral vector is derived, and
(v) the second plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the first plus-sense single-stranded viral vector is derived, and
(vi) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector comprise an RNA replicon which is able to replicate in plant cells, and
wherein
the virus from which the first plus-sense single-stranded viral vector is derived is a Potexvirus,
the virus from which the second plus-sense single-stranded viral vector is derived is a Tobamovirus, and
the at least one third plus-sense single-stranded viral vector is derived from a Potexvirus.

7. The mixture according to claim 6 , comprising
(i) the at least one first plus-sense single stranded RNA viral vector, and
(ii) the at least one second plus-sense single stranded RNA viral vector, and
(iii) the at least one third plus-sense single stranded RNA viral vector.

8. A plant or plant cell
comprising at least one first plus-sense single stranded RNA viral vector and at least one second plus-sense single stranded RNA viral vector, and, optionally, at least one third plus-sense single stranded RNA viral vector(s),
wherein
(i)the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector are derived from different plant viruses, and
(ii) the coat protein ORF of the virus from which the first vector is derived is completely deleted in the first plus-sense single stranded RNA viral vector, and
(iii) the coat protein ORF of the virus from which the second vector is derived is completely deleted in the second plus-sense single stranded RNA viral vector, and
(iv) the first plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the second plus-sense single-stranded viral vector is derived, and
(v) the second plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the first plus-sense single-stranded viral vector is derived, and
(vi) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector comprise an RNA replicon which is able to replicate in plant cells, and
wherein
the virus from which the first plus-sense single-stranded viral vector is derived is a Potexvirus, and
the virus from which the second plus-sense single-stranded viral vector is derived is a Tobamovirus, and
the at least one third plus-sense single-stranded viral vector is derived from a Potexvirus.

9. The plant according to claim 8 , wherein more than one plant cell in more than one tissue of the plant
comprises the at least one first plus-sense single stranded RNA viral vector and the at least one second plus-sense single stranded RNA vector, and, optionally, the at least one third plus-sense single stranded RNA viral vector(s).

10. A method for producing in a plant, or-plant tissue, or plant cell a heterooligomeric polypeptide and/or two or more polypeptides, comprising providing to at least one plant cell
a)at least one first plus-sense single stranded RNA viral vector, and
b)at least one second plus-sense single stranded RNA viral vector, and
c)optionally, at least one third plus-sense single stranded RNA viral vector,
wherein
(i)the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector are derived from different plant viruses, and
(ii) the coat protein ORF of the virus from which the first vector is derived is completely deleted in the first plus-sense single stranded RNA viral vector, and
(iii) the coat protein ORF of the virus from which the second vector is derived is completely deleted in the second plus-sense single stranded RNA viral vector, and
(iv) the first plus-sense single stranded RNA viral vector comprises a fu

Assignees

• Fraunhofer Ges Forschung
• Rheinisch Westfaelische Technische Hochschule Aachen

Inventors

• Commandeur Ulrich
• Dickmeis Christina
• Fischer Rainer

Classifications

• C12N2770/40043

  viral genome or elements thereof as genetic vector · CPC title

• C12N2770/00043

  viral genome or elements thereof as genetic vector · CPC title

• C12N15/8242 Primary

  with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits · CPC title