Simultaneous gene silencing and suppressing gene silencing in ihe same cell

Patent metadata

Field	Value
Publication number	US-11560571-B2
Application number	US-202017064672-A
Country	US
Kind code	B2
Filing date	Oct 7, 2020
Priority date	Dec 27, 2011
Publication date	Jan 24, 2023
Grant date	Jan 24, 2023

Abstract

The present invention relates to genetically modified cells that are capable of optimal transgene expression by co-expressing a silencing suppressor whilst at the same time are also capable of silencing a gene, such as a naturally occurring gene of the cell. The present invention also relates to methods of producing the modified cells, as well as relates to processes for obtaining a genetically modified cell with a desired property.

First claim

The invention claimed is:

1. A plant cell comprising,
i) a polynucleotide of interest which encodes a target RNA,
ii) a first exogenous polynucleotide encoding a double stranded RNA (dsRNA) molecule which comprises a stem-loop and a first nucleotide sequence which is complementary to a region of the target RNA encoded by the polynucleotide of interest,
iii) a second exogenous polynucleotide encoding a silencing suppressor polypeptide,
iv) a third exogenous polynucleotide, different to the first and second exogenous polynucleotides and the polynucleotide of interest, which encodes an RNA of interest, and
v) a reduced level of the target RNA encoded by the polynucleotide of interest and/or the amount of a protein encoded by the target RNA when compared to a corresponding cell lacking the first exogenous polynucleotide,

wherein each exogenous polynucleotide is operably linked to one or more promoters that are capable of directing expression of the polynucleotide in the cell, wherein the first and second exogenous polynucleotides form part of one DNA construct, and wherein the silencing suppressor polypeptide comprises amino acids having the sequence set forth in any one of SEQ ID NOs 39 to 51, or a sequence which is at least 95% identical to the sequence set forth in any one of SEQ ID NOs 39 to 51.

2. The plant cell of claim 1 , wherein the polynucleotide of interest is an endogenous gene of the cell or a gene of a pathogen of the cell.

3. The plant cell of claim 1 , wherein the first and second exogenous polynucleotides form part of one DNA construct which is integrated into the genome of the cell.

4. The plant cell of claim 1 , wherein the first, second and third exogenous polynucleotides form part of one DNA construct which is integrated into the genome of the cell.

5. The plant cell of claim 1 , wherein at least the second exogenous polynucleotide is integrated into the genome of the cell.

6. The plant cell of claim 1 , wherein the cell comprises at least a 25% reduction in the level of the target RNA encoded by the polynucleotide of interest and/or amount of protein encoded by the target RNA when compared to a corresponding cell lacking the first exogenous polynucleotide.

7. The plant cell of claim 1 , wherein the dsRNA molecule, or a processed RNA product thereof, comprises at least 19 consecutive nucleotides which is at least 95% identical to the complement of a region of the target RNA, and wherein the region of the target RNA is i) within a 5′ untranslated region of the target RNA, ii) within a 5′ half of the target RNA, iii) within a protein-encoding open-reading frame of the target RNA, iv) within a 3′ half of the target RNA, or v) within a 3′ untranslated region of the target RNA.

8. The plant cell of claim 1 , wherein the dsRNA molecule is a microRNA (miRNA) precursor and/or wherein the processed RNA product thereof is a miRNA.

9. The plant cell of claim 1 , wherein the third exogenous polynucleotide encodes a protein or microRNA precursor.

10. The plant cell of claim 1 , wherein the cell further comprises at least one, at least two, at least three, at least four or at least five additional, different exogenous polynucleotides each encoding different RNAs of interest.

11. The plant cell of claim 10 , wherein the additional, different exogenous polynucleotides form part of one DNA construct.

12. The plant cell of claim 1 , wherein the cell further comprises at least one, at least two, at least three, at least four or at least five additional, different exogenous polynucleotides each independently encoding different dsRNA molecules which comprise different nucleotide sequences which are complementary to a region of different target RNAs encoded by different polynucleotides of interest, and/or different nucleotide sequences which are complementary to different regions of the same target RNA.

13. The plant cell of claim 12 , wherein the additional polynucleotides form part of the same DNA construct.

14. The plant cell of claim 1 , wherein the first exogenous polynucleotide encodes more than one miRNA which independently comprise different nucleotide sequences which are complementary to a region of different target RNAs encoded by different polynucleotides of interest, and/or different nucleotide sequences which are complementary to different regions of the same target RNA.

15. The plant cell of claim 1 , wherein the exogenous polynucleotides are operably linked to different promoters.

16. The plant cell of claim 1 , wherein the second exogenous polynucleotide was introduced into the cell on a vector other than a viral vector.

17. The plant cell of claim 1 , wherein the dsRNA molecule is a hairpin RNA.

18. The plant cell of claim 1 , wherein the silencing suppressor polypeptide comprises amino acids having a sequence which is at least 96% identical to the sequence set forth in any one of SEQ ID NOs 39 to 51.

19. The plant cell of claim 1 , wherein the silencing suppressor polypeptide comprises amino acids having the sequence set forth in any one of SEQ ID NOs 39 to 51.

20. A process for selecting a plant cell with a desired property resulting from an increased level of an RNA of interest and/or amount of protein encoded by the RNA of interest, and a reduced level of target RNA encoded by a first polynucleotide of interest and/or amount of the protein encoded by the target RNA, the process comprising:
i) obtaining one or more plant cells comprising,
(a) a polynucleotide of interest which encodes a target RNA,
b) a first exogenous polynucleotide encoding a double stranded RNA (dsRNA) molecule which comprises a stem-loop and a first nucleotide sequence which is complementary to a region of the target RNA encoded by the polynucleotide of interest,
c) a second exogenous polynucleotide encoding a candidate silencing suppressor polypeptide,
d) a third exogenous polynucleotide, different to the first and second exogenous polynucleotides and the polynucleotide of interest, which encodes an RNA of interest, and
e) a reduced level of the target RNA encoded by the polynucleotide of interest and/or the amount of a protein encoded by the target RNA when compared to a corresponding cell lacking the first exogenous polynucleotide,
wherein each exogenous polynucleotide is operably linked to one or more promoters that are capable of directing expression of the polynucleotide in the cell, wherein the first and second exogenous polynucleotides form part of one DNA construct, and wherein the candidate silencing suppressor polypeptide comprises amino acids having the sequence set forth in any one of SEQ ID NOs 39 to 51, or a sequence which is at least 95% identical to the sequence set forth in any one of SEQ ID NOs 39 to 51,

ii) analysing the cell(s) for the desired property,
iii) if the cell(s) does not have the desired property, substituting the second exogenous polynucleotide encoding the candidate silencing suppressor polypeptide with a second exogenous polynucleotide encoding a different candidate silencing suppressor polypeptide comprising amino acids having the sequence set forth in any one of SEQ ID NOs 1 or 39 to 51, or having a sequence which is at least 95% identical to the sequence set forth in any one of SEQ ID NOs 1 or 39 to 51, and analysing the resultant cell(s) for the desired property,
iv) if necessary, repeating step iii) until the desired property is obtained, and
v) selecting a plant cell with the desired property.

Assignees

• Wood Craig Christopher
• Naim Fatima
• Singh Surinder Pal
• Waterhouse Peter Michael
• Commw Scient Ind Res Org

Inventors

• Wood Craig Christopher
• Naim Fatima
• Singh Surinder Pal
• Waterhouse Peter Michael

Classifications

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2750/12022

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

• C12N2320/50

  Methods for regulating/modulating their activity · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N15/8216 Primary

  Methods for controlling, regulating or enhancing expression of transgenes in plant cells · CPC title