Methods and compositions for the production of siRNAs

Patent metadata

Field	Value
Publication number	US-10508276-B2
Application number	US-201715804258-A
Country	US
Kind code	B2
Filing date	Nov 6, 2017
Priority date	Jan 11, 2013
Publication date	Dec 17, 2019
Grant date	Dec 17, 2019

Abstract

The technology described herein relates to siRNAs, e.g., methods and compositions relating to the production of siRNAs in bacterial cells.

First claim

What is claimed herein is:

1. A method of producing one or more siRNA species which can inhibit the expression of a target RNA, the method comprising:
culturing a bacterial cell comprising:
a. a viral siRNA-binding polypeptide and
b. a dsRNA comprising a nucleic acid sequence substantially complementary to a target RNA
under conditions suitable for the production of siRNAs.

2. The method of claim 1 , further comprising a second step of isolating the viral siRNA-binding polypeptide and eluting the siRNAs bound to the viral siRNA-binding polypeptide.

3. The method of claim 2 , further comprising purifying the siRNAs eluted from the viral siRNA-binding polypeptide by HPLC.

4. The method of claim 1 , further comprising contacting the cell with one or more modified nucleotides before or during the culturing step.

5. The method of claim 1 , wherein the dsRNA is exogenous to the bacterial cell.

6. The method of claim 1 , wherein the viral siRNA-binding polypeptide comprises a purification tag.

7. The method of claim 1 , wherein the viral siRNA-binding polypeptide is encoded by a nucleic acid.

8. The method of claim 1 , wherein the viral siRNA-binding polypeptide is selected from the group consisting of:
p19 polypeptide; tombusvirus p19 polypeptide; B2 polypeptide; HC-Pro polypeptide; p38 polypeptide; p122 polypeptide; p130 polypeptide; p21 polypeptide; p1b polypeptide; and NS3 polypeptide.

9. The method of claim 1 , wherein the dsRNA is greater than 21 nucleotides in length.

10. The method of claim 1 , wherein the dsRNA is a hairpin RNA.

11. The method of claim 1 , wherein the bacterial cell expresses an RNase III polypeptide.

12. The method of claim 1 , wherein the bacterial cell expresses an RNase III polypeptide encoded by an exogenous nucleic acid sequence.

13. The method of claim 1 , wherein the bacterial cell is an Escherichia coli cell.

14. The method of claim 1 , wherein at least one of the viral siRNA-binding polypeptide and the dsRNA are constitutively expressed.

15. The method of claim 1 , wherein at least one of the viral siRNA-binding polypeptide and the dsRNA are inducibly expressed.

16. The method of claim 1 , wherein the DNA encoding at least one of the viral siRNA-binding polypeptide or the dsRNA is part of a plasmid.

17. The method of claim 1 , wherein the dsRNA comprises nucleic acid sequences substantially complementary to a multiplicity of target RNAs.

Assignees

• Childrens Medical Center

Inventors

• Lieberman Judy
• Huang Linfeng

Classifications

• C12N2330/50

  Biochemical production, i.e. in a transformed host cell · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N15/111 Primary

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

• A61K31/713

  Double-stranded nucleic acids or oligonucleotides · CPC title