High potency siRNAS for reducing the expression of target genes

• US 8,524,680 B2
• Filed: 08/31/2009
• Issued: 09/03/2013
• Est. Priority Date: 02/01/2002
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Brown, David, Ford, Lance P., Jarvis, Richard A., Pallotta, Vince, Pasloske, Brittan L.
• Primary Examiner(s): WHITEMAN, BRIAN A
• Application Number:
  US12/550,625
• Publication Number:
  US 20100221789A1
• Time in Patent Office: 1,464 Days
• Field of Search: 514/44.A
• US Class Current: 514/44.A
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 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 Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/333 Modified A
• CPC Class Codes: C12N 2310/334 Modified C
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/336 Modified G
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity
• CPC Class Codes: C12Y 102/01012 Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)

Abstract

The present invention provides improved methods of attenuating gene expression through the phenomenon of RNA interference. The invention provides methods of synthesis of double stranded RNAs (dsRNAs) of increased potency for use as small interfering RNA (siRNA). Surprisingly and unexpectedly, siRNAs made by the methods of the invention are significantly more potent than previously available siRNAs.

Claims

1. A method for attenuating the expression of a target gene in a cell comprising:

introducing siRNA into the cell in an amount sufficient to attenuate expression of the target gene

wherein the siRNA is from 15 to 30 nucleotides in length and contains a duplex of from 15 to 30 contiguous nucleotides, and the siRNA is made by a method comprising;

obtaining a first polynucleotide template comprising a first promoter operatively linked to a first target sequence that has 5′ and

3′

ends and that is substantially identical to at least a portion of the target gene;

obtaining a second polynucleotide template comprising a second promoter operatively linked to a second target sequence that has 5′ and

3′

ends and that is substantially the reverse complement of the first target sequence of the first template;

enzymatically incorporating nucleotides into RNA by contacting the first template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the first template to form a first RNA product;

enzymatically incorporating nucleotides into RNA by contacting the second template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the second template to form a second RNA product;

annealing the first and second RNA products to form a siRNA containing a duplex of from 15 to 30 contiguous nucleotides, and

contacting the siRNA with a single strand specific ribonuclease,

wherein the siRNA has a sequence that is substantially identical to at least a portion of the target gene,

wherein the nucleotides of at least one incorporating step comprise at least one modified nucleotide analog selected from the group consisting of alpha-S ATP, alpha-S CTP, alpha-S GTP, and alpha-S UTP,

wherein the at least one modified nucleotide analog is incorporated into the siRNA, and

wherein fewer molecules of the siRNA are effective in achieving attenuation of gene expression when compared to the number of standard siRNA molecules required to achieve the same level of attenuation of target gene expression.

2. The method of claim 1, wherein the single strand specific ribonuclease is RNase T1, RNase A, RNase Sa, RNase Sa2, or RNase Sa3.

3. The method of claim 1, wherein the first template further comprises an overhang encoding sequence joined to the 3′

end of the first target sequence and the second template further comprises an overhang encoding sequence joined to the 3′

end of the second target sequence.

4. The method of claim 3, wherein the first and second overhang encoding sequences each comprise TT.

5. The method of claim 3, wherein the first two nucleotides of the 5′

end of the first target sequence are GG and the last two nucleotides of the 3′

end of the first target sequence are CC.

6. The method of claim 3, wherein the first two nucleotides of the 5′

end of the first target sequence are GA and the last two nucleotides of the 3′

end of the first target sequence are TC.

7. The method of claim 1, wherein the first target sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

8. The method of claim 1, wherein the first promoter is a T7, T3, or SP6 promoter.

9. The method of claim 1, wherein the second promoter is a T7, T3, or SP6 promoter.

10. The method of claim 1, wherein the first promoter and the second promoter are the same promoter.

11. The method of claim 1, wherein the first promoter and the second promoter are different promoters.

12. The method of claim 1, wherein the first template further comprises a first leader sequence of about 10 nucleotides positioned between the first promoter and the first target sequence.

13. The method of claim 12, wherein the second template further comprises a second leader sequence of about 10 nucleotides positioned between the second promoter and the second target sequence.

14. The method of claim 13, wherein the second leader sequence is substantially non-complementary to the first leader sequence.

15. The method of claim 1, wherein the cell is comprised within a tissue.

16. The method of claim 1, wherein the cell is comprised within an organism.

17. The method of claim 16, wherein the organism is a plant, animal, protozoan, virus, bacterium, or fungus.

18. The method of claim 17, wherein the organism is an animal.