Double-stranded ribonucleic acid with increased effectiveness in an organism

• US 7,786,290 B2
• Filed: 06/14/2004
• Issued: 08/31/2010
• Est. Priority Date: 06/13/2003
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Limmer, Stefan, John, Matthias, Hadwiger, Philipp, Vornlocher, Hans-Peter, Woppmann, Claudia
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US10/560,336
• Publication Number:
  US 20070275465A1
• Time in Patent Office: 2,269 Days
• Field of Search: 435/6, 435/91.1, 435/91.31, 514/44, 536/23.1, 536/24.5
• US Class Current: 536/24.5
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2310/14 interfering N.A.
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

The present invention relates to a method for the targeted selection of a double-stranded ribonucleic acid (dsRNA) consisting of two single strands that exhibits increased effectiveness in inhibiting the expression of a target gene by means of RNA interference, wherein at least end of the dsRNA comprises a nucleotide overhang of 1 to 4 unpaired nucleotides in length; wherein the unpaired nucleotide adjacent to the terminal nucleotide pair comprises a purine base; and wherein the terminal nucleotide pair on both ends of the dsRNA is a G-C pair, or at least two of the last four consecutive terminal nucleotide pairs are G-C pairs.

Claims

1. A double-stranded ribonucleic acid (dsRNA), consisting of first and second single RNA strands, wherein the first single RNA strand is an antisense RNA strand, the second single RNA strand is a sense RNA strand, wherein the antisense RNA strand is complementary to a target gene or a portion thereof, the dsRNA having increased effectiveness in inhibiting the expression of a target gene by means of RNA interference, wherein the dsRNA comprises first and second double-stranded ends and at least one single-stranded overhang which is 2 to 4 nucleotides in length, wherein the unpaired nucleotide of the single-stranded overhang that is directly adjacent to the terminal nucleotide base pair comprises a purine base, wherein a single-stranded overhang is located at the 3′

-end of the antisense strand, wherein the overhang comprises the sequence 5′

-GC-3′

; and

wherein the terminal base pair of the first double-stranded end comprises a guanine-cytosine (G-C) base pair or the four consecutive terminal base pairs of the first double-stranded end comprises at least two G-C base pairs;

wherein the terminal base pair of the second double-stranded end comprises a guanine-cytosine (G-C) base pair or the four consecutive terminal base pairs of the second double-stranded end comprises at least two G-C base pairs;

wherein the region of the antisense strand that is complementary to the targer gene is 19-28 nucleotides in length excluding the following dsRNAs;

2. The dsRNA of claim 1, wherein each nucleotide overhang independently consists of 2 unpaired nucleotides.

3. The dsRNA of claim 1, wherein at least half of the unpaired nucleotides comprise a purine base.

4. The dsRNA of claim 1, wherein the unpaired nucleotide adjacent to the terminal nucleotide base pair comprises a guanine (G).

5. The dsRNA of claim 1, wherein the unpaired nucleotide adjacent to the terminal nucleotide base pair comprises an adenine (A) base.

6. The dsRNA of claim 1, wherein said nucleotide overhang comprising the sequence 5′

-GC-3′

consists of the sequence 5′

-GC-3′

.

7. The dsRNA of claim 1, wherein the region of the antisense strand that is complementary to the target gene is 19 to 24 nucleotides in length.

8. The dsRNA of claim 1, wherein the antisense strand is 20 to 28 nucleotides in length.

9. The dsRNA of claim 1, wherein the antisense strand is 21 nucleotides in length.

10. The dsRNA of claim 1, wherein at least one of the RNA strands comprises at least one chemically modified nucleotide.

11. The dsRNA of claim 10, wherein the chemically modified nucleotide comprises a non-natural base.

12. The dsRNA of claim 10, wherein the chemically modified nucleotide comprises a 2′

modification.

13. The dsRNA of claim 12, wherein the 2′

modification is selected from the group consisting of a 2′

-amino modification, a 2′

-alkyl modification, a 2′

-O -methyl modification, a 2′

-O-ethyl modification, a 2′

-O-propyl modification, a 2′

-O-allyl modification, a 2′

-O-aminoalkyl modification, and a 2′

-deoxy-2′

-fluoro modification.

27. A pharmaceutical composition for inhibiting the expression of a target gene by means of RNA interference, comprising a dsRNA of claim 1, or a salt, prodrug or hydrate thereof;

and a pharmaceutically acceptable carrier.

28. A method for inhibiting the expression of a target gene in a cell, comprising:

(a) introducing into the cell a dsRNA of claim 1, or a salt, prodrug or hydrate thereof; and

(b) maintaining the cell for a time sufficient to obtain degradation of a mRNA transcript of the target gene.

29. The method of claim 28, wherein the cell is a mammalian cell.

30. The method of claim 29, wherein the cell is a human cell.

31. The method of claim 28, wherein the target gene is selected from the group consisting of 11-hyroxysteroid dehydrogenase-1, acetyl-CoA-carboxylase-2, acyl CoA:

DAG acyltransferase1, Adenosine A2 receptor, akt, AML-ETO, amyloid beta precursor protein (APP), ApoAl, ApoB, ApoM, APS (adaptor protein with pleckstrin homology and src homology 2 domains, a-synuclein, Aurora A, Aurora B, beta-1 integrin subunit, beta-amyloid converting enzyme (BACE), Bax, beta-catenin, Bcl2, Bcl-XL, Bcr- abl, caspase 8, caspase-3, C CR2, CD40, CD40L, cdk2, chk1, chk2, clottingfactorVII, collagen, CD132, CTLA4, cyclin E, Dhcr24, Dipeptidylpeptidase-IV, E-Cadherin, Eg5/KSP, EGF, EGFR1, EWS-Flil, FAS-fatty acid synthase, FoxA-3, FoxO-1, Fructose-1,6-bisphosphate, Glucose-6-phophate, GM3 synthase, HDAC (histone deacetylase 1-6,9), Her-2/erb2, HIF1, HMG CoA reductase, hormone sensitive lipase, huntingtin, IKK1, IKK2, LDLR, MDR1, Microsomal Triglyceride Transfer Protein, MMP1, MMP2, MMP9, MyD88, sodium voltage gated type X alpha polypeptide (NaVl.8), NFkB, p38 map kinase mitogen activated protein kinase, p85a regulatory subunit of PI3-kinase, PEPCK, plkl, PTEN, PTP-1B, PU.1, raf, ras, Resistin, SCAP, SERBP-2, SHIP-2, SMAD7, SREBP1C, STAT1, stearoyl-CoA desaturase-1, TERT, TGF-beta-1, TGF-beta-1R1, Topoisomerase I, Topoisomerase II, VEGF, VEGFR1, VEGFR2, VLA1, VLA4, and vanilloid receptor (VR1).

14. A method for the targeted selection of a double-stranded ribonucleic acid (dsRNA), consisting of first and second single RNA strands, wherein the first single RNA strand is an antisense RNA strand, the second single RNA strand is a sense RNA strand, wherein the antisense RNA strand is complementary to a target gene or a portion thereof, the dsRNA having increased effectiveness in inhibiting the expression of a target gene by means of RNA interference, comprising the steps of:

(a) selecting a dsRNA comprising first and second double-stranded ends and at least one single-stranded overhang which is 2 to 4 nucleotides in length;

(b) selecting a dsRNA comprising first and second double-stranded ends, wherein the unpaired nucleotide of the single-stranded overhang that is directly adjacent to the terminal nucleotide base pair comprises a purine base, wherein a single-stranded overhang is located at the 3′

-end of the antisense strand, and wherein the overhang comprises the sequence 5′

-GC-3′

; and

(c) selecting a dsRNA comprising first and second double-stranded ends, wherein terminal base pair of the first double-stranded end comprises a guanine-cytosine (G-C) base pair or the four consecutive terminal base pairs of the first double-stranded end comprises at least two G-C base pairs;

wherein the terminal base pair of the second double-stranded end comprises a guanine-cytosine (G-C) base pair or the four consecutive terminal base pairs of the second double-stranded end comprises at least two G-C base pairs;

wherein the region of the antisense strand that is complementary to the target gene is 19-28 nucleotides in length;

excluding the following dsRNAs;

15. The method of claim 14, wherein each nucleotide overhang independently consists of 2 unpaired nucleotides.

16. The methods of claim 14, wherein at least half of the unpaired nucleotides comprise a purine base.

17. The method of claim 14, wherein the unpaired nucleotide adjacent to the terminal nucleotide base pair comprises a guanine (G) base.

18. The method of claim 14, wherein the unpaired nucleotide adjacent to the terminal nucleotide base pair comprises an adenine (A) base.

19. The method of claim 14, wherein said nucleotide overhang comprising the sequence 5′

-GC-3′

consists of the sequence 5′

-GC-3′

.

20. The method of claim 14, wherein the region of the antisense strand that is complementary to the target gene is 19 to 24 nucleotides in length.

21. The method of claim 14, wherein the antisense strand is 20 to 28 nucleotides in length.

22. The method of claim 14, wherein the antisense strand is 21 nucleotides in length.

23. The method of claim 14, wherein at least one of the RNA strands comprises at least one chemically modified nucleotide.

24. The method of claim 23, wherein the chemically modified nucleotide comprises a non-natural base.

25. The methods of claim 23, wherein the chemically modified nucleotide comprises a 2′

modification.

26. The method of claim 25, wherein the 2′

modification is selected from the group consisting of a 2′

-amino modification, a 2′

-alkyl modification, and a 2′

-O-methyl modification, a 2′

-O-ethyl modification, a 2′

-O-propyl modification, a 2′

-O-allyl modification, a 2′

-O-aminoalkyl modification, and a 2′

-deoxy-2′

-fluoro modification.