Compositions and methods for inhibiting expression of a target gene

• US 8,546,143 B2
• Filed: 09/29/2010
• Issued: 10/01/2013
• Est. Priority Date: 01/09/2001
• Status: Expired due to Term

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Kreutzer, Roland, Limmer, Stefan, Limmer, Sylvia, Hadwiger, Philipp
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US12/894,018
• Publication Number:
  US 20110111493A1
• Time in Patent Office: 1,098 Days
• Field of Search: 435/6, 435/91.1, 435/91.31, 435/455, 435/458, 536/23.1, 536/24.5, 536/24.31
• US Class Current: 435/455
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/06 Antimalarials
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity

Abstract

The present invention relates to a double-stranded ribonucleic acid (dsRNA) having a nucleotide sequence which is substantially identical to at least a part of a target gene and which is no more than 49, preferably less than 25, nucleotides in length, and which comprises a complementary (antisense) RNA strand having a 1 to 4 nucleotide overhang at the 3′-end and a blunt 5′-end. The invention further relates to a pharmaceutical composition comprising the dsRNA and a pharmaceutically acceptable carrier. The pharmaceutical compositions are useful for inhibiting the expression of a target gene, as well as for treating diseases caused by expression of the target gene, at low dosages (i.e., less than 5 milligrams, preferably less than 25 micrograms, per kg body weight per day). The invention also relates to methods for inhibiting the expression of a target gene, as well as methods for treating diseases caused by the expression of the gene.

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a target gene in a cell, comprising a complementary RNA strand and a sense RNA strand, wherein the sense RNA strand comprises a nucleotide sequence which is substantially identical to the corresponding part of the target gene, wherein the complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an mRNA transcript formed during expression of the target gene, wherein the complementary strand specifically hybridizes with the mRNA transcript, wherein the complementary RNA strand comprises a 3′

-end and a 5′

-end, wherein the 3′

-end has a nucleotide overhang of 1 to 4 nucleotides and wherein the dsRNA at the 5′

-end of the complementary RNA strand is blunt, and wherein the dsRNA is 20 to less than 25 base pairs in length and wherein the two RNA strands of the dsRNA are separate and non-linked.

2. The dsRNA of claim 1, wherein the nucleotide overhang is 1 or 2 nucleotides in length.

3. The dsRNA of claim 1, wherein the nucleotides of the nucleotide overhang are replaced with nucleoside thiophosphates.

4. The dsRNA of claim 1, wherein at least one of the complementary RNA strand and the sense RNA strand is 22 nucleotides in length.

5. The dsRNA of claim 1, wherein at least one of the complementary RNA strand and the sense RNA strand is 24 nucleotides in length.

6. The dsRNA of claim 1, wherein at least one of the complementary RNA strand and the sense RNA strand is 21 nucleotides in length.

7. The dsRNA of claim 1, wherein the target gene comprises EGFR.

8. The dsRNA of claim 1, wherein the target gene comprises MDR1.

9. The dsRNA of claim 1, wherein the target gene comprises MDR1 or EGFR.

10. A method of inhibiting the expression of the target gene in the cell, the method comprising:

(a) introducing into the cell the double-stranded ribonucleic acid (dsRNA) of claim 1 for inhibiting the expression of the target gene in the cell; and

(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the target gene, thereby inhibiting expression of the target gene in the cell.

11. The method of claim 10, wherein the nucleotide overhang is 1 or 2 nucleotides in length.

12. The method of claim 10, wherein the nucleotides of the nucleotide overhang are replaced with nucleoside thiophosphates.

13. The method of claim 10, wherein at least one of the complementary RNA strand and the sense RNA strand is 22 nucleotides in length.

14. The method of claim 10, wherein at least one of the complementary RNA strand and the sense RNA strand is 24 nucleotides in length.

15. The method of claim 10, wherein at least one of the complementary RNA strand and the sense RNA strand is 21 nucleotides in length.

16. The method of claim 10, wherein the target gene comprises EGFR.

17. The method of claim 10, wherein the target gene comprises MDR1.

18. The method of claim 10, wherein the target gene comprises MDR1 or EGFR.