Method and medicament for inhibiting the expression of a given gene
View Patent ↗The present invention provides methods and compositions for inhibiting gene expression using double stranded RNA molecules that are between 15 and 24 nucleotides in length and are complementary to a target gene sequence.
1. A method for inhibiting the expression of a target gene in a mammalian cell in vitro, comprising the steps of:
a) introducing into said cell in vitro an isolated oligoribonucleotide consisting of two separate RNA single strands forming a double-stranded structure (dsRNA), wherein said dsRNA is prepared by:
providing said two separate RNA single strands, wherein one strand of the dsRNA is complementary to an RNA transcript of at least part of said target gene and the other strand of the dsRNA is complementary to the first strand, and wherein the dsRNA is 21 base pairs in length,
hybridizing said two separate RNA single strands,
linking said hybridized strands, and
b) maintaining the cell produced in step a) for a time sufficient to obtain degradation of an RNA transcript of said target gene by dsRNA-mediated interference, thereby inhibiting the expression of said target gene in said cell,
wherein said separate RNA strands are further chemically linked, in addition to the cohesion caused by the nucleotide pairs in the double-stranded region, to reduce dissociation compared to non-linked RNA strands.
2. The method of claim 1 , wherein said target gene comprises a mammalian gene.
3. The method of claim 1 , wherein said two separate complementary strands are fully complementary to each other.
4. The method of claim 1 , wherein one strand of the dsRNA is fully complementary to an RNA transcript of at least part of said target gene.
5. The method of claim 1 , wherein said RNA transcript of said target gene is a primary or processed RNA transcript.
6. The method of claim 1 , wherein at least one of said strands comprises at least one chemically modified nucleotide.
7. The method of claim 1 , wherein said chemical linkage is formed by a covalent bond or hydrogen bound.
8. The method of claim 1 , wherein said chemical linkage is a covalent linkage.
9. The method of claim 1 , wherein said covalent linkage comprises a C18 linker group.
10. The method of claim 1 , wherein said chemical linkage is a labile linkage.
11. The method of claim 10 , wherein said labile linkage comprises a disulfide bridge.
12. The method of claim 1 , wherein said chemical linkage comprises a covalent linkage that is labile.
13. The method of claim 1 , wherein one of the single strands is complementary to the other of the single strands, wherein the two separate single strands hybridize to each other to form the double-stranded structure, and wherein the one of the single strands is also chemically linked to the other of the single strands.
14. The method of claim 1 , wherein inhibiting the expression of said target gene further comprises inhibiting expression at a concentration of the dsRNA that is lower by one order of magnitude than a concentration required for a corresponding single-stranded oligoribonucleotide to inhibit expression.