Compositions and methods for inhibiting expression of a target gene
View Patent ↗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.
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a target gene in a cell in vitro, 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 less than 25 base pairs in length and wherein the two RNA strands of the dsRNA are separate and non-linked, and wherein the target gene comprises a sequence of SEQ ID NO:51.
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. A method of inhibiting the expression of a target gene in a cell in vitro, the method comprising:
(a) introducing into the cell a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a target gene in a cell in vitro, wherein the dsRNA comprises 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 4nucleotides and wherein the dsRNA at the 5′-end of the complementary RNA strand is blunt, and wherein the dsRNA less than 25 base pairs in length and wherein the two RNA strands of the dsRNA are separate and non-linked, and wherein the target gene comprises a sequence of SEQ ID NO:51; 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.
5. The method of claim 4 , wherein the nucleotide overhang is 1 or 2 nucleotides in length.
6. The method of claim 4 , wherein the nucleotides of the nucleotide overhang are replaced with nucleoside thiophosphates.