Methods and compositions for therapeutic use of RNA interference
The present invention provides methods and compositions for attenuating expression of a target gene in vivo. In general, the method includes administering RNAi constructs (such as small-interfering RNAs (i.e., siRNAs) that are targeted to particular mRNA sequences, or nucleic acid material that can produce siRNAs in a cell), in an amount sufficient to attenuate expression of a target gene by an RNA interference mechanism, e.g., in a sequence-dependent, PKR-independent manner. In particular, the subject method can be used to alter the growth, survival or differentiation of cells for therapeutic and cosmetic purposes.
1 . A method for attenuating expression of a target gene of a mammalian cell in vivo, comprising administering a small-interfering RNA formulated in a supramolecular complex comprising at least one cyclodextrin-containing polymer, wherein
said small-interfering RNA comprises two separate complementary strands, a first strand which hybridizes to the target gene, and a second strand which is complementary to said first strand and forms a duplex therewith, and
said small-interfering RNA attenuates expression of the target gene.
2 . The method of claim 1 , wherein the small-interfering RNA is 19-30 base pairs long.
3 . The method of any of claims 1 or 2 , wherein the supramolecular complex is a multi-dimensional polymer network including linear polymers.
4 . The method of any of claims 1 or 2 , wherein the supramolecular complex is a multi-dimensional polymer network including branched polymers.
5 . The method of claim 1 , wherein said supramolecular complex comprises α-cyclodextrin-containing polymers.
6 . The method of claim 5 , wherein said β-cyclodextrin-containing polymers are imidazole-terminated β-cyclodextrin-containing polymers.
7 . The method of claim 1 , wherein said supramolecular complex comprises cyclodextrin-modified poly(ethylenimine) polymers.
8 . The method of claim 7 , wherein said supramolecular complex comprises cyclodextrin-modified poly(ethylenimine) and has a structure of the formula:
wherein
R represents, independently for each occurrence, H, lower alkyl, a cyclodextrin moiety, or
and
m, independently for each occurrence, represents an integer from 2-10,000.
9 . The method of claim 1 , wherein the supramolecular complexes are aggregated into particles having an average diameter of between 20 and 500 nm.
10 . The method of claim 9 , wherein said particles have an average diameter of between 20 and 200 nm.
11 . The method of claim 1 , wherein the supramolecular complex further comprises a targeting ligand.
12 . The method of claim 11 , wherein said targeting ligand is galactose.
13 . The method of claim 11 , wherein said targeting ligand is transferrin.
14 . The method of claim 1 , wherein at least one strand of the small-interfering RNA comprises an overhang of about 1 to about 6 nucleotides.
15 . The method of claim 14 , wherein both strands of the small-interfering RNA comprise an overhang of about 1 to about 6 nucleotides.
16 . The method of claim 15 , wherein both strands of the small-interfering RNA comprise a 3′ overhang of 2 nucleotides.
17 . A method for attenuating expression of a target gene of a liver cell in vivo, comprising administering a small-interfering RNA formulated in a supramolecular complex comprising at least one cyclodextrin-containing polymer, wherein
said small-interfering RNA comprises two separate complementary strands, a first strand which hybridizes to the target gene, and a second strand which is complementary to said first strand and forms a duplex therewith, said small-interfering RNA being 19-30 base pairs long and having 3′ overhangs that are two nucleotides in length on both of said first and second strands, and
said small-interfering RNA attenuates expression of a target gene through an RNA interference mechanism,
and wherein said supramolecular complex comprises a galactose targeting ligand and is aggregated into particles having an average diameter of between 20 and 200 nm.