RNAI CONSTRUCTS AND USES THEREOF
The invention relates to improved double-stranded RNAi constructs (sometimes referred to as “solo-rxRNA”) and uses thereof. The construct comprises a structure formed in some aspects of the invention by two identical single-stranded polynucleotides, with the structure having two double-stranded stem regions (each having less than 21 base pairs) and a loop or bulge having about 4 to 11 nucleotides on each strand. The construct is resistant to cleavage by Dicer or other Dicer-like RNase III enzymes and is capable of being loaded into a RISC complex to effect RNA interference. In addition, the nucleotides of the present hairpin constructs may be modified to greatly enhance functionality, such as stability and specificity.
1 . A polynucleotide construct comprising two identical single-stranded polynucleotides, wherein each single-stranded polynucleotide comprises a 5′-stem sequence having a 5′-end, a 3′-stem sequence having a 3′-end, and a linker sequence linking the 5′-stem sequence and the 3′-stem sequence, wherein:
(1) the 5′-stem sequence of a first single-stranded polynucleotide hybridizes with the 3′-stem sequence of a second single-stranded polynucleotide to form a first double-stranded stem region;
(2) the 5′-stem sequence of the second single-stranded polynucleotide hybridizes with the 3′-stem sequence of the first single-stranded polynucleotide to form a second double-stranded stem region; and,
(3) the linker sequences of the first and the second single-stranded polynucleotides form a loop or bulge connecting the first and the second double-stranded stem regions, wherein the 5′-stem sequence and at least a portion of the linker sequence form an antisense sequence complementary to a transcript of a target gene, wherein said polynucleotide construct mediates sequence-dependent gene silencing of expression of the target gene.
2 . The polynucleotide construct of claim 1 , wherein the 5′-stem sequence, the loop, and at least a portion of the 3′-stem sequence collectively form the antisense sequence complementary to the transcript of the target gene.
3 . The polynucleotide construct of claim 1 , wherein the antisense sequence is about 15-21 nucleotides in length, about 17-21 nucleotides in length, about 19-21 nucleotides in length, about 17-18 nucleotides in length or about 16-18 nucleotides in length.
4 . The polynucleotide construct of claim 1 , wherein each of the single-stranded polynucleotides is about 15-49 nucleotides in length, about 33-35 nucleotide in length, or about 25-27 nucleotides in length, and/or wherein each of the first and second double-stranded stem regions is less than about 21 base pairs in length, less than about 20 base pairs in length, about 5-15 base pairs in length, or about 11-14 base pairs in length.
5 - 8 . (canceled)
9 . The polynucleotide construct of claim 1 , wherein each of the double-stranded regions is at least 8, 9, 10, 11 or 12 base pairs in length.
10 . The polynucleotide construct of claim 1 , wherein the linker sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length.
11 - 14 . (canceled)
15 . The polynucleotide construct of claim 1 , wherein at least one nucleotide is modified to improve resistance to nucleases, serum stability, target specificity, blood system circulation, tissue distribution, tissue penetration, cellular uptake, potency, and/or cell-permeability of the polynucleotide.
16 . The polynucleotide construct of claim 15 , wherein the modified nucleotides are modified on the sugar moiety, the base, and/or the phosphodiester linkage.
17 - 23 . (canceled)
24 . The polynucleotide construct of claim 15 , wherein the modification is a 2′-O-alkyl or 2′-halo group.
25 . The polynucleotide construct of claim 24 , wherein the modification comprises:
(1) a 2′-O-methyl modification of one or more pyrimidine nucleotides (C or U);
(2) a 2′-O-methyl modification of one or more nucleotides within the loop;
(3) a 2′-O-methyl modification of at least 30% of all nucleotides;
(4) a 2′-O-methyl modification of all nucleotides in the 3′-end stem region;
(5) a 2′-O-methyl modification of all nucleotides 3′ to the loop;
(6) a hydrophobic modification of one or more bases, optionally wherein the hydrophobic modification comprises an isobutyl group;
(7) one or more phosphate modifications, optionally wherein the phosphate modifications are phosphorothioate modifications; and/or
(8) one or more 2′-fluoro modifications, optionally wherein at least one C or U nucleotide in positions 2-10 of the first single-stranded polynucleotide has a 2′-fluoro modification.
26 - 46 . (canceled)
47 . A pharmaceutical composition comprising the polynucleotide construct of claim 1 , and a pharmaceutically acceptable salt, diluent, excipient, or carrier.
48 . (canceled)
49 . A method of inhibiting expression of a target gene with a polynucleotide construct of claim 1 , wherein the polynucleotide construct mediates antisense sequence-dependent reduction in expression of the target gene.
50 - 63 . (canceled)
64 . A polynucleotide construct comprising a first single-stranded polynucleotide and a second single-stranded polynucleotide, each comprising a 5′-stem sequence having a 5′-end, a 3′-stem sequence having a 3′-end, and a linker sequence linking the 5′-stem sequence and the 3′-stem sequence, wherein:
(1) the 5′-stem sequence of the first single-stranded polynucleotide hybridizes with the 3′-stem sequence of the second single-stranded polynucleotide to form a first double-stranded stem region;
(2) the 5′-stem sequence of the second single-stranded polynucleotide hybridizes with the 3′-stem sequence of the first single-stranded polynucleotide to form a second double-stranded stem region; and,
(3) the linker sequences of the first and the second single-stranded polynucleotides form a loop or bulge connecting the first and the second double-stranded stem regions, wherein the loop is at least 3 nucleotides in length, wherein the 5′-stem sequence and at least a portion of the linker sequence for the first single-stranded polynucleotide form a first antisense sequence complementary to a transcript of a first target gene, and the 5′-stem sequence and at least a portion of the linker sequence for the second single-stranded polynucleotide form a second antisense sequence complementary to a transcript of a second target gene, and, wherein the polynucleotide construct mediates sequence-dependent gene silencing of expression of the first and second target genes.
65 - 76 . (canceled)
77 . A single-stranded polynucleotide of less than 35 nucleotides in length that forms a hairpin structure, wherein the hairpin includes a double-stranded stem and a single-stranded loop, the double-stranded stem having a 5′-stem sequence having a 5′-end, and a 3′-stem sequence having a 3′-end; and the 5′-stem sequence and at least a portion of the loop form an antisense sequence complementary to a transcript of a target gene, wherein the polynucleotide mediates sequence-dependent gene silencing of expression of the target gene.
78 . The single-stranded polynucleotide of claim 77 , wherein the 5′-stem sequence, the loop, and at least a portion of the 3′-stem sequence collectively form the antisense sequence complementary to the transcript of the target gene.
79 - 81 . (canceled)
82 . A method of treating a patient for a disease characterized by overexpression of a target gene, comprising administering to the patient a therapeutically effective amount of a polynucleotide construct of claim 1 , wherein the polynucleotide construct mediates antisense sequence-dependent reduction in expression of the target gene.
83 . (canceled)
84 . The polynucleotide construct of claim 1 , wherein the linker sequence of each single-stranded polynucleotide is 8 nucleotides in length, and wherein the 3′-end stem region of each single-stranded polynucleotide is highly modified with 2′-O-methyl modifications.
85 . The polynucleotide construct of claim 64 , wherein the 5′-stem sequence, the loop, and at least a portion of the 3′-stem sequence collectively form the antisense sequence complementary to the transcript of the target gene.
86 . The polynucleotide construct of claim 64 , wherein at least one nucleotide is modified and wherein the modification comprises:
(1) a 2′-O-methyl modification of one or more pyrimidine nucleotides (C or U);
(2) a 2′-O-methyl modification of one or more nucleotides within the loop;
(3) a 2′-O-methyl modification of at least 30% of all nucleotides;
(4) a 2′-O-methyl modification of all nucleotides in the 3′-end stem region;
(5) a 2′-O-methyl modification of all nucleotides 3′ to the loop;
(6) a hydrophobic modification of one or more bases, optionally wherein the hydrophobic modification comprises an isobutyl group;
(7) one or more phosphate modifications, optionally wherein the phosphate modifications are phosphorothioate modifications; and/or
(8) one or more 2′-fluoro modifications, optionally wherein at least one C or U nucleotide in positions 2-10 of the first single-stranded polynucleotide has a 2′-fluoro modification.
87 . The polynucleotide construct of claim 64 , wherein the linker sequence of each single-stranded polynucleotide is 8 nucleotides in length, and wherein the 3′-end stem region of each single-stranded polynucleotide is highly modified with 2′-O-methyl modifications.