COMPOSITIONS AND METHODS FOR MODULATING GENE EXPRESSION
Aspects of the invention provide single stranded oligonucleotides for activating or enhancing expression of a target gene. Further aspects provide compositions and kits comprising single stranded oligonucleotides for activating or enhancing expression of a target gene. Methods for modulating expression of a target gene using the single stranded oligonucleotides are also provided. Further aspects of the invention provide methods for selecting a candidate oligonucleotide for activating or enhancing expression of a target gene.
1 . A single stranded oligonucleotide having a sequence 5′-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length, wherein the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a target gene listed in Table 4.
2 . The single stranded oligonucleotide of claim 1 , wherein the oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
3 . The single stranded oligonucleotide of claim 1 , wherein the oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
4 . The single stranded oligonucleotide of claim 1 , wherein the oligonucleotide is 8 to 30 nucleotides in length.
5 . The single stranded oligonucleotide of claim 1 , wherein the oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
6 . The single stranded oligonucleotide of claim 1 , wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue.
7 . The single stranded oligonucleotide of claim 6 , wherein the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5° C. compared with an oligonucleotide that does not have the at least one nucleotide analogue.
8 . The single stranded oligonucleotide of claim 1 , wherein at least one nucleotide of the oligonucleotide comprises a 2′ O-methyl.
9 . The single stranded oligonucleotide of claim 1 , wherein each nucleotide of the oligonucleotide comprises a 2′ O-methyl.
10 . The single stranded oligonucleotide of claim 1 , wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
11 . The single strand oligonucleotide of claim 10 , wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
12 . The single stranded oligonucleotide of claim 1 , wherein each nucleotide of the oligonucleotide is a LNA nucleotide.
13 . The single stranded oligonucleotide of claim 1 , wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2′-fluoro-deoxyribonucleotides.
14 . The single stranded oligonucleotide of claim 1 , wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2′-O-methyl nucleotides.
15 . The single stranded oligonucleotide of claim 1 , wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
16 . The single stranded oligonucleotide of claim 1 , wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides.
17 . The single stranded oligonucleotide of claim 13 , wherein the 5′ nucleotide of the oligonucleotide is a deoxyribonucleotide.
18 . The single stranded oligonucleotide of claim 1 , wherein the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2′-O-methyl nucleotides.
19 . The single stranded oligonucleotide of claim 18 , wherein the 5′ nucleotide of the oligonucleotide is a LNA nucleotide.
20 . The single stranded oligonucleotide of claim 1 wherein the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5′ and 3′ ends of the deoxyribonucleotides.
21 . The single stranded oligonucleotide of claim 1 , further comprising phosphorothioate internucleotide linkages between at least two nucleotides.
22 . The single stranded oligonucleotide of claim 21 , further comprising phosphorothioate internucleotide linkages between all nucleotides.
23 . The single stranded oligonucleotide of claim 1 , wherein the nucleotide at the 3′ position of the oligonucleotide has a 3′ hydroxyl group.
24 . The single stranded oligonucleotide of claim 1 , wherein the nucleotide at the 3′ position of the oligonucleotide has a 3′ thiophosphate.
25 . The single stranded oligonucleotide of claim 1 , further comprising a biotin moiety conjugated to the 5′ nucleotide.
26 . A single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a target gene listed in Table 4, wherein the oligonucleotide has at least one of:
a) a sequence that is 5′X-Y-Z, wherein X is any nucleotide and wherein X is anchored at the 5′ end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length;
b) a sequence that does not comprise three or more consecutive guanosine nucleotides;
c) a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length to the second nucleotide sequence, that are between 50 kilobases upstream of a 5′-end of an off-target gene and 50 kilobases downstream of a 3′-end of the off-target gene;
d) a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops; and/or
e) a sequence that has greater than 60% G-C content.
27 . The single stranded oligonucleotide of claim 26 , wherein the oligonucleotide has the sequence 5′X-Y-Z and wherein the oligonucleotide is 8-50 nucleotides in length.
28 . A composition comprising a single stranded oligonucleotide of claim 1 and a carrier.
29 . A composition comprising a single stranded oligonucleotide of claim 1 in a buffered solution.
30 . A composition of claim 28 , wherein the oligonucleotide is conjugated to the carrier.
31 . The composition of claim 30 , wherein the carrier is a peptide.
32 . The composition of claim 30 , wherein the carrier is a steroid.
33 . A pharmaceutical composition comprising a composition of claim 28 and a pharmaceutically acceptable carrier.
34 . A kit comprising a container housing the composition of claim 28 .
35 . A method of increasing expression of a target gene in a cell, the method comprising delivering the single stranded oligonucleotide of claim 1 into the cell.
36 . The method of claim 35 , wherein delivery of the single stranded oligonucleotide into the cell results in a level of expression of a target gene that is at least 50% greater than a level of expression of the target gene in a control cell that does not comprise the single stranded oligonucleotide.
37 . A method increasing levels of a target gene in a subject, the method comprising administering the single stranded oligonucleotide of claim 1 to the subject.
38 . A method of treating a condition associated with decreased levels of a target gene in a subject, the method comprising administering the single stranded oligonucleotide of claim 1 to the subject.
39 . The method of claim 38 , wherein the target gene is listed in Table 4.
40 . The method of claim 39 , wherein the condition is listed in Table 4 or otherwise disclosed herein.