Oligonucleotide compounds for targeting huntingtin mRNA
This disclosure relates to novel huntingtin targets. Novel oligonucleotides for the treatment of Huntington's disease are also provided.
1. An RNA molecule that is between 15 and 35 bases in length, comprising a region of complementarity which is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1).
2. The RNA molecule of claim 1 , wherein said RNA molecule is single stranded (ss) RNA or double stranded (ds) RNA.
3. A pharmaceutical composition for inhibiting the expression of Huntingtin (HTT) gene in an organism, comprising the dsRNA of claim 2 and a pharmaceutically acceptable carrier.
4. An RNA molecule that is between 15 and 35 bases in length, comprising a region of complementarity which is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3),
wherein the RNA molecule comprises a 5′ end, a 3′ end and has complementarity to a target, wherein:
(1) the RNA molecule comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro -ribonucleotides;
(2) the nucleotides at positions 2 and 14 from the 5′ end are not 2′-methoxy -ribonucleotides;
(3) the nucleotides are connected via phosphodiester or phosphorothioate linkages; and
(4) the nucleotides at positions 1-6 from the 3′ end, or positions 1-7 from the 3′ end, are connected to adjacent nucleotides via phosphorothioate linkages.
5. The RNA of claim 4 , wherein said dsRNA is double stranded RNA, said dsRNA having a 5′ end, a 3′ end and complementarity to a target, and comprising a first oligonucleotide and a second oligonucleotide, wherein:
(1) the first oligonucleotide comprises a sequence set forth as SEQ ID NO:1;
(2) a portion of the first oligonucleotide is complementary to a portion of the second oligonucleotide;
(3) the second oligonucleotide comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides;
(4) the nucleotides at positions 2 and 14 from the 3′ end of the second oligonucleotide are 2′-methoxy-ribonucleotides; and
(5) the nucleotides of the second oligonucleotide are connected via phosphodiester or phosphorothioate linkages.
6. The nucleic acid of claim 5 comprising one or more of the following:
the second oligonucleotide is linked to a hydrophobic molecule at the 3′ end of the second oligonucleotide;
the linkage between the second oligonucleotide and the hydrophobic molecule comprises polyethylene glycol or triethylene glycol;
the nucleotides at positions 1 and 2 from the 3′ end of second oligonucleotide are connected to adjacent nucleotides via phosphorothioate linkages; and
the nucleotides at positions 1 and 2 from the 3′ end of second oligonucleotide, and the nucleotides at positions 1 and 2 from the 5′ end of second oligonucleotide, are connected to adjacent ribonucleotides via phosphorothioate linkages.
7. The nucleic acid of claim 5 , wherein the nucleotides at positions 1 and 2 from the 3′ end of second oligonucleotide, and the nucleotides at positions 1 and 2 from the 5′ end of second oligonucleotide, are connected to adjacent ribonucleotides via phosphorothioate linkages.
8. A method for inhibiting expression of HTT gene in a cell, the method comprising:
(a) introducing into the cell a double-stranded ribonucleic acid (dsRNA) that is between 15 and 35 bases in length, comprising a region of complementarity which is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3); and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the HTT gene, thereby inhibiting expression of the HTT gene in the cell.
9. A method of treating or managing Huntington's disease comprising administering to a patient in need of such treatment or management a therapeutically effective amount of said dsRNA that is between 15 and 35 bases in length, comprising a region of complementarity which is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3).
10. The method of claim 9 comprising one or more of the following:
said dsRNA is administered to the brain of the patient;
said dsRNA is administered by intrastriatal infusion;
administering the dsRNA of claim 2 to the brain causes a decrease in HTT gene mRNA in the striatum; and
administering the dsRNA of claim 2 to the brain causes a decrease in HTT gene mRNA in the cortex.
11. The dsRNA of claim 2 , wherein said modified nucleotide is a terminal nucleotide linked to a phosphatidylcholine derivative.
12. A di-branched RNA compound comprising two RNA molecules that are between 15 and 35 bases in length, comprising a region of complementarity which is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3), wherein the two RNA molecules are connected to one another by one or more moieties independently selected from a linker, a spacer and a branching point.
13. The dsRNA of claim 2 , comprising a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity which is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1).
14. The dsRNA of claim 2 , comprising a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity which is complementary to at least 10, 11, 12 or 13 contiguous nucleotides of SEQ ID NO:1.
15. The dsRNA of claim 2 , comprising a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity contains no more than 3 mismatches with SEQ ID NO:1.
16. The dsRNA of claim 2 , comprising a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity is fully complementary to SEQ ID NO:1.
17. The dsRNA of claim 2 , wherein the dsRNA is between 30 and 35 base pairs in length.
18. The dsRNA of claim 2 , wherein the dsRNA is blunt-ended.
19. The dsRNA of claim 2 , wherein the dsRNA comprises at least one single stranded nucleotide overhang.
20. The dsRNA of claim 2 , wherein the dsRNA comprises naturally occurring nucleotides.
21. The dsRNA of claim 2 , wherein the dsRNA comprises at least one modified nucleotide.
22. The dsRNA of claim 21 , wherein the modified nucleotide is selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group.
23. The dsRNA of claim 21 , wherein the modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2 ′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, at least one 2′-O-methyl modified nucleotide and at least one nucleotide comprising a 5′-phosphorothioate group.
24. The dsRNA of claim 2 , wherein the dsRNA comprises a cholesterol moiety.