Anti-C9ORF72 oligonucleotides and related methods
The present disclosure provides antisense compounds, methods, and compositions for silencing C9ORF72 transcripts. The present disclosure provides antisense compounds, methods, and compositions for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with C9ORF72 in a subject in need thereof. Also contemplated are antisense compounds and methods for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with C9ORF72.
1. An antisense oligonucleotide comprising a region of complementarity to a C9ORF72 sense transcript sequence of 5′ GAGUCGCGCGCUAGGGGC 3′ (SEQ ID NO: 9), wherein the antisense oligonucleotide comprises internucleotide linkages from 5′ to 3′ of sooosssssssssooos, wherein each s is a phosphorothioate linkage and each o is a phosphodiester linkage.
2. The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide is 18 to 80 nucleotides in length.
3. The antisense oligonucleotide of claim 1 , comprising the formula:
A-B-C,
wherein:
A comprises from about 0 to about 8 modified nucleotides;
B comprises from about 4 to about 18 deoxyribonucleic acid (DNA) nucleotides and/or DNA-like nucleotides; and
C comprises from about 0 to about 8 modified nucleotides;
and the overall length of the antisense oligonucleotide is about 18 to about 30 nucleotides.
4. The antisense oligonucleotide of claim 1 , comprising a nucleic acid sequence with at least 90% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10 (GCCCCTAGCGCGCGACTC).
5. The antisense oligonucleotide of claim 1 , comprising a sequence modification pattern of X s X o X o X o X s X s X s X s X s X s X s X s X s X o X o X o X s X, wherein
s represents a phosphorothioate internucleotide linkage;
o represents a phosphodiester internucleotide linkage; and
X is an adenosine, a guanosine, a cytidine, or a thymine comprising a 2′-O-(2-methoxyethyl) modification.
6. An antisense oligonucleotide comprising the sequence G s C o C o C o C s T s A s G s C s G s C s G s C s G o A o C o T s C, wherein
s represents a phosphorothioate internucleotide linkage;
o represents a phosphodiester internucleotide linkage;
A is an adenosine comprising a 2′-O-(2-methoxyethyl) modification;
G is a guanosine comprising a 2′-O-(2-methoxyethyl) modification;
C is a cytidine comprising a 2′-O-(2-methoxyethyl) modification;
T is a thymine comprising a 2′-O-(2-methoxyethyl) modification; and
each cytosine is a 5-methylcytosine.
7. A method for inhibiting expression of C9ORF72 gene in a cell, the method comprising:
(a) introducing into the cell an antisense oligonucleotide of claim 1 ; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the transcript of the C9ORF72 gene, thereby inhibiting expression of the C9ORF72 gene in the cell.
8. A method of treating or managing Amyotrophic Lateral Sclerosis (ALS) comprising administering to a patient in need of such treatment or management a therapeutically effective amount of the antisense oligonucleotide of claim 1 .
9. A method of reducing the level of a dipeptide repeat protein in a patient, comprising administering to a patient in need of such reduction a therapeutically effective amount of the antisense oligonucleotide of claim 1 .
10. The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide is 18 to 30 nucleotides in length.
11. The antisense oligonucleotide of claim 1 , comprising at least one modified nucleotide with a modification of a ribose group.
12. The antisense oligonucleotide of claim 11 , wherein the modified nucleotide is a 2′-O-methyl, 2′-fluoro, 2′-H, 2′-O-(2-methoxyethyl) (MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH 2 , or a constrained nucleotide.
13. The antisense oligonucleotide of claim 12 , wherein the constrained nucleotide is a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNA NC ), an alpha-L-locked nucleic acid, or a tricyclo-DNA.
14. The antisense oligonucleotide of claim 1 , comprising at least one modified nucleotide with a modification of a nucleobase group.
15. The antisense of oligonucleotide of claim 14 , wherein each modification of the nucleobase group is 2-thiouridine, 4-thiouridine, N 6 -methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, or halogenated aromatic groups.
16. The antisense of oligonucleotide of claim 1 , wherein the antisense oligonucleotide is conjugated to a ligand.
17. The antisense oligonucleotide of claim 3 , wherein A comprises from about 2 to about 6 modified nucleotides, B comprises from about 6 to about 12 DNA nucleotides and/or DNA-like nucleotides, and C comprises from about 2 to about 6 modified nucleotides.
18. The antisense oligonucleotide of claim 3 , wherein B comprises about 8 DNA nucleotides and/or DNA-like nucleotides, and C comprises about 5 modified nucleotides.
19. The antisense oligonucleotide of claim 3 , wherein A comprises about 5 modified nucleotides, B comprises about 10 DNA nucleotides and/or DNA-like nucleotides, and C comprises about 5 modified nucleotides.
20. The antisense oligonucleotide of claim 3 , wherein A comprises from about 2 to about 6 2′-O-(2-methoxyethyl) (MOE) modified nucleotides, B comprises from about 6 to about 12 DNA-like nucleotides, and C comprises from about 2 to about 6 locked 2′-O-(2-methoxyethyl) (MOE) modified nucleotides.
21. The antisense oligonucleotide of claim 3 , wherein A comprises about 5 2′-O-(2-methoxyethyl) (MOE) modified nucleotides, B comprises about 8 DNA-like nucleotides, and C comprises about 5 locked 2′-O-(2-methoxyethyl) (MOE) modified nucleotides.
22. The antisense oligonucleotide of claim 3 , wherein A comprises about 5 2′-O-(2-methoxyethyl) (MOE) modified nucleotides, B comprises about 10 DNA-like nucleotides, and C comprises about 5 locked 2′-O-(2-methoxyethyl) (MOE) modified nucleotides.
23. The antisense oligonucleotide of claim 5 , wherein each cytidine is a 5-methylcytosine.
24. The method of claim 8 , wherein the antisense oligonucleotide is administered to the brain of the patient.
25. The method of claim 8 , wherein the antisense oligonucleotide is administered by intrathecal, intraventricular or intrastriatal injection or infusion.
26. The method of claim 25 , wherein the injection or infusion comprises administration using an Ommaya reservoir or intrathecal catheter.
27. The method of claim 8 , the antisense oligonucleotide is administered at a dose of between about 0.5 mg/mL to about 5.0 mg/mL.
28. The method of claim 8 , the antisense oligonucleotide is administered at a dose of about 11 mg/mL.
29. The method of claim 8 , the antisense oligonucleotide is formulated in Lactated Ringers Solution.
30. The method of claim 9 , wherein the antisense oligonucleotide is administered to the brain of the patient.
31. The method of claim 9 , wherein the antisense oligonucleotide is administered by intrathecal, intraventricular or intrastriatal injection, or infusion.
32. The method of claim 31 , wherein the injection or infusion comprises administration using an Ommaya reservoir or intrathecal catheter.
33. The method of claim 9 , wherein the dipeptide repeat protein comprises one or more of poly(GP), poly(GR), poly(GA), poly(PA), and poly(PR).