Induced exon inclusion in spinal muscle atrophy
View Patent ↗The invention relates to the use of an antisense compound for inducing exon inclusion as a treatment for Spinal Muscle Atrophy (SMA). More particularly it relates to inducing inclusion of exon 7 to restore levels of Survival Motor Neuron (SMN) protein encoded by the Survival Motor Neuron (SMN) gene.
1. An antisense oligonucleotide of 10-40 nucleotides in length which specifically hybridizes to a region within the SMN2 gene, such that the level of exon 7-containing SMN2 mRNA relative to exon 7-deleted SMN2 mRNA in the cell is enhanced, wherein the antisense oligonucleotide is a morpholino oligonucleotide, and wherein each nucleotide is a nucleotide having a formula:
wherein Nu is a nucleobase;
R 1 is a moiety of the formula (I):
or
R 1 is —N(CH 3 ) 2 ;
q is 0, 1, 2, 3 or 4;
R 2 is selected from the group consisting of hydrogen, C 1 -C 5 alkyl, and a formamidinyl moiety, and
R 3 is selected from the group consisting of hydrogen and C 1 -C 5 alkyl, or
R 2 and R 3 are joined to form a 5-7 membered heterocyclic ring optionally containing an oxygen hetero atom, where the ring may be optionally substituted with a substituent selected from the group consisting of C 1 -C 5 alkyl, phenyl, halogen, and aralkyl;
R 4 is selected from the group consisting of null, hydrogen, a C 1 -C 6 alkyl and aralkyl;
R x is selected from the group consisting of HO—, a nucleotide, and piperazinyl;
R y is selected from the group consisting of hydrogen, a C 1 -C 6 alkyl, a nucleotide, a, an amino acid, a formamidinyl moiety, and acyl; and,
R z is selected from the group consisting of null, hydrogen, a C 1 -C 6 alkyl, and acyl; and pharmaceutically acceptable salts thereof,
wherein at least one R 1 is of formula (I).
2. The antisense oligonucleotide of claim 1 , wherein at least one nucleotide has the formula:
wherein Rx, Ry, Rz, and Nu are as stated in claim 1 .
3. The antisense oligonucleotide of claim 1 , wherein the target region is within exon 7, intron 7, or exon 8 of the SMN2 gene.
4. The antisense oligonucleotide of claim 3 , wherein the antisense oligonucleotide comprises a sequence which is complementary to a target region within intron 7 of the SMN2 gene.
5. The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide comprises a sequence which is complementary to a portion of intron 7 and exon 8 of the SMN2 gene.
6. The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide comprises a sequence of from about 10 to about 30 nucleotides.
7. The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide comprises a sequence of from about 14 to about 21 nucleotides.
8. The antisense oligonucleotide of claim 1 , wherein the antisense oligonucleotide comprises a sequence selected from SEQ ID NOS: 2-8, 10-18, 20-25, 27-32, 34-41, 43-52, 54-61, 63-69, and 71-79.
9. A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in a cell, comprising contacting the cell with an antisense oligonucleotide of 10-40 nucleotides in length which specifically hybridizes to a region within the SMN2 gene, such that the level of exon 7-containing SMN2 mRNA relative to exon 7-deleted SMN2 mRNA in the cell is enhanced, wherein the antisense oligonucleotide is a morpholino oligonucleotide, and wherein each nucleotide is a nucleotide having a formula:
wherein Nu is a nucleobase;
R 1 is a moiety of the formula (I):
or
R 1 is —N(CH 3 ) 2 ;
q is 0, 1, 2, 3 or 4;
R 2 is selected from the group consisting of hydrogen, C 1 -C 5 alkyl, and a formamidinyl moiety, and
R 3 is selected from the group consisting of hydrogen and C 1 -C 5 alkyl, or
R 2 and R 3 are joined to form a 5-7 membered heterocyclic ring optionally containing an oxygen hetero atom, where the ring may be optionally substituted with a substituent selected from the group consisting of C 1 -C 5 alkyl, phenyl, halogen, and aralkyl;
R 4 is selected from the group consisting of null, hydrogen, a C 1 -C 6 alkyl and aralkyl;
R x is selected from the group consisting of HO—, a nucleotide, and piperazinyl;
R y is selected from the group consisting of hydrogen, a C 1 -C 6 alkyl, a nucleotide, a an amino acid, a formamidinyl moiety, and acyl; and,
R z is selected from the group consisting of null, hydrogen, a C 1 -C 6 alkyl, and acyl; and pharmaceutically acceptable salts thereof,
wherein at least one R 1 is of formula (I).
10. The method of claim 9 , wherein at least one nucleotide has the formula:
wherein Rx, Ry, Rz, and Nu are as stated in claim 9 .
11. The method of claim 10 , wherein Nu is thymine or uracil.
12. The method of claim 9 , wherein the target region is within exon 7, intron 7, or exon 8 of the SMN2 gene.
13. The method of claim 12 , wherein the antisense oligonucleotide comprises a sequence which is complementary to intron 7 of the SMN2 gene.
14. The method of claim 13 , wherein the antisense oligonucleotide comprises a sequence which is complementary to a portion of intron 7 and exon 8 of the SMN2 gene.
15. The method of claim 9 , wherein the antisense oligonucleotide comprises a sequence of from about 10 to about 30 nucleotides.
16. The method of claim 9 , wherein the antisense oligonucleotide comprises a sequence of from about 14 to about 21 nucleotides.
17. The method of claim 9 , wherein the antisense oligonucleotide comprises a sequence selected from SEQ ID NOS: 2-8, 10-18, 20-25, 27-32, 34-41, 43-52, 54-61, 63-69, and 71-79.
18. A method of treating spinal muscular atrophy (SMA) in a patient, comprising administering to the patient an antisense oligonucleotide according to claim 1 , thereby treating the patient.