Spinal muscular atrophy treatment via targeting SMN2 catalytic core
View Patent ↗The present invention is directed to methods and compositions for blocking the effect of the intronic inhibitory splicing region of intron 7 of the SMN2 gene. The compositions and methods of the instant invention include short oligonucleotide reagents (e.g., oligoribonucleotides) that effectively target sites in the SMN2 pre-mRNA, thereby modulating the splicing of SMN2 pre-mRNA to include exon 7 in the processed transcript. The short target regions are 8-mers and 5-mers and also include the identification of a single nucleotide base that is essential for initiating a long distance stearic inhibitory interactions as well as novel targets distant from intron 7 which block the intronic inhibitory splicing of the same. These short target regions and concomitant inhibitory blocking oligonucleotides are less expensive and easier to manufacture and are small enough to cross the blood brain barrier.
1. A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in a cell or cell extract, comprising:
contacting the cell or cell extract with an isolated oligonucleotide reagent comprising a nucleotide sequence at least 90% complementary over its entire length to a target region of the sequence 5′-CUGGUGUCCACAGAGGAC-3 (SEQ ID NO:3), wherein said oligonucleotide contains between about 10 nucleotide bases and about 20 nucleotide bases, and said nucleotide sequence is effective to hybridize to at least 10 contiguous bases of said target region, such that the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the cell or cell extract is enhanced.
2. The method of claim 1 , wherein the cell or cell extract is a spinal muscular atrophy (SMA) patient-derived neuronal cell, muscle cell or fibroblast, or extract thereof.
3. The method of claim 1 , wherein the cell or cell extract is selected from the group consisting of an embryonic stem cell, an embryonic stem cell extract, a neuronal stem cell and a neuronal stem cell extract.
4. A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in an organism, comprising administering to the organism an isolated oligonucleotide reagent comprising a nucleotide sequence at least 90% complementary over its entire length to a target region of the sequence 5′-CUGGUGUCCACAGAGGAC-3′ (SEQ ID NO:3), wherein said oligonucleotide contains between about 10 nucleotide bases and about 20 nucleotide bases, and said nucleotide sequence is effective to hybridize to at least 10 contiguous bases of said target region, such that the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the organism extract is enhanced.
5. The method of claim 4 , wherein the organism is a mammal.
6. The method of claim 5 , wherein the organism is a human.
7. The method of claim 6 , wherein the human has spinal muscular atrophy (SMA).
8. A method of treating spinal muscular atrophy (SMA) in a patient, comprising administering to the patient an isolated oligonucleotide reagent comprising a nucleotide sequence at least 90% complementary over its entire length to a target region of the sequence 5′-CUGGUGUCCACAGAGGAC-3′ (SEQ ID NO:3), wherein said oligonucleotide contains between about 10 nucleotide bases and about 20 nucleotide bases, and said nucleotide sequence is effective to hybridize to at least 10 contiguous bases of said target region in a dose effective to enhance the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in cells of the patient, such that SMA in the patient is treated.
9. A method for inhibiting an SMN2 pre-mRNA intronic splicing silencer site in a cell or cell comprising contacting the cell with an isolated oligonucleotide reagent comprising a nucleotide sequence at least 90% complementary over its entire length to a target region of the sequence 5′-CUGGUGUCCACAGAGGAC-3′ (SEQ ID NO:3), wherein said oligonucleotide contains between about 10 nucleotide bases and about 20 nucleotide bases, and said nucleotide sequence is effective to hybridize to at least 10 contiguous bases of said target region, such that the SMN2 intronic splicing silencer site is inhibited.
10. A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in a cell or cell extract, comprising contacting the cell or cell extract with an isolated oligonucleotide reagent comprising a nucleotide sequence at least 90% complementary over its entire length to a target region of the sequence 5′-CUGGUGUCCACAGAGGAC-3″ (SEQ ID NO:3), wherein said oligonucleotide contains between about 10 nucleotide bases and about 20 nucleotide bases, and said nucleotide sequence is effective to hybridize to at least 10 contiguous bases of said target region, such that the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the cell or cell extract is enhanced.
11. The method of claim 10 , wherein the cell or cell extract is a spinal muscular atrophy (SMA) patient-derived neuronal cell, muscle cell or fibroblast, or extract thereof.
12. The method of claim 10 , wherein the cell or cell extract is selected from the group consisting of an embryonic stem cell, an embryonic stem cell extract, a neuronal stem cell and a neuronal stem cell extract.
13. A method of enhancing the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in an organism, comprising administering to the organism an isolated oligonucleotide reagent comprising a nucleotide sequence at least 90% complementary over its entire length to a target region of the sequence 5′-CUGGUGUCCACAGAGGAC-3′ (SEQ ID NO:3), wherein said oligonucleotide contains between about 10 nucleotide bases and about 20 nucleotide bases, and said nucleotide sequence is effective to hybridize to at least 10 contiguous bases of said target region, such that the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the organism extract is enhanced.
14. The method of claim 13 , wherein the organism is a mammal.
15. The method of claim 14 , wherein the organism is a human.
16. The method of claim 15 , wherein the human has spinal muscular atrophy (SMA).
17. A method of treating spinal muscular atrophy (SMA) in a patient, comprising administering to the patient an isolated oligonucleotide reagent comprising a nucleotide sequence at least 90% complementary over its entire length to a target region of the sequence 5′-CUGGUGUCCACAGAGGAC-3′ (SEQ ID NO:3), wherein said oligonucleotide contains between about 10 nucleotide bases and about 20 nucleotide bases, and said nucleotide sequence is effective to hybridize to at least 10 contiguous bases of said target region in a dose effective to enhance the level of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in cells of the patient, such that SMA in the patient is treated.
18. A method for inhibiting an SMN2 pre-mRNA intronic splicing silencer site in a cell or cell comprising contacting the cell with an isolated oligonucleotide reagent comprising a nucleotide sequence at least 90% complementary over its entire length to a target region of the sequence 5′-CUGGUGUCCACAGAGGAC-3′ (SEQ ID NO:3), wherein said oligonucleotide contains between about 10 nucleotide bases and about 20 nucleotide bases, and said nucleotide sequence is effective to hybridize to at least 10 contiguous bases of said target region, such that the SMN2 intronic splicing silencer site is inhibited.