Compositions and methods for modulation of SMN2 splicing in a subject

Abstract

To provide compounds, compositions and methods for modulating splicing of SMN2mRNA in a subject.SOLUTION: Methods are provided comprising administering to a subject an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of nucleic acids encoding human SMN2pre mRNA, wherein the antisense compound is administered into the cerebral spinal fluid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided in the file entitled "20100617_CORE0086WOSEQ.txt," created on June 17, 2010, and 5 Kb in size. This information is incorporated herein by reference in its entirety. [Background technology]

[0002] Newly synthesized eukaryotic mRNA molecules, known as primary transcripts or pre-mRNAs, are processed prior to translation. Pre-mRNA processing involves the addition of a 5' methylated cap and a poly(A) tail of approximately 200–250 bases to the 3' end of the transcript. Processing of mRNA from pre-mRNA also often involves splicing of the pre-mRNA, which occurs during the maturation of 90-95% of mammalian mRNAs. Introns (or intervening sequences) are regions of the pre-mRNA (or the DNA encoding it) that are not included in the coding sequence of the mature mRNA. Exons are regions of the primary transcript that remain in the mature mRNA. Exons are spliced ​​together to form the mature mRNA sequence. Splice junctions are also referred to as splice sites, with the 5' end of the junction often referred to as the "5' splice site" or "splice donor site" and the 3' end of the junction often referred to as the "3' splice site" or "splice acceptor site." During splicing, the 3' end of the upstream exon is joined to the 5' end of the downstream exon. Thus, the unspliced ​​pre-mRNA has an exon / intron junction at the 5' end of the intron and an intron / exon junction at the 3' end of the intron. After the intron is removed, the exon is adjacent to a site or boundary in the mature mRNA, often referred to as an exon / exon junction. Ambiguous splice sites are splice sites that are used less frequently but can be used when the normal splice sites are blocked or unavailable. Alternative splicing, defined as splicing involving different combinations of exons, often results in multiple mRNA transcripts from a single gene.

[0003] Up to 50% of human genetic diseases resulting from point mutations result in abnormal pre-mRNA processing. Such point mutations can either disrupt existing splice sites or form new splice sites, resulting in mRNA transcripts containing different combinations of exons or mRNA transcripts containing deletions in exons. Point mutations can also result in the activation of subambiguous splice sites or alter regulatory cis-elements (such as i.e., splicing enhancers or silencers) can be disrupted (Cartegni et al., Nat. Rev. Genet., 2002, 3, 285-298; Drawczak et al., Hum. Genet., 1992, 90, 41-54). Antisense oligonucleotides have been used to target mutations that cause aberrant splicing in several genetic diseases in order to direct splicing to obtain the desired spliced ​​product (Kole, Acta Biochimica Polonica, 1997, 44, 231-238).

[0004] Antisense compounds can also be used to alter the ratio of naturally occurring alternative splice variants, such as the long and short forms of Bcl-x pre-mRNA (US Patent 6,172,216; US Patent 6,214,986; Taylor et al., Nat. Biotechnol. 1999, 17, 1097-1100), or to alter the ratio of naturally occurring alternative splice variants, such as the long and short forms of Bcl-x pre-mRNA (US Patent 6,172,216; US Patent 6,214,986; Taylor et al., Nat. Biotechnol. 1999, 17, 1097-1100). or forced skipping of specific exons containing premature stop codons (Wilton et al. (T al., Neuromuscul. Disord., 1999, 9, 330-338). US Patent 5,627,274 and WO 94 / 26887 disclose compositions and methods for combating aberrant splicing in pre-mRNA molecules containing mutations using antisense oligonucleotides that do not activate RNAse H.

[0005] Proximal spinal muscular atrophy (SMA) is characterized by loss of spinal motor neurons SMA is an early-onset, autosomal recessive disorder, and currently It is the leading cause of death among infants. The severity of SMA varies among patients and Type I SMA is the most severe type, occurring at birth or within six months of birth. It is a severe form of SMA and is fatal within two years. Children with type 1 SMA are unable to sit or walk. Type II SMA is an intermediate type in which patients can sit but cannot stand or walk. Patients with type III SMA, a chronic form of the disease, typically develop SMA after 18 months of age (Lefebvre et al., Hum. Mol. Genet., 1998, 7, 1531-1536).

[0006] The molecular basis of SMA results from the loss of both copies of the survival motor neuron gene 1 (SMN1), which is thought to be involved in snRNP biogenesis and recycling. SMN is a protein that is part of a multi-protein complex known as the SMN telomeric. A nearly identical gene, SMN2, is also known as the SMN centromere. However, it resides in a duplicated region on chromosome 5q13 and modifies disease severity. Expression of the normal SMN1 gene results in minimal expression of the survival motor neuron (SMN) protein. Although SMN1 and SMN2 potentially encode the same protein, SMN2 contains a translationally silent mutation at position +6 of exon 7, which results in inefficient inclusion of exon 7 in the SMN2 transcript. Thus, the predominant form of SMN2 is a truncated form lacking exon 7, which is unstable and inactive (Cartegni and Krainer, Nat. Genet., 2002, 30, 377-384). Expression of the SMN2 gene accounts for approximately 10-20% of the SMN protein and 80-90% of the SMN2 protein. % of unstable / non-functional SMN delta7 protein. SMN protein has a well-established function in spliceosome assembly and may mediate mRNA trafficking in neuronal axons and nerve terminals.

[0007] Antisense technology targets one or more specific target genes, including alternative splicing products. Antisense is an effective means for regulating the expression of gene products and is uniquely useful in numerous therapeutic, diagnostic, and research applications. The principle behind antisense technology is that antisense compounds that hybridize to a target nucleic acid modify gene expression activities such as transcription, splicing, or translation through one of a number of antisense techniques. The sequence specificity of antisense compounds makes them extremely attractive as tools for target validation and gene functioning, as well as therapeutic agents for selectively modifying the expression of disease-related genes.

[0008] Certain antisense compounds complementary to SMN2 are known in the art.See, for example, WO 2007 / 002390; US 61 / 168,885; Hua et al., American Journal of Human Genetics (April 2008) 82, 1-15; Singh et al., RNA Bio. 6:3, 1-10 (2009).Compared with such compounds and methods in the art, certain antisense compounds and methods disclosed herein have desirable characteristics.Chimeric peptide nucleic acid molecules designed to modify the splicing of SMN2 have been described (WO 02 / 38738; Cartegni and Krainer, Nat. Struct. Biol., 2003, 10, 120-125). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US Patent 6,172,216 [Patent Document 2] US Patent 6,214,986 [Patent Document 3] US Patent 5,627,274 [Patent Document 4] WO 94 / 26887

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[0010]

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[0011] In certain embodiments, the present invention provides a method for administering to a subject a nucleic acid encoding human SMN2 pre-mRNA. Antisense oligonucleotides containing antisense complementary to intron 7 of the nucleotide sequence The present invention provides a method comprising administering a compound, wherein the antisense compound is administered into cerebrospinal fluid. In certain embodiments, the administration is carried out into the subarachnoid space. In certain embodiments, the administration is carried out into the cerebrospinal fluid in the brain. In certain embodiments, the administration comprises bolus injection. In certain embodiments, the administration comprises infusion using a delivery pump.

[0012] In certain embodiments, the antisense compound is administered at a dose of 0.01 to 10 milligrams of antisense compound per kg of subject body weight. In certain embodiments, the dose is 0.01 to 10 milligrams of antisense compound per kg of subject body weight. In certain embodiments, the dose is 0.01 to 5 milligrams of antisense compound per kg of subject body weight. In this case, the dose is 0.05 to 1 milligram of antisense compound per kg of subject body weight. In certain embodiments, the dose is 0.01 to 0.5 milligrams of antibiotic per kg of subject body weight. In certain embodiments, the dose is 0.05 to 0.5 milligrams per kilogram of the subject's body weight. It is an antisense compound from Liguram.

[0013] In certain embodiments, the dose is administered daily. In certain embodiments, the dose is administered weekly. In certain embodiments, the antisense compound is administered continuously, In certain embodiments, the method includes an induction phase. In certain embodiments, the induction phase comprises administering at least one induction dose during a period of 10 days, and administering at least one maintenance dose during a maintenance phase. In certain embodiments, the induction phase comprises administering 0.05 to 5.0 milligrams of antisense compound per kg of body weight of the subject. In certain embodiments, the maintenance dose In certain cases, the antisense compound is administered at a dose of 0.01 to 1.0 milligrams per kg of subject body weight. In certain embodiments, the duration of the induction phase is at least one week. The duration is at least one week. In certain embodiments, each of the induction and maintenance administrations Each of the administrations comprises one injection. In certain embodiments, each of the induction administrations and Each of the maintenance doses independently comprises two or more injections. The antisense compound is administered at least twice over a treatment period of at least one week. In certain embodiments, the treatment period is at least one month. The treatment period is at least two months. In certain embodiments, the treatment period is at least four months. In certain embodiments, the induction phase is administered by one or more bolus injections. and maintenance doses are administered via an infusion pump.

[0014] In certain embodiments, the method includes evaluating the tolerability and / or efficacy of the antisense compound. In certain embodiments, the dosage or frequency of administration of the antisense compound is reduced according to an indication in which administration of the antisense compound is unacceptable. In certain embodiments, the dosage or frequency of administration of the antisense compound is maintained or reduced according to an indication in which administration of the antisense compound is effective. In certain embodiments, the dosage of the antisense compound is increased according to an indication in which administration of the antisense compound is ineffective. In certain embodiments, the frequency of administration of the antisense compound is decreased according to an indication in which administration of the antisense compound is effective. In certain embodiments, the frequency of administration of the antisense compound is increased according to an indication in which administration of the antisense compound is ineffective.

[0015] In certain embodiments, the method comprises combining an antisense compound with at least one other therapeutic agent. In certain embodiments, the antisense compound and at least one therapeutic agent are co-administered. In certain embodiments, the antisense compound is administered prior to the administration of at least one other treatment. The sense compound is administered after the administration of at least one other treatment. and at least one other treatment is valproic acid, riluzole, hydroxyurea, and butyrate. At least one other treatment comprises administering trichostatin-A. In certain embodiments, the at least one other treatment comprises administering stem cells. In certain embodiments, the at least one other treatment is a gene therapy. The antisense treatment is administered into the CSF and the antisense compound is administered systemically. In certain embodiments, the present invention provides a method for administering gene therapy to the CSF and an antisense compound systemically and to the CSF. administering the gene therapy to the CSF and then administering the antisense compound systemically. In certain such embodiments, the subject is an infant at the time of initial treatment. In certain such embodiments, the subject is under 2 years of age. In certain such embodiments, In certain such embodiments, the antisense compound is administered to the CNS of the subject until the subject is old enough for gene therapy. Continue administering.

[0016] In certain embodiments, the antisense compound is administered at a concentration of about 0.01 mg / ml, about 0.05 mg / ml, about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 5 mg / ml, about 10 mg / ml, about 50 mg / ml, or about 100 mg / ml.

[0017] In certain embodiments, the content of exon 7 of SMN2 mRNA in the motor neurons of the subject is increased. In certain embodiments, the content of exon 7 amino acids of SMN2 polypeptide in the motor neurons of the subject is increased.

[0018] In certain embodiments, the present invention provides a method for administering to a subject an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2. and thereby increasing the content of exon 7 of SMN2 mRNA in the motor neurons of the subject.

[0019] In certain embodiments, the present invention provides a method for administering to a subject an intravenous injection of a nucleic acid encoding human SMN2. Antisense compounds containing antisense oligonucleotides complementary to IL-7 were administered. and thereby increasing the content of exon 7 amino acids in the SMN2 polypeptide in the motor neurons of the subject.

[0020] In certain embodiments, the subject has SMA. In certain embodiments, the subject has SMA type I. In certain embodiments, the subject has SMA type II. In certain embodiments, the subject has SMA type III.

[0021] In certain embodiments, the first dose is administered in utero. In certain embodiments, the first dose is administered before the formation of the blood-brain barrier is complete. In certain embodiments, the first dose is administered within the first week of the subject's life. In certain embodiments, the first dose is administered within the first month of the subject's life. In certain embodiments, the first dose is administered within the first three months of the subject's life. In certain embodiments, the first dose is administered within the first six months of the subject's life. In certain embodiments, the first dose is administered when the subject is 1 to 2 years of age. In certain embodiments, In certain embodiments, the first dose is administered when the subject is 1 to 15 years of age. is administered when the subject is 15 years of age or older.

[0022] In certain embodiments, the subject is a mammal, hi certain embodiments, the subject is a human.

[0023] In certain embodiments, the method includes identifying a subject with SMA. In embodiments, the subject is monitored by measuring the electrical activity of one or more of the subject's muscles. In certain embodiments, the subject is identified by genetic testing to determine if the subject has a mutation in the subject's SMN1 gene. In certain embodiments, the subject is identified by muscle biopsy.

[0024] In certain embodiments, administration of the antisense compound reduces at least 10% of the In certain embodiments, the amount of SMN2 mRNA containing exon 7 is increased. In certain embodiments, the increase in the amount of SMN2 mRNA is at least 20%. In certain embodiments, the increase in the amount of SMN2 mRNA is at least 50%. The amount of SMN2 mRNA present is at least 70%.

[0025] In certain embodiments, administration of the antisense compound reduces at least 10% of the This results in an increase in the amount of SMN2 polypeptide having amino acid sequence 7. In certain embodiments, the increase in the amount of SMN2 polypeptide having amino acids from exon 7 is at least 20%. In certain embodiments, the increase in the amount of SMN2 polypeptide having amino acids from exon 7 is at least 50%. In certain embodiments, the increase in the amount of SMN2 polypeptide having amino acids from exon 7 is at least 70%.

[0026] In certain embodiments, administering the antisense compound reduces or eliminates SMA in a subject. In certain embodiments, administering an antisense compound improves at least one symptom of the disease. In certain embodiments, administering an antisense compound results in an improvement in the subject's motor function. In certain embodiments, administering an antisense compound results in a delay or reduction in the loss of motor function in the subject. In certain embodiments, administering an antisense compound results in an improvement in respiratory function. In certain embodiments, administering an antisense compound results in an improvement in survival.

[0027] In certain embodiments, at least one nucleoside of the antisense oligonucleotide In certain embodiments, at least one modified sugar moiety comprises a 2'-methionine. In certain embodiments, essentially each nucleoside of the antisense oligonucleotide comprises a modified sugar moiety. In certain embodiments, all nucleosides comprising a modified sugar moiety comprise the same sugar modification. In certain embodiments, each modified sugar moiety comprises a 2'-methoxyethyl sugar moiety. In certain embodiments, the antisense oligonucleotide Each nucleoside of the nucleotide comprises a modified sugar moiety. In certain embodiments, all of the nucleosides comprise the same sugar modification. In certain embodiments, each modified sugar moiety comprises a 2'-methoxyethyl sugar moiety. In certain embodiments, at least one internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

[0028] In certain embodiments, the antisense oligonucleotide consists of 10 to 25 linked nucleosides. In certain embodiments, the antisense oligonucleotide consists of 12 to 22 linked nucleosides. In certain embodiments, the antisense oligonucleotide consists of 15 to 20 linked nucleosides. In certain embodiments, the antisense oligonucleotide consists of 18 linked nucleosides.

[0029] In certain embodiments, the antisense oligonucleotide is at least 90% complementary to a nucleic acid encoding human SMN2. In certain embodiments, the antisense oligonucleotide is fully complementary to a nucleic acid encoding human SMN2. In certain embodiments, the oligonucleotide has a nucleobase sequence comprising at least 10 contiguous nucleobases of the nucleobase sequence SEQ ID NO: 1. In certain embodiments, the oligonucleotide has a nucleobase sequence comprising at least 15 contiguous nucleobases of the nucleobase sequence SEQ ID NO: 1. In certain embodiments, the oligonucleotide has a nucleobase sequence comprising the nucleobase sequence SEQ ID NO: 1. In certain embodiments, the oligonucleotide has a nucleobase sequence consisting of the nucleobase sequence SEQ ID NO: 1.

[0030] In certain embodiments, the antisense compound comprises a conjugate group or a terminal group.

[0031] In certain embodiments, the antisense compound consists of an antisense oligonucleotide.

[0032] In certain embodiments, the antisense compound is also administered systemically. In certain embodiments, the systemic administration is by intravenous injection or intraperitoneal injection. In certain embodiments, the systemic administration and the administration to the central nervous system are carried out simultaneously. In certain embodiments, the systemic administration and the administration to the central nervous system are carried out at different times.

[0033] In certain embodiments, the present invention provides for systemic administration of antisense compounds, either alone or in conjunction with delivery into the CSF. In certain embodiments, the pharmaceutical composition is administered subcutaneously. In certain embodiments, the pharmaceutical composition is administered intravenously. In certain embodiments, the pharmaceutical composition is administered by intramuscular injection.

[0034] In certain embodiments, the pharmaceutical composition is administered directly to the CSF (e.g., IT and / or is administered both ICV injection and / or infusion) and systemically.

[0035] In certain embodiments, the present invention provides for the administration of at least one antibody to a subject having at least one symptom associated with SMA, the antibody comprising an oligonucleotide consisting of 15 to 20 linked nucleosides and having a nucleobase sequence that is 100% complementary to SEQ ID NO. 7 over its entire length. The present invention provides a method for administering doses of an antisense compound, wherein each nucleoside is a 2'-MOE modified nucleoside; and wherein at least one dose is administered to the CSF. In certain such embodiments, the dose ranges from 0.5 mg / kg to 2 mg / kg. In certain embodiments, at least one dose is administered by bolus injection. In certain such embodiments, the dose is administered by bolus intrathecal injection. In certain such embodiments, at least one second dose is administered. In certain such embodiments, the second dose is administered at least two weeks after the first dose. In certain such embodiments, In certain embodiments, the second dose is administered at least 4 weeks after the first dose. The second dose is administered at least 8 weeks after the first dose. In certain embodiments, the second dose is administered at least 12 weeks after the first dose. In certain embodiments, the second dose is administered at least 16 weeks after the first dose. In certain embodiments, the second dose is administered at least 18 weeks after the first dose. In certain embodiments, the subject receives at least 20 weeks after the first dose. In certain embodiments, the subject is 2 years of age or younger. In certain embodiments, the subject is between 2 and 15 years of age. In certain embodiments, the subject is between 15 and 30 years of age. In certain embodiments, the subject is 30 years of age or older. In certain embodiments, the oligonucleotide reduces or slows the progression of at least one symptom associated with SMA. In certain embodiments, the oligonucleotide is ISIS 396443.

[0036] In certain embodiments, the present invention provides for the administration of at least one antibody to a subject having at least one symptom associated with SMA, the antibody comprising an oligonucleotide consisting of 15 to 20 linked nucleosides and having a nucleobase sequence that is 100% complementary to SEQ ID NO. 7 over its entire length. and administering at least one dose of an antisense compound systemically, wherein each nucleoside is a 2'-MOE modified nucleoside; and In certain such embodiments, at least one dose is administered by bolus injection. In certain embodiments, the dose is administered by bolus subcutaneous injection. In certain embodiments, the dose ranges from 0.5 mg / kg to 50 mg / kg. In certain embodiments, the dose ranges from 1 mg / kg to 10 mg / kg. In certain embodiments, the dose ranges from 1 mg / kg to 5 mg / kg. In certain embodiments, the dose ranges from 0.5 mg / kg to 1 mg / kg. In certain embodiments, at least one second dose is administered. In certain such embodiments, the second dose In certain embodiments, the second dose is administered at least two weeks after the first dose. In certain embodiments, the second dose is administered at least four weeks after the first dose. In certain embodiments, the second dose is administered at least four weeks after the first dose. In certain embodiments, the second dose is administered at least 8 weeks after the first dose. In certain embodiments, the second dose is administered at least 12 weeks after the first dose. In certain embodiments, the second dose is administered at least 16 weeks after the first dose. In certain embodiments, the second dose is administered at least 20 weeks after the first dose. In certain embodiments, the subject is 2 years of age or younger at the time of the first dose. In certain embodiments, the subject is in the range of 2 to 15 years of age. In certain embodiments, the subject is The subject is in the age range of 15 to 30 years. In certain embodiments, the subject is 30 years of age or older. In certain embodiments, the at least one symptom associated with SMA is reduced or its progression is slowed. In certain embodiments, the oligonucleotide is ISIS 396443.

[0037] In certain embodiments, the present invention provides an antisense antibody comprising an oligonucleotide consisting of 15-20 linked nucleosides and having a nucleobase sequence that is 100% complementary to SEQ ID NO. 7 over its entire length for a subject having at least one symptom associated with SMA. administering at least one dose to the CSF and at least one systemic dose of the compound; wherein each nucleoside is a 2'-MOE modified nucleoside. In certain such embodiments, the CSF dose ranges from 0.1 mg / kg to 5 mg / kg. In certain embodiments, the systemic dose ranges from 0.5 mg / kg to 50 mg / kg. In certain such embodiments, at least one CSF dose is administered by bolus injection. At least one CSF dose is administered by bolus intrathecal injection. In certain embodiments, at least one systemic dose is administered by bolus injection. In certain such embodiments, In certain embodiments, the CSF dose and the systemic dose are administered simultaneously. In certain embodiments, the CSF dose and the systemic dose are administered subcutaneously. The doses are administered at different times. In certain embodiments, the subject is 2 years of age or older at the time of the first dose. In certain embodiments, the subject is in the age range of 2 to 15 years. In certain embodiments, the subject is between 15 and 30 years of age. In certain embodiments, the subject is 30 years of age or older. In certain embodiments, the method reduces or slows the progression of at least one symptom associated with SMA. In certain embodiments, the oligonucleotide is ISIS 396443.

[0038] In certain embodiments, the present invention provides a method of administering to a subject having at least one symptom associated with SMA at least one systemic dose of an antisense compound comprising an oligonucleotide consisting of 15 to 20 linked nucleosides and having a nucleobase sequence that is 100% complementary to SEQ ID NO. 7 over its entire length, wherein each nucleoside is a 2'-MOE modified nucleoside; and administering at least one dose of a gene therapy agent. In certain embodiments, the systemic dose ranges from 0.5 mg / kg to 50 mg / kg. In embodiments, at least one systemic dose is administered by bolus injection. In certain embodiments, at least one systemic dose is administered by subcutaneous injection. In certain embodiments, the systemic dose and the gene therapy agent are administered simultaneously. In certain embodiments, the systemic dose and the gene therapy agent are administered at different times. In certain embodiments, the gene therapy agent is administered to the CSF. In certain such embodiments, the gene therapy agent is administered to the CSF. In certain such embodiments, the gene therapy agent is administered by intrathecal injection and / or intravenous infusion. In certain such embodiments, the gene therapy agent is administered by intracerebroventricular injection and / or intravenous infusion. In certain embodiments, the subject is 2 years of age or younger at the time of the first dose. In certain embodiments, the subject is in the age range of 2-15 years. In certain embodiments, the subject is in the age range of 15-30 years. In certain embodiments, the subject is 30 years of age or older. In certain embodiments, the treatment reduces or slows the progression of at least one symptom associated with SMA. In certain embodiments, the oligonucleotide is ISIS 396443.

[0039] In certain embodiments, the present invention provides a method of selecting a subject with at least one symptom associated with SMA and administering an antisense compound according to any of the methods described above. In certain such embodiments, at least one symptom of SMA is evaluated after administration. In certain such embodiments, at least one symptom of SMA is improved. In certain such embodiments, at least one symptom of SMA does not progress or progresses more slowly than in subjects who do not receive the antisense compound.

[0040] In certain embodiments, the invention provides an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2 for use in any of the methods described above. In certain embodiments, the present invention provides antisense compounds comprising the Survival Motor Neuron 1 (SMN1) gene for use in treating a disease or condition associated with SMN1. Such compounds are provided.

[0041] In certain embodiments, the invention provides the use of an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2 in the manufacture of a medicament for use in any of the methods described above. In certain embodiments, the medicament is for treating a disease or condition associated with survival motor neuron 1 (SMN1). This is what we want to achieve. [Brief explanation of the drawings]

[0042] [Figure 1] Figure 1 shows results from the duration of action study discussed in Example 4. In this example, the percentage of SMN2 containing exon 7 (y-axis) was assessed at 0, 2, 4, 6, and 8 weeks (x-axis) after the end of 7 days of treatment. The "0" week sample was taken 1 day after the end of treatment. Control refers to mice treated with saline. There was no difference in percent inclusion between control saline-treated mice at different time points from 0 to 6 months. [Figure 2] Figure 2 shows results from the duration of action study discussed in Example 4. In this example, the percentage of SMN2 containing exon 7 was assessed at 0, 0.5, 1, 2, 5, and 6 months after the end of 7 days of treatment. The "0" month sample was taken 1 day after the end of treatment. CON indicates saline-treated mice. There was no difference in % inclusion between control saline-treated mice at different time points from 0 to 6 months. [Figure 3] Figure 3 shows results from the experiment discussed in Example 6, which measured the effect of embryonic administration of ISIS 396443 on tail extent in the Taiwanese strain of SMA mice. Figure 3A shows the first such experiment, and Figure 3B shows the results from a repeated experiment testing different concentrations of the antisense compound as indicated, including data on normal mice for comparison. [Figure 4] Figure 4 shows the results from the Western blot examined in Example 7. The Y-axis is the percentage of SMN in the various samples that contains exon 7. [Figure 5] Figures 5 and 6 show results from the experiments discussed in Example 7. A number of SMA mice (Taiwanese strain) were evaluated after treatment with either the antisense compounds or a control oligonucleotide. [Figure 6]Figures 5 and 6 show results from the experiments discussed in Example 7. A number of SMA mice (Taiwanese strain) were evaluated after treatment with either the antisense compounds or a control oligonucleotide. [Figure 7] FIG. 7 shows the survival curves from the experiments discussed in Example 7. [Figure 8] FIG. 8 shows the results from an assessment of the number of motor neurons in different regions of the spinal cord after treatment with antisense compounds or control oligonucleotides, as discussed in Example 7. [Figure 9] FIG. 9 shows the results from evaluation of the complete SMN RNA (including exon 7) in animals treated with the antisense as discussed in Example 7. [Figure 10] Figure 10 shows survival curves from the experiment discussed in Example 7, in which animals were either (1) untreated, (2) given a single dose of antisense compound at birth (Day PO), or (3) given a first dose on PO and a second dose on day 21 (P21). [Figure 11] FIG. 11 shows survival curves from the experiment described in Example 7 comparing animals that received a second dose with animals that received only the first dose. [Figure 12] Figure 12 shows the results from the experiment discussed in Example 9. In this experiment, antisense compounds were administered to monkeys by infusion, and compound concentrations were assessed in different tissues 96 hours later. [Figure 13] Figure 13 shows survival curves from the experiment discussed in Example 12. In this experiment, different doses of antisense compound were administered to SMA mice by subcutaneous injection. DETAILED DESCRIPTION OF THE INVENTION

[0043] Detailed Description of the Invention It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to be limiting of the invention as claimed. As used herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" does not necessarily imply otherwise. Unless otherwise expressly stated, "and / or" means "and / or." Furthermore, the terms "including" and "including" The use of other forms such as "contained in" is not limiting. Similarly, terms such as "element" or "component" refer to elements and components that comprise a unit unless otherwise specified. It encompasses both components, and elements and constituents that contain more than one subunit.

[0044] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, publications, treatises, and treatises, are expressly incorporated by reference in their entirety for any purpose.

[0045] I. Definition Unless specific definitions are provided, the terms used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Certain such techniques and procedures can be found in the following references: (e.g., "Carbohydrate Modifications in Antisense Research," Edited by Sangvi and Cook, American Chemical Society, Washington, DC, 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990; and "Antisense Drug Technology, Principles, Strategies, and Applications"). Edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989), which are incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other documents referred to throughout the disclosure herein are incorporated by reference in their entirety.

[0046] Unless otherwise indicated, the following terms have the following meanings: "Nucleoside" means a compound containing a heterocyclic base moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides, modified nucleosides, and nucleosides having mimetic base and / or sugar groups. Nucleosides may be modified with any of a variety of substituents.

[0047] "Sugar moiety" means a naturally occurring sugar, or a modified sugar or sugar substitute.

[0048] "Natural sugar" means a ribofuranose moiety of DNA (2'-H) or RNA (2'-OH).

[0049] A "modified sugar" is a ribofuranose that contains at least one substituent other than the substituents of naturally occurring sugars. It means the ingredients.

[0050] "Sugar substitute" refers to any non-ribofuranose ring that can replace the sugar of a nucleoside. "sugar surrogate" refers to a structure. Examples of sugar surrogates include, but are not limited to, open ring systems, 6-membered rings, and sugars in which oxygen has been replaced with, for example, sulfur or nitrogen. For example, sugar surrogates include, but are not limited to, morpholino and 4'-thio-containing sugars.

[0051] "Nucleobase" refers to the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring or modified. In certain embodiments, a nucleobase may contain any atom or group capable of hydrogen bonding to a nucleobase of another nucleic acid.

[0052] "Nucleotide" means a nucleoside containing a phosphate linking group. When used herein, nucleoside includes nucleotides.

[0053] A "modified nucleoside" is a nucleoside that contains at least one modification compared to a naturally occurring RNA or DNA nucleoside. Such modifications may include modifications to the sugar moiety and / or the nucleus. It may be present in the acid-base.

[0054] "Bicyclic nucleoside" or "BNA" means a nucleoside in which the sugar moiety of the nucleoside contains a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic sugar moiety.

[0055] "4'-2' bicyclic nucleoside" means a bicyclic nucleoside comprising a furanose ring containing a bridge connecting two carbon atoms of the furanose ring, linking the 2' and 4' carbon atoms of the sugar ring.

[0056] "2'-modified" or "2'-substituted" refers to a nucleotide that contains a sugar that contains a substituent at the 2' position other than H or OH. It means leioside.

[0057] "2'-OMe" or "2'-OCH3" or "2'-O-methyl" respectively refer to the -OCH3 at the 2' position of the sugar ring. It refers to a nucleoside containing a sugar containing a group.

[0058] "MOE" or "2'-MOE" or "2'-OCH2CH2OCH3" or "2'-O-methoxyethyl" refers to a nucleoside containing a sugar containing a -OCH2CH2OCH3 group at the 2'-position of the sugar ring, respectively.

[0059] "Oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the nucleosides are modified. In the present specification, the oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

[0060] "Oligonucleoside" means an oligonucleotide that does not contain a phosphorus atom in any of its internucleoside linkages. As used herein, oligonucleotide includes oligonucleosides.

[0061] A "modified oligonucleotide" is an oligonucleotide that contains at least one modified nucleoside and / or By "oligonucleotide" is meant an oligonucleotide containing at least one modified internucleoside linkage.

[0062] "Internucleoside linkage" means the covalent bond between adjacent nucleosides of an oligonucleotide.

[0063] "Naturally occurring internucleoside linkage" means a 3'-5' phosphodiester linkage.

[0064] "Modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0065] "Oligomeric compound" means a compound comprising an oligonucleotide. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, an oligomeric compound further comprises one or more conjugate and / or terminal groups.

[0066] "Antisense compound" means an oligomeric compound, at least a portion of which is at least partially complementary to a target nucleic acid, wherein such hybridization occurs produces at least one antisense activity.

[0067] "Antisense oligonucleotide" means an antisense compound where the oligomeric compound is comprised of an oligonucleotide.

[0068] "Antisense activity" refers to the ability of an antisense compound to hybridize to its target nucleic acid. Antisense activity refers to any detectable and / or measurable effect that can contribute to the progression of a disease. In certain embodiments, such antisense activity is an increase or decrease in the amount of a nucleic acid or protein. In certain embodiments, such antisense activity is a change in the ratio of splice variants of a nucleic acid or protein. In certain embodiments, such antisense activity is a phenotypic change in a cell and / or subject.

[0069] "Detecting" or "measuring" antisense activity can be direct or indirect. For example, in certain embodiments, antisense activity is assessed by detecting and / or measuring the amount of a target nucleic acid or protein or the relative amount of a splice variant of a target nucleic acid or protein. In certain embodiments, antisense activity is detected by observing a phenotypic change in a cell or animal. The terms "detecting" and "measuring," in connection with any activity, reaction, or effect, indicate that a test for detecting or a test for measuring is performed. Such detection and / or measurement may include a value of zero. Thus, even if a test for detecting or a test for measuring results in no activity being found (zero activity), a step of detecting or measuring activity is still performed.

[0070] "Target nucleic acid" refers to any nucleic acid molecule whose expression, amount, or activity can be modulated by an antisense compound.

[0071] "Target mRNA" means a preselected RNA molecule that encodes a protein.

[0072] "Target pre-mRNA" refers to a preselected RNA transcript that has not been fully processed into mRNA. In particular, the pre-mRNA contains one or more introns.

[0073] "Target protein" means a protein encoded by a target nucleic acid.

[0074] "Modulation" refers to a change in function or activity. In certain embodiments, modulation refers to an increase in gene expression. In certain embodiments, modulation refers to a decrease in gene expression.

[0075] "Expression" refers to any of the functions and processes by which a gene's encoded information is converted into structures present and functioning in a cell.

[0076] "Nucleobase sequence" means the order of contiguous nucleobases in a 5' to 3' direction, regardless of any sugar, linkage and / or nucleobase modifications.

[0077] "Contiguous nucleobases" means nucleobases located immediately next to each other in a nucleic acid.

[0078] "Nucleobase complementarity" refers to the non-covalent complementation of two nucleobases through hydrogen bonds. It means the ability to

[0079] "Complementary" means that a first nucleic acid is capable of hybridizing to a second nucleic acid under stringent hybridization conditions. For example, an antisense compound is complementary to a target nucleic acid if the antisense compound is capable of hybridizing to the target nucleic acid under stringent hybridization conditions.

[0080] "Perfectly complementary" means that each nucleobase of a first nucleic acid has a corresponding complementary base with each counterpart of a second nucleic acid. This means that the nucleotides can be added together with the corresponding nucleobases at successive positions.

[0081] "Percent complementarity" of an antisense compound refers to the percentage of nucleobases of the antisense compound that are complementary to an equivalent length of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the antisense oligonucleotide that are complementary to nucleobases at corresponding consecutive positions in the target nucleic acid by the total length of the antisense compound.

[0082] "% identity" means the number of nucleobases in a first nucleic acid that are identical to nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

[0083] "Hybridize" refers to the annealing of complementary nucleic acids resulting in complementarity through the nucleobases.

[0084] A "mismatch" is an inability to pair with a nucleobase at a corresponding position in a second nucleic acid. means a nucleobase of a first nucleic acid.

[0085] "Identical nucleobase sequence" means having the same nucleobase sequence regardless of any chemical modification of the nucleosides.

[0086] "Differentially modified" or "differently modified" refers to nucleosides or internucleoside linkages that have different nucleoside modifications or internucleoside linkages from one another, including no modifications. Thus, for example, an MOE nucleoside and an unmodified DNA nucleoside are also "differently modified" even if the DNA nucleoside is unmodified. Similarly, DNA and RNA are "differentially modified" even though both contain naturally occurring, unmodified nucleosides. Identical nucleosides that contain different nucleobases are not differentially modified unless otherwise specified. For example, a nucleoside containing a 2'-OMe-modified sugar, an adenine nucleobase, a nucleoside containing a 2'-OMe-modified sugar, and a thymine nucleobase are not differentially modified.

[0087] "Identical modifications" means nucleosides and internucleoside linkages that are identical to each other (including unmodified nucleosides and internucleoside linkages). Thus, for example, two An unmodified DNA nucleoside of the formula (I) is one that has the "same modification" as if the DNA nucleoside were unmodified.

[0088] "Type of modification" or a "type" of nucleoside refers to a modification of a nucleoside and includes modified and unmodified nucleosides. Thus, unless otherwise specified, a "nucleoside having a first type of modification" refers to an unmodified nucleoside. It's okay to have it.

[0089] A "distinct region" of an oligonucleotide means a portion of the oligonucleotide in which the nucleosides and internucleoside linkages within a region all contain the same modification; and the nucleosides and / or internucleoside linkages of any adjacent portion all contain at least one Each contains one different modification.

[0090] "Motif" means the pattern of modified and / or unmodified nucleobases, sugars, and / or internucleoside linkages in an oligonucleotide.

[0091] A "fully modified oligonucleotide" means that each nucleobase, each sugar, and / or each internucleoside linkage is modified.

[0092] A "uniformly modified oligonucleotide" means that each nucleobase, each sugar, and / or each internucleoside linkage has the same modification throughout the modified oligonucleotide.

[0093] An "alternating motif" is a motif that combines at least four different regions of modified nucleosides in a pattern (AB) n A mwherein A is a first modification A indicates a nucleoside region with a different modification type; B indicates a nucleoside region with a different modification type. n is 2 to 15; and m is 0 or 1. Thus, in certain embodiments, Thus, the alternating motif includes 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more alternating regions. The region and each B region independently contain 1 to 4 nucleosides.

[0094] "Subject" means a human or non-human animal selected for treatment or therapy. .

[0095] "Subject in need thereof" means a subject identified as being in need of therapy or treatment. In such embodiments, the subject has or will develop SMA. It has one or more indications.

[0096] "Administering" means providing a pharmaceutical agent or composition to a subject, and this concept includes, but is not limited to, administration by a medical professional and self-administration.

[0097] "Parenteral administration" means administration via injection or infusion.

[0098] Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.

[0099] "Systemic administration" means administration to an area other than the intended site of activity. Examples of systemic administration are subcutaneous administration, intravenous administration, and intraperitoneal administration.

[0100] "Subcutaneous administration" means administration just below the skin.

[0101] "Intravenous administration" means administration into a vein.

[0102] "Cerebrospinal fluid" or "CSF" means the fluid that fills the spaces around the brain and spinal cord.

[0103] "Administration into the cerebrospinal fluid" means any administration that delivers a substance directly into the CSF. do.

[0104] "Intraventricular" or "ICV" means administration into the ventricular system of the brain.

[0105] "Intrathecal" or "IT" refers to administration into the CSF beneath the arachnoid membrane that covers the brain and spinal cord. IT injections are made through the outer membrane of the spinal cord into the subarachnoid space, injecting a pharmaceutical agent into the sheath that surrounds the spinal cord.

[0106] The "induction phase" begins administration and allows the steady-state concentration of the active pharmaceutical agent to reach the target tissue. For example, the induction phase refers to the administration phase when a steady-state concentration of an antisense This is the administration phase when the oligonucleotide is achieved in the liver.

[0107] "Maintenance phase" refers to the administration phase after steady-state drug concentrations in the target tissue are achieved.

[0108] "Duration" refers to the period during which activity or reduction continues. For example, the induction phase The period is the period during which the induction dose is administered.

[0109] "Maintenance dose" means a dose administered in a single administration during the maintenance phase. As used herein, "induction dose" means a dose administered in a single administration during the induction phase.

[0110] "Co-administration" means administering two or more pharmaceutical agents to a subject. The two or more pharmaceutical agents may be in a single pharmaceutical composition or in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered via the same or separate routes of administration. Co-administration includes simultaneous or sequential administration.

[0111] "Treatment" refers to a method of treating a disease. In certain embodiments, treatment includes, but is not limited to, surgical treatment, clinical treatment, and physical intervention, such as assisted breathing, feeding tubes, and physical therapy aimed at increasing strength.

[0112] "Treatment" refers to the application of one or more specific procedures used to cure or ameliorate a disease. In certain embodiments, the specific procedure is the administration of one or more pharmaceutical agents.

[0113] "Amelioration" means a decrease in the severity of at least one indicator of a symptom or disease. In certain embodiments, amelioration includes a decrease in the progression of one or more indicators of a symptom or disease. The severity of indicators can be determined by subjective or objective measures known to those skilled in the art.

[0114] "Inhibiting the onset of" means inhibiting the onset of a symptom or disease in a subject at risk of developing a disease or condition. In certain embodiments, a subject at risk of developing a disease or condition is given a treatment similar to the treatment given to a subject who already has the disease or condition.

[0115] "Delaying the onset of" means delaying the onset of a symptom or disease in a subject at risk of developing the disease or condition.

[0116] "Slowing the progression of" means that the severity of at least one symptom of a disease or condition worsens. This means moving more slowly.

[0117] "Exon 7 amino acids" refers to the portion of the SMN protein that corresponds to exon 7 of the SMN RNA. This means that the amino acids of exon 7 are present in the SMN protein expressed from SMN RNA in which exon 7 has not been removed during splicing.

[0118] "SMN protein" means the normal, full-length survival motor neuron protein. SMN may be expressed from either the SMN1 gene or the SMN2 gene, so long as exon 7 is present in the mature mRNA and the amino acids of exon 7 are present in the SMN protein.

[0119] "Dose" means a defined amount of a pharmaceutical agent provided in a single administration or over a defined period of time. In certain embodiments, a dose may be administered in the form of two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous, intrathecal, or ICV administration is preferred, the desired dose requires a dose that cannot be easily provided by a single injection. In such embodiments, two or more injections may be used. In a continuous infusion setting, the dose can be expressed as the amount of pharmaceutical agent delivered per unit time.

[0120] "Dosage unit" refers to the form in which a pharmaceutical agent is provided. In certain embodiments, the dosage unit is a vial containing lyophilized oligonucleotide. The dosage unit is a vial containing the reconstituted oligonucleotide.

[0121] "Therapeutically effective amount" means an amount of a pharmaceutical agent that confers a therapeutic benefit on an animal.

[0122] "Pharmaceutical composition" means a mixture of substances suitable for administration to an individual, including a pharmaceutical agent. For example, a pharmaceutical composition may comprise a modified oligonucleotide and a sterile aqueous solution.

[0123] "Acceptable safety profile" means a pattern of side effects that is within clinically acceptable limits.

[0124] "Side effects" refers to physiological responses other than the desired effects that can be attributed to treatment.

[0125] 1. Specific modified oligonucleotides In certain embodiments, the present invention provides antisense oligonucleotides that contain one or more modifications compared to naturally occurring oligomers, e.g., DNA or RNA oligonucleotides. The present invention provides methods and compositions related to modified antisense oligonucleotides. Such modified antisense oligonucleotides can have one or more desirable properties. Certain such modifications may be used to enhance the affinity of an antisense oligonucleotide for its target nucleic acid, for example, by increasing its resistance to one or more nucleases. and / or alter the pharmacokinetics or tissue distribution of the oligonucleotide, thereby altering the antisense activity of the antisense oligonucleotide. In certain embodiments, such modified antisense oligonucleotides contain one or more modified nucleotides. nucleosides and / or one or more modified nucleoside linkages and / or one or more conjugate groups.

[0126] a. Certain modified nucleosides In certain embodiments, the antisense oligonucleotide comprises one or more modified nucleotides. Modified nucleosides include modified nucleosides. Such modified nucleosides may include modified sugars and / or modified nucleobases. In certain embodiments, the incorporation of such modified nucleosides into oligonucleotides results in increased stability, including (but not limited to) increased affinity for the target nucleic acid and / or increased resistance to nuclease degradation, and / or improved toxicity and / or improved incorporation properties of the modified oligonucleotide.

[0127] i. Specific nucleobases The base moiety of naturally occurring nucleosides is a heterocyclic base, typically a purine or pyrimidine. The purine nucleobases are adenine (A) and guanine (G), and the pyrimidine nucleobases are thymine (T), cytosine (C), and uracil (U). In addition to "unmodified" or "natural" nucleobases, numerous modified nucleobases or nucleobase mimetics known to those of skill in the art are amenable for incorporation into preferred compounds herein. In certain embodiments, the modified nucleobase is a nucleobase that is structurally quite similar to the parent nucleobase, such as, for example, a 7-deazapurine, 5-methylcytosine, or G-clamp. In certain embodiments, the nucleobase mimics include more complex structures, such as, for example, tricyclic phenoxazine nucleobase mimics. Methods for preparing the modified nucleobases described above are also described. and known to those skilled in the art.

[0128] ii. Certain modified sugars and sugar substitutes The antisense oligonucleotides of the present invention may optionally contain one or more nucleosides in which the sugar moiety is modified compared to the natural sugar. Oligonucleotides containing such sugar-modified nucleosides may have enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. Such modifications include the addition of substituents, or the addition of nucleotides to form bicyclic nucleic acids (BNAs). Bridging ring atoms that are not geminal (two atoms of the same kind bonded to one atom), S, N(R), or C(R1)(R)2 (R = H, C1-C 12 Ribosyl ring (by alkyl or protecting group) Oxygen atom substitutions and combinations thereof, such as, for example, 2'-F-5'-methyl substituted nucleosides (see PCT International Application No. 2008 / 101157, and other disclosed 5',2'-bis substituted nucleosides). (Released on August 21, 2008) or a ribose having S by a substituent at the 2'-position. Substitution of ring oxygen atoms (see published U.S. patent application US2005-0130923, published June 16, 2005) ), or selective 5'-substitution of BNA (see PCT International Application WO 2007 / 134181, November 22, 2007 , wherein LNA is substituted, for example, with a 5'-methyl or 5'-vinyl group. However, the present invention is not limited to these.

[0129] Examples of nucleosides having modified sugar moieties include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ), you can also choose from the respective R m and R n is independent and H or substituted or unsubstituted C1-C 10 It is alkyl.

[0130] Examples of bicyclic nucleic acids (BNAs) include, but are not limited to, those having a ribosyl group between the 4' and 2' ring atoms. In certain embodiments, the nucleosides provided herein include nucleosides containing a bridge. The antisense compounds include one or more BNA nucleosides, where the bridge has the following formula: 4'-β-D-(CH2)-O-2' (β-D-LNA); 4'-(CH2)-S-2'; 4'-α-L-(CH2)-O-2' (α-L-LNA); 4'-(CH2)2-O-2' (ENA); 4'-C(CH3)2-O-2' (see PCT / US2008 / 068922); 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)--O-2' (see PCT / US2008 / 068922, published July 15, 2008). 4'-CH2-N(OCH3)-2' (see PCT / US2008 / 064591); 4'-CH2-ON(CH3)-2' (see published US patent application US2004-0171570 published September 2, 2004) 4'-CH2-N(R)-O-2' (see U.S. Patent 7,427,672 issued September 23, 2008); 4'-CH2-C(CH3)-2' and 4'-CH2-C-(=CH2)-2' (see PCT / US2008 / 066154); and R is independently H, C1-C 12 alkyl, or a protecting group.

[0131] In certain embodiments, the present invention includes modified nucleosides that contain a modified sugar moiety that is not a bicyclic sugar moiety. Certain such modified nucleosides are known. In certain embodiments, the sugar ring of the nucleoside may be modified at any position. Examples of sugar modifications useful in the present invention include compounds containing a sugar substituent selected from the following: OH, F, O-alkyl, S-alkyl, N-alkyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are C1-C2 groups that may be substituted or unsubstituted. 10 Alkyl or is C2~C 10 Examples include, but are not limited to, alkenyl and alkynyl. In certain such embodiments, such a substituent is at the 2' position of the sugar.

[0132] In certain embodiments, the modified nucleoside comprises a substituent at the 2' position of the sugar. In the present invention, such substituents are the following: halide (including but not limited to F) (not present), allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ), or O-CH2-C(=O)-N(R m )(R n ), where R m and R n each independently represents H or a substituted Substituted or unsubstituted C1-C 10 It is alkyl.

[0133] In certain embodiments, modified nucleosides suitable for use in the present invention are: 2-methoxyethoxy, 2'-O-methyl (2'-O-CH3), 2'-fluoro (2'-F).

[0134] In certain embodiments, the modified nucleoside has at the 2'-position: O[(CH) n O] m CH3 , O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, OCH2C(=O)N(H)CH3, and O(CH2 ) n ON[(CH2) n CH3]2, where n and m are from 1 to about 10. Other 2'-sugar substituents include: C1 to C 10Alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclo Alkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, and groups for improving pharmacokinetic properties. These include groups for improving the pharmacodynamic properties of the oligomeric compound, or groups for improving the pharmacodynamic properties of the oligomeric compound, and other substituents with similar properties.

[0135] In certain embodiments, the modified nucleoside comprises a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitutions are described as improving binding affinity compared to unmodified nucleosides and other modified nucleosides, such as 2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides that target gene expression also have a promising future for in vivo applications. It has been shown to be an antisense inhibitor (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides or Nucleotides, 1997, 16, 917-926).

[0136] In certain embodiments, the 2'-sugar substituent is in either the arabino (up) or ribo (down) position. In certain such embodiments, the 2'-arabino modification is 2'-F arabino (FANA Other positions on the sugar, especially the 3' position of the sugar of the 3'-terminal nucleoside, or the 2'-5'-linked nucleoside, Similar modifications can also be made to the 5' position of oligonucleotides and the 5' terminal nucleotide.

[0137] In certain embodiments, nucleosides suitable for use in the present invention have sugar substitutes, such as cyclobutyl, in place of the ribofuranosyl sugar. Representative US patents that teach the preparation of such modified sugar structures include, but are not limited to, US: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is incorporated by reference herein in its entirety, but is not limited to these.

[0138] In certain embodiments, the present invention provides nucleosides that include modifications at the 2'-position of the sugar. In certain embodiments, the present invention provides nucleosides that include modifications at the 5'-position of the sugar. In some embodiments, the present invention provides nucleosides containing modifications at the 2'- and 5'-positions of the sugar. In certain embodiments, the modified nucleosides may be useful for incorporation into oligonucleotides. In certain embodiments, the modified nucleosides are incorporated into oligonucleotides at the 5'-terminus of the oligonucleotide.

[0139] b. Specific internucleoside linkages The antisense oligonucleotides of the present invention may optionally contain one or more modified nucleic acids. These two main classes of linking groups are Typical phosphorus-containing bonds include phosphodiester (P=O), phosphodiester (P=O), Representative non-phosphorus-containing linking groups include, but are not limited to, phototriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylenemethylamino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Oligonucleotides with non-phosphorus linking groups can be prepared by the addition of a methyl group to an oligonucleotide. They are called nucleosides. Compared to natural phosphodiester bonds, modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides. In certain embodiments, linkages with chiral atoms can be prepared as racemic mixtures, as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

[0140] The antisense oligonucleotides described herein contain one or more antisense Some antisense compounds contain symmetric centers, resulting in enantiomers, diastereomers, and other stereoisomeric structures that can be specified in terms of absolute stereochemistry as (R) or (S), such as sugar anomers, or (D) or (L), such as amino acids. The antisense compounds provided herein include all such possible isomers, as well as their racemic and optionally pure forms.

[0141] In certain embodiments, the antisense oligonucleotide comprises at least one modified nucleotide. It has a leoside internucleoside bond. In certain embodiments, the antisense oligonucleotide has at least two modified internucleoside bonds. In certain embodiments, the antisense o ligonucleotide has at least three modified internucleoside bonds. In certain embodiments the antisense oligonucleotide has at least ten modified internucleoside bonds. In certain embodiments, each internucleoside bond of the antisense oligonucleotide is a modified internucleoside bond. In certain embodiments, such modified internucleoside bonds are phosphorothioate bonds.

[0142] c. Length In certain embodiments, the present invention provides antisense oligonucleotides of any length within various ranges. In certain embodiments, the present invention includes or consists of an antisense compound or antisense oligonucleotide comprising an X-Y linked nucleoside, where X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X < Y. For example, in certain embodiments, the present invention is: 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23 ​​、8~24、8~25、8~26、8~27、8~28、8~29、8~30、9~10、9~11、9~12、9~13、9~14、9~15、9~16、9~17、9~18、9~19、9~20、9~21、9~22、9~23、9~24、9~25、9~26、9~27、9~28、9~29、9~30、10~11、10~12、10~13、10~14、10~15、10~16、10~17、10~18、10~19、10~20、10~21、10~22、10~23、10~24、10~25、10~26、10~27、10~28、10~29、10~30、11~12、11~13、11~14、11~15、11~16、11~17、11~18、11~19、11~20、11~21、11~22、11~23、11~24、11~25、11~26、11~27、11~28、11~29、11~30、12~13、12~14、12~15、12~16、12~17、12~18、12 ~19、12~20、12~21、12~22、12~23、12~24、12~25、12~26、12~27、12~28、12~29、12~30、13~14、13~15、13~16、13~17、13~18、13~19、13~20、13~21、13~22、13~23、13~24、13~25、13~26、13~27、13~28、13~29、13~30、14~15、14~16、14~17、14~18、14~19、14~20、14~21、14~22、14~23、14~24、14~25、14~26、14~27、14~28、14~29、14~30、15~16、15~17、15~18、15~19、15~20、15~21、15~22、15~23、15~24、15~25、15~26、15~27、15~28、15~29、15~30、16~17、16~18、16~19、16~20、16~21、16~22、16~23、16~24、16~25、16~26、16~27、16~28、16~29、16~30、17~18、17~19、17~20、17~21、17~22、17~23、17~24、17~25、17~26、17~27、17~28、17~29、17~30、18~19、18~20、18~21、18~22、18~23、18~24、18~25、18~26、18~27、18~28、18~29、18~30、19~20、19~21、19~22、19~23、19~24、19~25、19~26、19~29、19~28、19~29、19~30、20~21、20~22、20~23、20~24、20~25、20~26、20~27、20~28、20~29、20~30、21~22、21~23、21~24、21~25、21~26、21~27、21~28、21~29、21~30、22~23、22~24、22~25、22~26、22~27、22~28、22~29、22~30、23~24、23~25、23~26、23~27、23~28、23~29、23~30、24~25、24~26、24~27、24~28、24~29、24~30、25~26、25~27、25~28、25~29、25~30、26~27、26~28、26~29、26~30、27~28、27~29、27~30、28~29、28~30、or antisense compounds or antisense oligonucleotides comprising or consisting of 29 to 30 linked nucleosides.

[0143] In certain embodiments, the antisense compounds or antisense oligonucleotides of the present invention are 15 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the present invention are 16 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the present invention are 17 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the present invention are 18 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the present invention are 19 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the present invention are 20 nucleosides in length.

[0144] d. Specific oligonucleotide motifs In certain embodiments, antisense oligonucleotides have chemically modified subunits arranged in a specific direction along their length. In certain embodiments, antisense oligonucleotides of the present invention are fully modified. In certain embodiments, antisense oligonucleotides of the present invention are homogeneously modified. In certain embodiments, antisense oligonucleotides of the present invention are homogeneously modified, and each nucleoside contains a 2'-MOE sugar moiety. In certain embodiments, antisense oligonucleotides of the present invention are homogeneously modified. The nucleosides are uniformly modified, and each nucleoside contains a 2'-OMe sugar moiety. In certain embodiments, the antisense oligonucleotides of the present invention are uniformly modified, and each nucleoside contains a morpholino sugar moiety.

[0145] In certain embodiments, the oligonucleotides of the invention comprise alternating motifs. In certain such embodiments, the alternating modifications include 2'-MOE, 2'-F, bicyclic sugar-modified nucleosides, and the like. and DNA (unmodified 2'-deoxy). In certain such embodiments, each alternating region comprises a single nucleoside.

[0146] In certain embodiments, oligonucleotides of the invention comprise one or more blocks of a first type of nucleoside and one or more blocks of a second type of nucleoside.

[0147] In certain embodiments, one or more alternating regions in an alternating motif include For example, oligomeric compounds of the invention may contain one or more regions of any of the following nucleoside motifs: stomach: Nu1Nu1Nu2Nu2Nu1Nu1; Nu1Nu2Nu2Nu1Nu2Nu2; Nu1Nu1Nu2Nu1Nu1Nu2; Nu1Nu2Nu2Nu1Nu2Nu1Nu1Nu2Nu2; Nu1Nu2Nu1Nu2Nu1Nu1; Nu1Nu1Nu2Nu1Nu2Nu1Nu2; Nu1Nu2Nu1Nu2Nu1Nu1; Nu1Nu2Nu2Nu1Nu1Nu2Nu2Nu1Nu2Nu1Nu2Nu1Nu1; Nu2Nu1Nu2Nu2Nu1Nu1Nu2Nu2Nu1Nu2Nu1Nu2Nu1Nu1; or Nu1Nu2Nu1Nu2Nu2Nu1Nu1Nu2Nu2Nu1Nu2Nu1Nu2Nu1Nu1; wherein Nu1 is a first type of nucleoside and Nu2 is a second type of nucleoside. In certain embodiments, one of Nu1 and Nu2 is a 2'-MOE nucleoside and the other of Nu1 and Nu2 is: a 2'-OMe modified nucleoside, BNA, and unmodified DNA or RNA. nucleosides.

[0148] 2. Oligomeric compounds In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, the oligomeric compounds consist solely of oligonucleotides. In certain embodiments, the oligomeric compounds consist of oligonucleotides and one or more conjugates and / or terminal groups. Such conjugates and / or terminal groups can be added to oligonucleotides having any of the chemical motifs described above. Thus, for example, oligomeric compounds including oligonucleotides having regions of alternating nucleosides may include terminal groups.

[0149] a. Specific Complex Groups In certain embodiments, the oligonucleotides of the invention comprise one or more conjugate groups. Generally, the conjugate group modifies one or more properties of the oligomeric compound to which it is attached, including, but not limited to, pharmacodynamics, pharmacokinetics, stability, binding, adsorption, cellular distribution, cellular uptake, charge, and clearance. Groups are commonly used in chemistry and are attached to parent compounds, such as oligomeric compounds, including oligonucleotides, directly or via selective conjugate-binding moieties or groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. Specific conjugate groups have been previously described, for example, cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654;Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0150] In certain embodiments, the conjugate group comprises an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbutan, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folic acid, benzothiadiazole, chlorothiazide, diazepine, indomethacin, barbiturates, cephalosporins, sulfa drugs, antidiabetic agents, antibacterial agents, or antibiotic agents. Oligonucleotide-drug conjugates and their preparations are described in US Pat. Described in patent application 09 / 334,130.

[0151] Representative US patents describing the preparation of oligonucleotide conjugates include US: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,41 4,077;5,486,603;5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830 ;5,112,963;5,214,136;5,082,830;5,112,963;5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241;5,391,723;5,416,203;5,451,46 3; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941.

[0152] The conjugate groups can be attached to either or both the termini (terminal conjugate groups) and / or any internal portion of the oligonucleotide.

[0153] b. Terminal group In certain embodiments, the oligomeric compound comprises a terminal group at one or both ends. In certain embodiments, the terminal group may comprise any of the conjugate groups described below. In certain embodiments, the terminal group may comprise an additional nucleoside and / or an inverted abasic nucleoside. In certain embodiments, the terminal group is a stabilizing group.

[0154] In certain embodiments, oligomeric compounds contain one or more terminal stabilizing groups that enhance properties such as, for example, nuclease resistance. Stabilizing groups include cap structures. The term "cap structure" or "terminal cap moiety," as used herein, refers to a chemical modification attached to one or both termini of an oligomeric compound. Certain such terminal modifications can protect oligomeric compounds having terminal nucleic acid moieties from exonuclease degradation and aid in intracellular delivery and / or localization. The cap may be present at the 5'-end (5'-cap) or the 3'-end (3'-cap), or may be present at both ends. (For further details, see Wincott et al., International PCT Publication No. WO 97 / 26270; Beaucage and Tyer, 1993, Tetrahedron 49, 1925; U.S. Patent Application Publication No. US 2005 / 0020525; and WO 03 / 004602).

[0155] In certain embodiments, one or more additional nucleosides are present in the oligomeric compound. Such additional terminal nucleosides may be added to one or both ends of an oligonucleotide, and such additional terminal nucleosides are referred to herein as terminal nucleosides. A suitable terminal nucleoside is a terminal (3' and / or 5') overhang. In the configuration of a double-stranded antisense compound, such a terminal nucleoside may or may not be complementary to the target nucleic acid. In certain embodiments, the terminal group is a non-nucleoside terminal group. Such a non-terminal group may be any terminal group other than a nucleoside.

[0156] c. Oligomeric compound motif In a particular embodiment, the oligomeric compounds of the present invention comprise the motif: T1-(Nu1) n1-(Nu2) n2 -(Nu1) n3 -(Nu2) n4 -(Nu1) n5 -T2, Contains, where: Nu1 is the first type of nucleoside; Nu2 is the first type of nucleoside; each of n1 and n5 independently represents 0 to 3; The sum of n2+n4 is in the range of 10 to 25; n3 is 0 to 5; and Each of T1 and T2 is independently H, a hydroxyl protecting group, a selective binding complex group or a capping group.

[0157] In certain such embodiments, the sum of n2+n4 is 13 or 14; n1 is 2; n3 is 2 or 3; and n5 is 2. In certain such embodiments, the oligomeric compounds of the invention comprise a motif selected from Table A.

[0158] [Table 1]

[0159] Table A is intended to illustrate, but not limit, the present invention. The oligomeric compounds shown in Table A each contain 20 nucleosides. Oligomeric compounds containing more or fewer nucleosides may contain one or more of n1 through n5. Nu1 and Nu2 can be easily prepared by selecting different numbers of nucleosides for each. In certain embodiments, Nu1 and Nu2 are: 2'-MOE, 2'-OMe, DNA, and bicyclic nucleosides, respectively. The compound is selected from the group consisting of cyclohexyl methyl esters, ...

[0160] 3. Antisense In certain embodiments, the oligomeric compounds of the present invention are antisense compounds. Thus, in such embodiments, the oligomeric compounds hybridize to a target nucleic acid, resulting in antisense activity.

[0161] a. Hybridization In certain embodiments, the present invention provides methods for assaying antibodies under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, and assays where the assay is an in vitro assay. Under conditions as described in (a), antisense compounds are provided that specifically hybridize to target nucleic acids when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compounds to non-target nucleic acid sequences.

[0162] Thus, "stringent hybridization conditions" or "stringent conditions" refer to conditions under which an antisense compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent. "Stringent conditions" under which an antisense oligonucleotide will hybridize to a target sequence are dependent on and will vary under different conditions, and are determined by the nature and composition of the antisense oligonucleotide and the assay in which the antisense oligonucleotide is studied.

[0163] Nucleotide incorporation, affinity, and modification are more mismatched compared to unmodified compounds. Matches are equally permissive, and a particular nucleic acid sequence is more permissive than other nucleic acid sequences. It is understood in the art that the hydroxyl group may be better tolerated than the hydroxyl group. Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between an antisense oligonucleotide and a target nucleic acid, for example, by determining the melting temperature (Tm). Tm or ΔTm can be calculated by techniques well known to those skilled in the art. For example, by the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443), those skilled in the art can increase the melting temperature of an RNA:DNA duplex by Nucleotide modifications can be evaluated for their ability to inhibit the synthesis of nucleotides.

[0164] b. Pre-mRNA processing In certain embodiments, the antisense compounds provided herein are complementary to a pre-mRNA. In certain embodiments, such antisense compounds alter the splicing of the pre-mRNA. In certain such embodiments, the antisense compounds are complementary to the target pre-mRNA. The ratio of one variant of the corresponding mature mRNA to another variant of the mature mRNA is changed. In certain such embodiments, the ratio of one variant of a protein expressed from a target pre-mRNA to another variant of the protein is altered. Specific oligomeric compounds and nucleobase sequences that can be used to alter pre-mRNA splicing are described, for example, in the following publications: US 6,210,892; US 5,627,274; US 5,665,593; US 5,916,808; US 5,976,879; US 2006 / 0172962; US 2007 / 002390; US 2005 / 0074801; US ​​2007 / 0105807; US 2005 / 0054836; WO 2007 / 090073; WO 2007 / 047913; Hua et al., PLoS Biol 5(4):e73; Vickers et al., J. Immunol. 2006 Mar 15, 176(6):3652-61; and Hua et al., American Journal of Human Genetics (April 2008) 82, 1-15), each of which can be In certain embodiments, antisense sequences that alter splicing have been modified according to the motifs of the present invention.

[0165] Antisense is an effective method for modifying the expression of one or more specific gene products. Antisense compounds are useful tools and are uniquely useful in numerous therapeutic, diagnostic, and research applications. Provided herein are antisense compounds useful for modifying gene expression through antisense mechanisms, including target-specific antisense mechanisms. In one aspect, the antisense compounds provided herein modify the splicing of target genes. Such modulation includes promoting or inhibiting exon inclusion. The antisense compounds can enhance or inhibit cis-splicing control elements present in pre-mRNA molecules, including exon splicing enhancers, exon splicing silencers, intron splicing enhancers, and intron splicing silencers. Further provided herein are antisense compounds that target cis-splicing. Disruption of regulatory elements is thought to alter splicing site selection, which can result in changes in the composition of splice products.

[0166] Eukaryotic pre-mRNA processing is a complex process that requires multiple signals and protein factors to achieve proper mRNA splicing. Exon definition by the genome requires more than the canonical splicing signals that define intron-exon boundaries. One such additional signal is a cis-acting Sex-regulated enhancer and silencer sequences are provided by splicing donor sequences. Exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs) and intronic splicing silencers, which suppress or enhance the use of splicing sites or splicing acceptor sites, Several splicing sites (ISSs) have been identified, depending on their site and mode of action (Yeo et al. 2004, Proc. Natl. Acad. Sci. USA 101(44):15700-15705). The binding of specific proteins (trans factors) to these regulatory sequences affects the splicing process, either promoting or suppressing the use of specific splicing sites, and modifying the ratio of spliced ​​products (Scamborova et al. 2004, Mol. Cell. Biol. 24(5):1855-1869; Hovhannisyan and Carstens 2005, Mol. Cell. Biol. 25(1):250-263; Minovitsky et al. 2005, Nucleic Acids Res. 33(2):714-724).

[0167] 4. Pharmaceutical Compositions In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compounds. In certain embodiments, such pharmaceutical compositions comprise a sterile saline solution and one or more antisense compounds. In certain embodiments, such pharmaceutical compositions consist of a sterile saline solution and one or more antisense compounds.

[0168] In certain embodiments, the antisense compounds may be mixed with pharmaceutically acceptable active and / or inactive substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions depend on a number of criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0169] In certain embodiments, antisense compounds are prepared by combining such oligomeric compounds with suitable The antisense compound can be used in pharmaceutical compositions by combining with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in parenterally delivered compositions. Thus, in certain embodiments, pharmaceutical compositions comprising an antisense compound and a pharmaceutically acceptable diluent are used in the methods described herein. In certain embodiments, the pharmaceutically acceptable diluent is PBS.

[0170] Pharmaceutical compositions containing antisense compounds include any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions containing antisense compounds include one or more oligonucleotides that, upon administration to an animal, including a human, can (directly or indirectly) provide a biologically active metabolite or residue thereof. Thus, for example, the disclosure also relates to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0171] Prodrugs include oligomeric compounds that contain a nucleotide at one or both termini that is cleaved by endogenous nucleases in the body to form the active antisense oligomeric compound. Additional nucleosides may also be included.

[0172] Lipid-based vectors have been used in nucleic acid therapy in a variety of ways, e.g. In one method, the nucleic acid is incorporated into preformed liposomes or lipoplexes consisting of a mixture of cationic and neutral lipids. DNA complexes containing cationic or poly-cationic lipids are formed in the absence of neutral lipids.

[0173] Certain preparations are described in Akinc et al., Nature Biotechnology 26, 561-569 (01 May 2008 ), which is incorporated herein by reference in its entirety.

[0174] 5. Administration to Subjects In certain embodiments, a pharmaceutical composition comprising one or more antisense compounds is administered to a subject. In certain embodiments, such a pharmaceutical composition is administered by injection. In certain embodiments, such a pharmaceutical composition is administered by infusion.

[0175] In certain embodiments, the pharmaceutical composition is administered into the CSF by injection or infusion. In certain such embodiments, the pharmaceutical composition is administered by direct injection or infusion into the spinal cord. In certain embodiments, the pharmaceutical composition is administered by injection or infusion into the brain. In certain embodiments, the pharmaceutical composition is administered intrathecally rather than into the spinal cord tissue itself. Without being limited by theory, in certain embodiments, the antisense compound is released into the surrounding CSF and penetrates into the spinal cord parenchyma. An additional advantage of intrathecal delivery is that the intrathecal route is similar to lumbar puncture administration (i.e., spinal tap), which is already commonly used in humans.

[0176] In certain embodiments, the pharmaceutical composition is administered by intracerebroventricular (ICV) injection or infusion. Intraventricular administration of a pharmaceutical composition containing one or more antisense compounds, or Delivery can be into one or more of the ventricles, which are either in the cerebrospinal fluid CSF is a clear fluid that fills the ventricles, resides in the subarachnoid space, and surrounds the brain and spinal cord. CSF is produced by the choroid plexus and transports blood by the brain into the ventricles. The choroid plexus is created through the flow or transport of tissue fluid. The choroid plexus is located at the base of the lateral ventricles and the third and These structures line the roof of the fourth ventricle. Specific studies have shown that these structures can produce 400-600 ccs of fluid per day, which is consistent with filling the central nervous system cavity four times a day. In adults, this volume of fluid is calculated to be 125-150 ml (4-5 oz). CSF is continuously produced, circulated, and absorbed. Specific studies have shown that the It has been shown that approximately 430-450 ml (almost 2 cups) of CSF can be produced daily. From a quantitative calculation, production is approximately 0.35 ml per minute in adults and 0.5 ml per minute in human infants. It is estimated that the CSF reaches 0.15 ml per minute. The choroid plexus of the lateral ventricles produces the majority of CSF. It flows through the foramen of Monro into the third ventricle, where it is enriched with products from the third ventricle, and continues down through the aqueduct of Sylvius into the fourth ventricle, which adds more CSF. The fluid then travels through the foramina of Magendie and the foramina of Luschka into the subarachnoid space. The fluid then circulates throughout the base of the brain, descends around the spinal cord, and ascends to the cerebral hemispheres. CSF enters the bloodstream via the arachnoid villi and intracranial venous sinuses.

[0177] In certain embodiments, such pharmaceutical compositions are administered systemically, subcutaneously, intravenously, or by intramuscular injection.

[0178] In certain embodiments, the pharmaceutical composition is administered directly to the CSF (e.g., IT and / or is administered systemically (ICV injection and / or infusion).

[0179] In certain embodiments, systemically administered antisense compounds enter neurons, particularly those that cross the blood-brain barrier. In young subjects (e.g., subjects in utero and / or newborn subjects), the blood-brain barrier is not fully formed and can penetrate. Systemically administered antisense compounds are useful in subjects with intact blood-brain barriers. Even in the body, antisense compounds can be taken up by nerve cells. For example, antisense compounds can be taken up by neurons at or near neuromuscular junctions (retrograde uptake). In certain embodiments, such retrograde uptake results in antisense activity within neurons, including (but not limited to) motor neurons, and antisense activity within neurons provides therapeutic effects.

[0180] In certain embodiments, systemic administration provides a therapeutic effect due to antisense activity occurring within cells and / or tissues other than neurons. While evidence suggests that functional SMN within neurons is required for normal neuronal function, other The effects of reduced functional SMN in cells and tissues have not been fully characterized. In certain embodiments, antisense activity in non-neuronal cells is This results in restoration of SMN function in the cells, which in turn produces a therapeutic effect.

[0181] In certain embodiments, improved SMN function in non-neuronal cells provides improved neuronal cell function, regardless of whether SMN function within neurons is improved. For example, in certain embodiments, systemic administration of a pharmaceutical composition of the present invention results in antisense activity in muscle cells. Such antisense activity in muscle cells can provide benefits to motor neurons associated with muscle cells or neurons in general. In such embodiments, muscle cells with restored SMN function may be characterized by improved neuronal viability. and / or can provide factors that improve function. In certain embodiments, such antisense activity is independent of the benefit from antisense activity resulting from antisense compounds within neurons. In certain embodiments, systemic administration of pharmaceutical compositions of the present invention can be used to target other non-neuronal cells, including cells not immediately associated with neurons. In non-neuronal cells, such antisense activity occurs. Transence activity can improve neuronal function. For example, Antisense activity in cells (e.g., liver cells) can produce factors in those cells that improve neuronal function. Note: The term "antisense activity" includes direct and indirect activity, so even if the antisense compound does not enter the neuron, a benefit to neuronal function is still "antisense activity."

[0182] In certain embodiments, systemic administration of the pharmaceutical composition produces a therapeutic effect independent of direct or indirect antisense activity within neurons. Typically, in the setting of SMA, Neuronal function is reduced, resulting in significant symptoms. Additional symptoms may result from reduced SMN activity in other cells. Certain such symptoms may be due to reduced SMN activity in other cells. The relative severity of symptoms resulting from neuronal function may mask this. In certain embodiments, systemic administration results in restored or improved SMN function in non-neuronal cells. In certain such embodiments, such restored or improved SMN function in non-neuronal cells has a therapeutic effect. For example, in certain cases, subjects with SMA have reduced growth. Such growth The decrease in growth may not result in a decrease in neuronal cell function. In fact, the decrease in growth may cause a decrease in cell function in other organs, such as the pituitary gland, and / or may result in SMN deficiency throughout the body. In such embodiments, the whole body Administration of steroids may result in improved SMN activity in pituitary and / or other cells. In certain cases, administration to the CSF may be necessary to achieve adequate nutritional support. The effect of the present invention is to restore neuronal function and allow the subject to live longer; however, as the subject lives longer, one or more symptoms may develop that were previously unknown because the subject typically dies before such symptoms develop. Symptoms can be fatal. In certain embodiments, the presenting symptoms are treated by systemic administration. Regardless of the mechanism, in certain embodiments, the decline in neuronal function A variety of SMA symptoms can be treated by systemic administration, including, but not limited to, symptoms previously masked by more severe symptoms associated with SMA.

[0183] In certain embodiments, systemic administration of the pharmaceutical compositions of the present invention increases SMN activity in muscle cells. In certain embodiments, such improvement in SMN activity in muscle cells results in an increase in Improving SMN activity only in the muscle is insufficient to produce a therapeutic effect. It has been reported that the effect of ATP on the function of SMN in muscle is sufficient (e.g., Gravrilina, et al., Hum Mol Genet 2008 17(8):1063-1075). In certain embodiments, the present invention provides a method for improving SMN function in muscle. In certain cases, the therapeutic effect can be attributed to improved SMN function in other cells (alone or in combination with muscle cells). In certain embodiments, improvement of SMN function in muscle alone can provide benefit.

[0184] In certain embodiments, systemic administration results in improved survival.

[0185] 6. Spinal Muscular Atrophy (SMA) SMA is a genetic disorder characterized by the destruction of spinal motor neurons. SMA results from the homozygous loss of both functional copies of the SMN1 gene. However, the SMN2 gene has the potential to encode the same protein as SMN1 and, therefore, may be involved in SMA. Overcome the patient's gene deletion. SMN2 contains a translationally silent mutation (C → T) at position +6 of exon 7, which results in incomplete inclusion of exon 7 in the SMN2 transcript. Therefore, the predominant form of SMN2, which lacks exon 7, is unstable and inactive. Thus, therapeutic compounds increase the proportion of SMN2 transcripts containing exon 7. SMN2 splicing can be modified to prevent the splicing of SMN2, and is useful for the treatment of SMA. is.

[0186] In certain embodiments, the present invention provides antisense compounds complementary to the pre-mRNA encoding SMN2. In certain such embodiments, the antisense compounds alter the splicing of SMN2. Specific sequences and regions useful for altering the splicing of SMN2 can be found in PCT / US06 / 024469, which discloses its use for any purpose. In certain embodiments, oligomeric compounds having any of the motifs described herein are complementary to intron 7 of SMN2. Specific such nucleobase sequences are exemplified in the non-limiting table below.

[0187] [Table 2]

[0188] The antisense compounds of the present invention can be used to modify the expression of SMN2 in a subject, such as a human. In certain embodiments, the subject has spinal muscular atrophy. In certain such subjects, the SMN1 gene is absent or does not contain sufficient amounts of functional SMN1. In certain embodiments, the antisense compounds of the present invention effectively modify the splicing of SMN2, resulting in increased exon 7 inclusion in the SMN2 mRNA and ultimately in an SMN2 protein that contains amino acids corresponding to exon 7. Such alternative SMN2 proteins resemble the wild-type SMN protein. Antisense compounds of the invention that effectively modulate the expression of SMN2 mRNA or protein expression product are considered active antisense compounds.

[0189] Modulation of SMN2 expression can be measured in animal cells, animal tissues, or body fluids that may or may not contain animal organs (e.g., saliva, serum, CSF). Methods for obtaining samples for analysis, such as tissues (e.g., biopsies) or organs, and methods for preparing samples to allow analysis are well known to those skilled in the art. Methods for analyzing protein levels are described above and are well known to those skilled in the art. The effect of treatment can be assessed by measuring biomarkers associated with target gene expression in the aforementioned fluids, tissues, or organs collected by common clinical methods known in the art from animals contacted with one or more compounds of the invention.

[0190] The body fluid, organ or tissue is treated with an effective amount of one or more of the antisense compounds of the present invention or Also contemplated are methods of contacting a body fluid, organ, or tissue with one or more of the compositions. The compounds of the present invention can be contacted with cells of a body fluid, organ, or tissue, resulting in modulation of SMN2 expression in the cells. The degree of modification can be determined by monitoring the modifying effect of the compound or composition on the target nucleic acid or its products by methods common to those skilled in the art.

[0191] The present invention also provides antisense compounds described herein for use in any of the methods described herein above. For example, the present invention provides antisense compounds comprising antisense oligonucleotides complementary to a nucleic acid encoding human SMN2 for use in treating a disease or condition associated with survival motor neuron protein (SMN), such as spinal muscular atrophy (SMA). As a further example, the present invention provides antisense compounds comprising antisense oligonucleotides complementary to a nucleic acid encoding human SMN2 for use in treating a disease or condition associated with survival motor neuron protein (SMN) by administering the antisense compound directly into the central nervous system (CNS) or CSF. For use, antisense compounds are provided, including antisense oligonucleotides complementary to a nucleic acid encoding human SMN2.

[0192] The present invention also provides the use of an antisense compound as described herein in the manufacture of a medicament for use in any of the methods as described herein. For example, the present invention provides an antisense compound for the treatment of a disease such as survival motor neuron protein (SMP) deficiency syndrome (SMA). The present invention relates to the use of human SMN2 in the manufacture of a medicament for treating a disease or condition associated with SMN. By way of further example, the present invention provides for the use of antisense compounds comprising antisense oligonucleotides complementary to nucleic acids encoding survival motor neuron proteins (SMNs). The present invention provides the use of an antisense compound, including an antisense oligonucleotide complementary to a nucleic acid encoding human SMN2, in the manufacture of a medicament for treatment by continuous administration.

[0193] In certain embodiments, oligomeric compounds having any of the motifs described herein have a nucleobase sequence complementary to exon 7 of SMN2.

[0194] In certain embodiments, oligomeric compounds having any of the motifs described herein have a nucleobase sequence complementary to intron 6 of SMN2.

[0195] In certain embodiments, antisense compounds comprise antisense oligonucleotides having a nucleobase sequence comprising at least 10 nucleobases of the following sequence: TCACTTTCATAATGCTGG (SEQ ID NO: 1). In certain embodiments, antisense oligonucleotides have a nucleobase sequence comprising at least 11 nucleobases of such a sequence. In certain embodiments, antisense oligonucleotides have a nucleobase sequence comprising at least 12 nucleobases of such a sequence. In certain embodiments, antisense oligonucleotides have a nucleobase sequence comprising at least 13 nucleobases of such a sequence. In certain embodiments, antisense oligonucleotides have a nucleobase sequence comprising at least 14 nucleobases of such a sequence. In certain embodiments, antisense oligonucleotides have a nucleobase sequence comprising at least 15 nucleobases of such a sequence. In certain embodiments, antisense oligonucleotides have a nucleobase sequence comprising at least 16 nucleobases of such a sequence. In certain embodiments, antisense oligonucleotides have a nucleobase sequence comprising at least 17 nucleobases of such a sequence. In certain embodiments, antisense oligonucleotides have a nucleobase sequence comprising the nucleobases of such a sequence. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence consisting of the nucleobases of such a sequence. In certain embodiments, the antisense oligonucleotide consists of 10 to 18 linked nucleosides and has a nucleobase sequence that is 100% identical to an equal length portion of the following sequence: TCACTTTCATAATGCTGG (SEQ ID NO: 1). It has.

[0196] 7. Specific Subjects In certain embodiments, the subject has one or more indicators of SMA. In certain embodiments, the subject has reduced electrical activity in one or more muscles. In certain embodiments, the subject has a mutated SMN1 gene. is absent or unable to produce functional SMN protein. In certain embodiments, the subject is diagnosed by genetic testing. In certain embodiments, the subject is identified by muscle biopsy. In certain embodiments, the subject is diagnosed by sitting upright. In certain embodiments, the subject is unable to sit and / or walk. In certain embodiments, the subject requires assistance with breathing and / or eating. In some embodiments, the subject is monitored by electrophysiological measurements of muscle and / or muscle biopsies. It is identified by:

[0197] In certain embodiments, the subject has SMA Type I. In certain embodiments, the subject has SMA Type II. In certain embodiments, the subject has SMA Type III. In certain embodiments, the subject has SMA Type I. In certain embodiments, the subject is diagnosed with SMA in utero. In certain embodiments, the subject is diagnosed with SMA within one week of birth. In certain embodiments, a subject is diagnosed with SMA by 3 months of age. In certain embodiments, a subject is diagnosed with SMA by 6 months of age. In certain embodiments, a subject is diagnosed with SMA by 1 year of age. In certain embodiments, a subject is diagnosed with SMA between 1 and 2 years of age. In certain embodiments, Subjects are diagnosed with SMA between the ages of 1 and 15. In certain embodiments, subjects are diagnosed with SMA when the subject is 15 years of age or older.

[0198] In a particular embodiment, the first dose of the pharmaceutical composition according to the present invention is administered intrauterinely. In certain such embodiments, the first dose is administered before the blood-brain barrier is fully developed. In certain embodiments, the first dose is administered systemically intrauterinely to the subject. In certain embodiments, the first dose is administered in utero after the formation of the blood-brain barrier. In certain embodiments, the first dose is administered to the CSF.

[0199] In certain embodiments, the first dose of the pharmaceutical composition according to the invention is administered when the subject is less than one week of age. In certain embodiments, the pharmaceutical composition according to the present invention is administered when the subject is under one month of age. In certain embodiments, the first dose of the pharmaceutical composition according to the present invention is administered when the subject is under 3 months of age. In certain embodiments, the first dose of the pharmaceutical composition according to the present invention is administered when the subject is under 6 months of age. In certain embodiments, the first dose of the pharmaceutical composition according to the present invention is administered when the subject is under 1 year of age. In certain embodiments, the first dose of the pharmaceutical composition according to the present invention is administered when the subject is under 2 years of age. In certain embodiments, the first dose of the pharmaceutical composition according to the present invention is In certain embodiments, the first dose of a pharmaceutical composition according to the present invention is administered when the subject is 15 years of age or older.

[0200] 8. Specific Doses In certain embodiments, the present invention provides dosages and administration frequencies. In certain embodiments, the pharmaceutical composition is administered as a bolus injection. In certain such embodiments, the bolus injection dose is 0.01 to 25 milligrams of antisense compound per kilogram of subject body weight. In certain such embodiments, the bolus injection dose is 0.01 to 10 milligrams of antisense compound per kilogram of subject body weight. In certain such embodiments, the dose is 0.05 to 5 milligrams of antisense compound per kilogram of subject body weight. In certain embodiments, the dose is 0.1 to 2 milligrams of antisense compound per kilogram of subject body weight. In certain embodiments, the dose is 0.5 to 1 milligram of antisense compound per kilogram of subject body weight. In certain embodiments, such doses are administered twice a month. In certain embodiments, such doses are administered monthly. In certain embodiments, such doses are administered every two months. Such doses are administered every six months. In certain embodiments, such doses are administered by bolus injection into the CSF. In certain embodiments, such doses are administered by intrathecal bolus injection. In certain embodiments, such doses are administered by bolus systemic injection (e.g., subcutaneous injection, intramuscular injection, or intravenous injection). In certain embodiments, subjects receive a bolus injection into the CSF and a bolus systemic injection. In such embodiments, the CSF bolus and systemic bolus doses are identical to each other. In certain embodiments, the CSF and systemic doses may be administered at different frequencies. In certain embodiments, the present invention provides a method for administering at least one bolus intrathecal injection. and at least one bolus subcutaneous injection.

[0201] In certain embodiments, the pharmaceutical composition is administered by continuous infusion. Such continuous infusion can be achieved by an infusion pump that delivers the pharmaceutical composition into the CSF. In embodiments, such infusion pumps deliver pharmaceutical compositions IT or ICV. In such particular embodiments, the dose administered is In certain embodiments, the administered dose ranges from 0.05 to 25 milligrams of antisense compound per kilogram of subject body weight per day. In certain embodiments, the administered dose ranges from 0.1 to 10 milligrams of antisense compound per kilogram of subject body weight per day. In certain embodiments, the administered dose ranges from 0.5 to 10 milligrams of antisense compound per kilogram of subject body weight per day. In certain embodiments, the administered dose is 0.5 to 5 milligrams of antisense compound per kilogram of subject body weight per day. In certain embodiments, the administered dose is 1 to 5 milligrams of antisense compound per kilogram of subject body weight per day. In certain embodiments, the invention provides a dosing regimen comprising an infusion into the CNS and at least one bolus systemic injection. In certain embodiments, the invention provides a dosing regimen comprising an infusion into the CNS and at least one bolus subcutaneous injection. In certain embodiments, the dose, whether bolus or infusion, is prepared to achieve or maintain a concentration of 0.1 to 100 micrograms of antisense compound per gram of CNS tissue. In certain embodiments, the dose, whether bolus or infusion, is prepared to achieve or maintain a concentration of 1 to 10 micrograms of antisense compound per gram of CNS tissue. In certain embodiments, the dose, whether bolus or infusion, is prepared to achieve or maintain a concentration of 0.1 to 1 micrograms of antisense compound per gram of CNS tissue. The concentration of the antisense compound is adjusted to achieve or maintain a concentration of the antisense compound of 0.1% or more.

[0202] In certain embodiments, administration to a subject is divided into an induction phase and a maintenance phase. In certain such embodiments, the dose administered during the induction phase is greater than the dose administered during the maintenance phase. In certain embodiments, the dose administered during the induction phase is less than the dose administered during the maintenance phase. In certain embodiments, the induction phase is administered by bolus injection. The initial phase is achieved by continuous infusion, and the maintenance phase is achieved by continuous infusion.

[0203] In certain embodiments, the present invention provides for systemic administration of antisense compounds, alone or in combination with delivery into the CSF. In certain embodiments, the dose for systemic administration is 0.1 mg / kg to 200 mg / kg. In certain embodiments, the dose for systemic administration is 0.1 mg / kg to 100 mg / kg. In certain embodiments, the dose for systemic administration is 0.5 mg / kg to 100 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 100 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 50 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 25 mg / kg. In certain embodiments, the dose for systemic administration is 0.1 mg / kg to 25 mg / kg. In certain embodiments, the dose for systemic administration is 0.1 mg / kg to 10 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 10 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 5 mg / kg. In certain embodiments involving both systemic and CSF delivery, the doses for the two routes are determined independently.

[0204] a. Calculation of appropriate human dose In certain embodiments, the subject is a human. In certain embodiments, the human dose is calculated or estimated from data from animal studies such as those described herein. In certain embodiments, the human dose is calculated or estimated from data from monkey and / or mouse studies such as those described herein. In certain embodiments, the human dose is calculated or estimated from data from mouse studies such as those described herein. In certain embodiments, the appropriate human dose is determined using pharmacokinetic data from mice in conjunction with knowledge of brain weight and / or cerebrospinal fluid (CSF) turnover rate. For example, the brain weight of a mouse is approximately 0.4 g, which is It is approximately 2% of body weight. In humans, the average brain weight is 1.5 kg, which is The CSF concentration is approximately 2.5% of the total CSF concentration. In certain embodiments, administration into the CSF results in the elimination of a portion of the compound through uptake into brain tissue and subsequent metabolism. By using the ratio of human brain weight to mouse brain weight as a scaling factor, an estimate of removal and clearance through brain tissue can be calculated. Furthermore, the CSF turnover rate can be used to estimate the removal of a compound from the CSF to the blood. Mouse CSF turnover rate The CSF turnover rate in humans is approximately 10-12 times per day (0.04 mL produced at 0.325 μl / min). The CSF turnover rate in humans is approximately 4 times per day (100-160 mL produced at 350-400 μl / min). Clearance, and therefore dosage requirements, depend on brain weight removal, scaling, and and / or CSF turnover scaling. Using this technique, it is possible to estimate the human equivalent dose, which approximates the dose in mice. Thus, the effect of CSF turnover on tissue metabolism can be estimated based on brain weight and CSF turnover rate. Human doses that account for the differences can be extrapolated. Such methods of calculation and extrapolation are known to those skilled in the art.

[0205] By way of non-limiting example, in certain embodiments, the mg / kg mouse dose may be multiplied by a factor of about 0.25 to about 1.25, depending on the determined clearance and elimination of the particular compound. Thus, for example, in certain embodiments, the human dose equivalent to a 0.01 mg dose for a 20 g mouse ranges from about 8.75 mg to about 43.75 mg total dose for a 70 kg human. Similarly, in certain embodiments, Thus, the human dose equivalent to a 0.01 mg dose for a 4 g newborn mouse ranges from about 1.9 mg to about 9.4 mg total dose for a 3 kg newborn human. These exemplary doses are merely illustrative of how one of skill in the art can determine an appropriate human dose and are not intended to limit the invention.

[0206] In certain embodiments, a human dose for systemic delivery (administered alone or via CSF delivery) is The dose (whether administered in combination with other drugs) is calculated or estimated from data from animal studies such as those described herein. Typically, the appropriate human dose (mg / kg) for systemic administration is 0.1 to 10 times the effective dose in animals. Thus, for example, a subcutaneous administration of 50 μg in a 2 kg neonatal mouse is a dose of 25 mg / kg. The corresponding dose in humans is expected to be 2.5 mg / kg to 250 mg / kg. For a 3 kg neonate, the corresponding dose is 2.5 mg / kg to 250 mg / kg. The dose ranges from 7.5 mg to 750 mg. For a 25 kg child, the corresponding dose ranges from 62.5 mg to 6250 mg.

[0207] 9. Treatment Prescription In certain embodiments, the above-described dosages, administration frequencies, administration routes, induction and maintenance phases, and timing of the first administration are combined to provide a dosing regimen for a subject with SMA. Such a dosing regimen can be selected and adjusted to achieve improvement in one or more symptoms of SMA. and / or reduce or avoid toxicity or side effects that may be attributable to administration of the pharmaceutical composition. In certain embodiments, the subject is in utero or a newborn. In such embodiments, administration of pharmaceutical compositions, particularly by continuous infusion, presents particular challenges. Thus, in certain embodiments, the present invention provides for administration of pharmaceutical compositions by bolus administration while the subject is in utero or very young, followed by continuous infusion via an implanted infusion pump when the subject is older and placement of such a pump is more practical. Furthermore, in certain embodiments, as the subject grows, the absolute dose is increased to achieve the same or similar dose:body weight ratio. The following table is intended to illustrate treatment regimens, but is not intended to limit the possible combinations that may be readily achieved by one of ordinary skill in the art.

[0208] [Table 3]

[0209] In certain embodiments, the dosage regimen includes systemic administration alone or in combination with administration into the CSF (e.g., regimen 3, supra). The following table further illustrates such regimens.

[0210] [Table 4]

[0211] These treatment regimens are intended to be illustrative, but not limiting, of the present invention. Those skilled in the art will be able to select an appropriate combination of dosage and delivery based on various factors, such as the severity of the symptoms and the general health and age of the subject, with reference to the present disclosure.

[0212] 10. Co-administration In certain embodiments, the pharmaceutical compositions of the present invention are administered in combination with at least one other pharmaceutical composition for treating SMA and / or one or more symptoms associated with SMA. In certain embodiments, such other pharmaceutical compositions are selected from trichostatin-A, valproic acid, riluzole, hydroxyurea, and butyrate or a butyrate derivative. In certain embodiments, the pharmaceutical composition of the present invention is co-administered with trichostatin-A. In certain embodiments, the pharmaceutical composition of the present invention is co-administered with a quinazoline derivative, as described, for example, in Thurmond, et al., J. Med Chem. 2008, 51, 449-469. In certain embodiments, the pharmaceutical composition of the present invention and at least one other pharmaceutical composition In certain embodiments, the pharmaceutical composition of the present invention and at least one are co-administered at different times.

[0213] In certain embodiments, the pharmaceutical composition of the present invention is co-administered with a gene therapy agent. In such certain embodiments, the gene therapy agent is administered into the CSF and the pharmaceutical composition of the present invention is co-administered with a gene therapy agent. In certain such embodiments, the gene therapy agent is administered to the CSF. and administering the pharmaceutical compositions of the present invention to the CSF and systemically. In certain embodiments, the pharmaceutical composition of the present invention and the gene therapy agent are co-administered at the same time. In certain embodiments, the pharmaceutical composition of the present invention and the gene therapy agent are co-administered at different times. Specific gene therapy approaches have been reported (e.g., Coady et al., PLoS ONE 2008 3(10): e3468; Passini et al., J Clin Invest 2010 Apr 1, 120(4): 1253-64).

[0214] In certain embodiments, the pharmaceutical composition of the present invention is co-administered with at least one other treatment for SMA. In certain embodiments, such other treatment for SMA is surgery. In certain embodiments, such other treatment is physical therapy, including (but not limited to) exercises designed to strengthen the muscles needed for breathing, e.g., cough therapy. In certain embodiments, the other treatment is physical intervention, such as a feeding tube or a device to assist breathing.

[0215] In certain embodiments, the pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical compositions that reduce undesirable side effects of the pharmaceutical compositions of the present invention.

[0216] 11. Phenotypic Effects In certain embodiments, administration of at least one pharmaceutical composition of the present invention results in a patient receiving In certain embodiments, such phenotypic changes include: Increased absolute amount of SMN mRNA containing exon 7; increased ratio of SMN mRNA containing exon 7 to SMN mRNA lacking exon 7; increased absolute amount of SMN protein containing exon 7; Increased ratio of exon 7-containing SMN protein to exon 7-deleted SMN protein; improved muscle strength, improved electrical activity of at least one muscle; improved breathing; weight gain; and In certain embodiments, at least one phenotypic change is detected in the motor neurons of the subject. In certain embodiments, administration of at least one pharmaceutical composition of the present invention results in the subject being able to do sit-ups, stand up, and / or walk. In certain embodiments, administration of at least one pharmaceutical composition of the present invention results in the subject being able to eat, drink, and walk without assistance. In certain embodiments, the therapeutic effect is assessed by electrophysiological assessment of muscles. In certain embodiments, administration of a pharmaceutical composition of the present invention improves at least one symptom of SMA and has little or no inflammatory effect. In certain such embodiments, the absence of inflammatory effect is determined by the absence of a significant increase in Aif1 levels upon treatment.

[0217] In certain embodiments, administration of at least one pharmaceutical composition of the present invention delays the onset of at least one symptom of SMA. In certain embodiments, administration of at least one pharmaceutical composition of the present invention delays the progression of at least one symptom of SMA. In certain embodiments, administration of at least one pharmaceutical composition of the present invention reduces the severity of at least one symptom of SMA. .

[0218] In certain embodiments, administration of at least one pharmaceutical composition of the present invention results in a desired In certain embodiments, a therapeutic regimen is identified that produces desirable relief of symptoms while avoiding undesirable side effects.

[0219] 12. Dosage Unit In certain embodiments, the pharmaceutical compositions of the present invention are prepared as dosage units for administration. Particular such dosage units are at concentrations selected from 0.01 mg to 100 mg. In certain such embodiments, the pharmaceutical compositions of the present invention are prepared in dosage units of 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, In certain embodiments, the pharmaceutical composition comprises a dose of antisense compound selected from 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, and 200 mg. In certain embodiments, the pharmaceutical composition comprises a dose of antisense compound selected from 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 25 mg, and 50 mg of oligonucleotide. Includes dosage.

[0220] 13. Kit In certain embodiments, the present invention provides kits comprising at least one pharmaceutical composition. In certain embodiments, such kits further comprise a delivery means, such as a syringe or an infusion pump.

[0221] Non-limiting disclosure and incorporation by reference While the particular compounds, compositions and methods described herein are specifically described according to certain embodiments, the following examples serve only to illustrate and are not intended to limit the compounds described herein. Accession numbers, and the like, described herein are hereby incorporated by reference in their entirety.

[0222] The sequence listing accompanying this application refers to it as either "RNA" or "DNA" where appropriate. The sequences of each gene are identified, but in fact, the sequences are modified with any combination of chemical modifications. Those skilled in the art will recognize the term "RNA" to describe modified oligonucleotides. " or "DNA" is, in certain instances, arbitrary. For example, an oligonucleotide containing a nucleoside containing a 2'-OH sugar moiety and a thymine base can be described as a DNA with a modified sugar (2'-OH as opposed to the natural 2'-H of DNA) or an RNA with a modified base (thymine (methylated uracil) as opposed to the natural uracil of RNA).

[0223] Thus, the nucleic acid sequences presented herein, including but not limited to those in the Sequence Listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to nucleic acids with modified nucleobases. By further example and without limitation, an oligomeric compound having the nucleobase sequence "ATCGATCG" is intended to encompass nucleic acid salts, including but not limited to compounds containing RNA bases, whether modified or unmodified. The term "AUCGAUCG" encompasses any oligomeric compound having a base sequence, including those having the sequence "AUCGAUCG" and those having several DNA bases and several RNA bases, such as "AUCGATCG." What we have, and “AT me CGAUCG”, where me C denotes a cytosine base containing a methyl group at the 5-position. [Example]

[0224] Example 1 – Antisense compounds targeting SMN2 The following oligonucleotides were synthesized using standard techniques as previously reported:

[0225] [Table 5]

[0226] Example 2 – Smn− / − SMN transgenic mice The therapeutic efficacy and safety of the antisense compounds described above can be tested in appropriate animal models, such as those that are most likely to resemble human disease. The species includes animal species that naturally develop or can be induced to develop a particular disease at a high frequency.

[0227] In particular, animal models for SMA are known. As mentioned above, the molecular basis of SMA, an autosomal recessive neuromuscular disorder, is the homozygous loss of the survival motor neuron gene 1 (SMN1). A nearly identical copy of the SMN1 gene, called SMN2, is found in humans and modifies the severity of the disease. In contrast to humans, mice have a single gene (Smn) as the counterpart of SMN1. Homozygous loss of this gene results in embryonic and causes massive cell death, indicating that the Smn gene product This means that SMN2 is required for the survival and function of SMN. Introduction of two copies of SMN2 into SMN-deficient mice rescues embryonic lethality, resulting in mice with an SMA phenotype (Monani et al., Hum. Mol. Genet. (2000) 9 :333-339. High copy numbers of SMN2 rescue the mice because sufficient SMN protein is produced in motor neurons. Similarly, Hsieh-Li, et al., Nat. Genet. (2000), who reported the generation of a transgenic mouse line expressing human SMN2, 24 :66-70. In particular, the Smn- / - background, as well as the spinal cord and skeletal muscle of SMA patients, Transgenic mice carrying SMN2 in their spinal cords exhibit pathological changes in the spinal cord and skeletal muscles. The severity of the pathological changes in these mice was determined by the gene encoded by exon 7. The amount of SMN protein containing the region that is involved in the SMN deletion correlated with the amount of SMN protein. The mice are designated Smn − / + and are a model of a less severe form of SMA, type III. .

[0228] The severity of the SMA phenotype is a function of the copy number of human SMN2 in the mice. The "Taiwan" strain has four copies of human SMN2, resulting in mice with a moderate to severe SMA phenotype resembling Type I or Type II.

[0229] Delta-7 mice (Smn - / - , hSMN2 + / + , SMNΔ7 + / + ) also lack mouse Smn and express human SMN2. Delta7 mice have a more severe phenotype and develop a phenotype shortly after birth. Death typically occurs around 15 to 20 days after birth.

[0230] Example 3 - In vivo administration of systemic antisense compounds in Smn- / -SMN2 (Taiwanese strain) Give Taiwanese mice were treated with saline or 35 mg / kg of 396443 or a mismatched antisense oligonucleotide control by intraperitoneal injection once daily for 5 days and sacrificed on day 7. Livers and kidneys were harvested, and RNA was isolated using standard techniques. SMN2 with and without exon 7 was visualized by RT-PCR. Administration of ISIS 396443 resulted in a substantial increase in exon 7 inclusion in SMN2 from kidney and liver compared with saline- and mismatched control-treated animals.

[0231] Example 4 - In vivo administration of intracerebroventricular (ICV) antisense compounds in Smn- / -SMN2 (Taiwanese strain) Taiwanese mice were injected intravenously with either saline or 150 μg of ISIS 396443 daily for 7 days. Mice were sacrificed on day 8, and RNA was extracted from the brain and spinal cord. RT-PCR analysis demonstrated a substantial increase in exon 7 inclusion in SMN2 in brain and spinal cord samples from animals treated with ISIS 396443. These results suggest that exon 7 removal contributes to the development of SMA. Because of its association with the phenotype, antisense oligonucleotides targeting SMN have been used to treat It has been shown that ICV treatment with steroids can relieve SMA symptoms.

[0232] Dose-response Taiwanese mice were treated with either saline or 10, 50, 100, or 150 μg of ISIS 396443. Each mouse was injected ICV with either 10 μg or 10 μg of the ... The 50 μg, 100 μg, and 150 μg groups all showed substantial exon 7 inclusion.

[0233] Response Period To determine the duration of effect, 24 mice were injected ICV with 50 μg of ISIS 396443 daily for 7 days. Four mice were sacrificed at the final dose (time 0), and four mice were sacrificed at 1 week, 2 weeks, 4 weeks, and 8 weeks after the final dose. All treated mice showed substantial exon 7 inclusion by RT-PCR at week 8, along with efficacy. , as shown in Figure 1, showed no difference from the other groups: These results indicate that ISIS 396443 administered ICV at 50 μg per day for 7 days is effective for at least 8 weeks after treatment.

[0234] This experiment was repeated for a longer period. Type III mice were treated with ISIS 396443 by ICV infusion at 50 μg / day for 7 days. Mice were then resuspended at the end of the 7-day infusion period. The mice were sacrificed at 0, 0.5, 1, 2, 4, and 6 months after the initial injection. The results were collected and analyzed by Northern blot. As shown, the efficacy of ISIS 396443 infusion was sustained for 6 months after infusion. There are several possible explanations for this effect: it may reflect the stability of ISIS 396443, the stability of the modified SMN protein, and / or the dose was very high. It is possible that the remaining dose continues to provide benefit even after the compound is metabolized and lost. Thus, these data may support lower or occasional dosing.

[0235] Example 5 - Administration of antisense compounds by continuous intracerebroventricular (ICV) infusion Using a micro-osmotic pump (Azlet Osmotic Pumps, Cupertino, CA, USA), ISIS 396443 was delivered into the cerebrospinal fluid (CSF) via the right lateral ventricle to adult type-III Smn+ / - mice or Smn- / - SMA mice (Taiwanese strain) carrying the human SMN2 transgene. A sex study showed that ISIS 396443 reduced the risk of HIV infection by approximately 10% compared with approximately 10% in saline-treated mice. Intracerebroventricular (ICV) infusion of increased SMN2 exon 7 inclusion in the spinal cord by approximately 90%. Western blotting and immunohistochemical analyses demonstrated robust increases in the levels of human transgenic SMN protein in spinal motor neurons. The results indicate that removal of exon 7 is associated with the SMA phenotype and that CNS infusion of the antisense oligonucleotide ISIS 396443 can rescue SMA symptoms.

[0236] Example 6 - Embryo Administration A single ICV injection of either 20 μg or 10 μg of ISIS 396443 was administered to embryonic Taiwanese mice on gestational day 15 (E15). Animals were sacrificed on postnatal day 7 (P7). RNA was isolated from the lumbar spinal cord and analyzed by RT-PCR. Single embryonic administration of ISIS 396443 resulted in substantial embryonic development. These results suggest that exon 7 removal is associated with the SMA phenotype. Therefore, in utero treatment with the antisense oligonucleotide ISIS396443 may be effective in preventing SMA It has been shown that it can relieve symptoms.

[0237] The above experiment was repeated and the animals were sacrificed at 11 weeks of age. Untreated Taiwanese mice developed necrotic tails, which developed over time. A single embryonic injection of 20 μg of ISIS 396443 As shown in Figure 3A, this significantly delayed the onset of tail degradation. These results suggest that embryonic treatment with antisense oligonucleotides targeting SMN delays the onset of SMA. It is shown that this can be done.

[0238] These results were confirmed in another study using identical conditions, except that the doses tested were 20 μg and 10 μg of ISIS 396443 and that normal mice were included for comparison. The results from that experiment are shown in Figure 3B.

[0239] Example 7 – In vivo administration in the Delta-7 mouse model Heterozygote (SMN + / - , hSMN2 + / + , SMNΔ7 + / + ) Mating pairs and then giving birth On day 0 (P0), neonates were treated with ISIS396443 (18-mer, SEQ ID NO. 1), ISIS396449 (15-mer, SEQ ID NO. 2), ISIS387954 (20-mer, SEQ ID NO. 7), or a scrambled control ASO (ISIS439273; 18-mer). Mice were administered a total dose of 8 μg into the lateral ventricles (10 μg per lateral ventricle). All injections were performed as described (Passini et al., J. Virol. (2001) 75 :12382-12392) and was performed using a finely drawn glass pipette needle. After injection, the toes of the newborn pups were amputated and genotyped (Le et al., Hum. Mol. Genet. (2005) 14 :845-857), SMA(SMN - / - , hSMN2 + / + , SMNΔ7 + / + ) Mouse, heterozygous mice, and wild-type (SMN + / + , hSMN2 + / + , SMNΔ7 + / + ) mice were identified. The fetuses were removed and seven newborns served as controls for fetal size relative to survival. To generate a control group, some fetuses were not injected.

[0240] The widely known 18-mer was injected into the thoracic spinal cord of SMA mice 14 days after injection. It was detected in the spinal cord, including the lumbar, cervical, and ventricular regions. Localization studies confirmed that the majority of cells targeted by ISIS 396443 in the spinal cord were motor neurons. No signal was detected in control and untreated mice.

[0241] Western blotting analysis at 14 days showed that the amount of SMN in the brain and spinal cord was This was 40-60% of wild-type levels compared to 10% in untreated SMA controls. The signal above background was observed in control mice treated with a scrambled ASO. The results of the Western blot are presented in Figure 4.

[0242] Compared with untreated SMA mice or SMA mice treated with scrambled ASOs, SMA mice treated with SMA ASOs, regardless of ASO length, showed significant increases in body weight, locomotor function (righting reflex and grip strength), and coordination (hindlimb splay). No significant increases in body weight, locomotor function (righting reflex and grip strength), or coordination were found in SMA mice treated with scrambled ASO compared to control mice. The results are presented in Figures 5 and 6.

[0243] Importantly, as shown in Figure 7, regardless of ASO length, SMA mice treated with ASOs have a significant increase in median survival. Survival is calculated from birth and is compared to untreated SMA controls. The survival time was 31.5 days (15-mer), 27.0 days (18-mer), and 28.0 days (20-mer) compared with 16.0 days for the 15-mer. In contrast, SMA mice treated with the 18-mer scrambled control showed improved survival. These results suggest that antisense oligonucleotides targeting SMN treatment may be effective. Treatment has been shown to increase the lifespan of subjects with SMA.

[0244] The SMA ASO also increases the number of motor neuron cells in the spinal cord, as shown in Figure 8.

[0245] SMN RNA was measured by RT-PCR. SMA ASO-treated compared to untreated SMA mice. Animals treated with the 20-mer ASO had increased SMN RNA levels. Results from mice treated with the 20-mer ASO compared to untreated SMA mice are shown in Figure 9.

[0246] To determine whether survival was further increased by administering a second dose, the above experiment was repeated. The results were repeated with an additional dose of 20 μg on day 21. The results are shown in Figure 10. Shows the effect of the first dose of 8 μg on day 0. On day P21, half of the treated mice received a second treatment.

[0247] The effect of a second treatment compared to mice receiving only the first treatment is shown in Figure 11. The results suggest that a second ICV treatment with antisense oligonucleotide further increases survival.

[0248] Example 8 – Activity in SMA type III mice Two antisense compounds and one control compound were tested in a mouse model of SMA The compounds are listed in the table below.

[0249] [Table 6]

[0250] Taiwanese strain of SMA type III mice was obtained from The Jackson Laboratory (Bar Harbor, Maine). These mice lack mouse SMN and are homozygous for human SMN2. These mice were described in Hsieh-Li HM, et al., Nature Genet. 24, 66-70, 2000.

[0251] Mice were treated with 3, 10, 30, or 100 μg of ISIS 396443 or ISIS 449220 per day, or with 30 or 100 μg of the control compound ISIS 439272 per day in phosphate-buffered saline (PBS). Control mice were treated with PBS only (dose 0). All treatments were performed with Azlet 1007D It was administered by intracerebroventricular (ICV) infusion using an osmotic pump. There were five animals per dose, and two of the mice from the highest dose of ISIS 449220 died before the end of the study. Animals were sacrificed on day 9 (two days after the final dose), and brain and lumbar sections of the spinal cord were collected from each animal. Real-time PCR was performed on each sample. The amount of human SMN2 message containing exon 7 ((+) exon 7) and the exon The amount of human SMN2 message without exon 7 ((-) exon 7) was determined. Real-time PCR was also performed to determine the expression levels of allograft inflammatory factor (AIF1) and glyceraldehyde 3-phosphate dehydrogenase (GADPH).

[0252] Expression levels for (+) exon 7 and (-) exon 7 relative to GADPH levels The normalized expression levels were compared with the GADPH-normalized expression levels obtained from PBS-treated control mice. The resulting fold-control values ​​are shown in Table 17 below. The data show that all five mice in each group survived except for the highest dose of ISIS 449220, which showed three survivors. The mean fold increase of the control mice is shown.

[0253] Administration of ISIS 396443 resulted in a significant increase in exon 7 follicles. On day 1, ISIS396443 resulted in almost double (1.8-fold) the amount of SMN2 mRNA remaining in exon 7. The increased activity in the brain and lumbar spinal cord was more than doubled when compared to untreated controls.

[0254] [Table 7]

[0255] Allograft inflammatory factor (AIF1) expression was examined as a measure of inflammation. After normalizing samples to (GADPH), the AIF1 ratio for each treatment group was divided by the value for the PBS control. ISIS 396443 did not result in an increase in AIF1, even at high doses. ISIS 449220 resulted in an increase in AIF1 in both the brain and lumbar spinal cord. The data in Table 18 are consistent with those of the highest dose of ISIS 449220, where three surviving mice were shown. , The mean fold over control for all five mice in each group is shown.

[0256] [Table 8]

[0257] Example 9 – Administration to monkeys Cynomolgus monkeys were used to evaluate the distribution of ISIS 395443 at different doses and by different routes of administration. ISIS 396443 was administered to two monkeys. One monkey had a 24-hour bolus administration. One monkey received 3 mg via ICV infusion, and the other received 3 mg via IT infusion. Both infusions were delivered over a 24-hour period. The monkeys were sacrificed 96 hours after the end of the infusion period, and tissues were collected. ISIS 396443 concentrations were measured in samples from the cervical, thoracic, and lumbar regions of the spinal cord. The results are summarized in the table below.

[0258] [Table 9]

[0259] A cynomolgus monkey weighs approximately 3 kg, so this dose is approximately 1 mg / kg.

[0260] To further evaluate the distribution of ISIS 39644, the 26 monkeys were divided into 6 groups as shown in the table below.

[0261] [Table 10]

[0262] The infusion rate was 100 μL / hour for all groups. Group 1 received normal saline. All monkeys received a total of 3 mg of ISIS 39644 in saline, except for one monkey that received only ISIS 39644. The monkeys were sacrificed 5 days after the end of the infusion and tissues were collected.

[0263] The concentration of ISIS 39644 in tissue samples from monkeys was assessed using standard techniques. A summary of the results is provided in the graph in FIG.

[0264] Samples were also evaluated histologically, which showed no adverse effects of treatment and confirmed the presence of ISIS 396443. There was no evidence of Purkinje cell loss.

[0265] Rapid infusion appeared to have more ISIS 396443 than slower infusion. These results suggest that faster infusion rates or bolus injection may be preferred in certain embodiments. Bolus administration has certain practical advantages over infusion and is therefore the preferred method of administration into the CSF in certain embodiments. In the present study, the preferred method of administration into the CSF is by bolus IT injection.

[0266] Example 10 – Generation of a mouse model of severe SMA and ICV treatment We generated mice with a severe SMA phenotype (sSMA mice). Homozygous sSMA mice , have two copies of human SMN2 and no mouse SMN. Average lifespan is approximately 10 days. SMA mice also have smaller, shorter tails. Heterozygotes carry mouse SMN and develop normally.

[0267] To study the effect of antisense compounds in these sSMA mice, 20 μg of ISIS 396443 was injected ICV on day P1. Treatment resulted in a median survival of 9.9 days (saline-treated controls). RT-PCR analysis revealed that full-length SMN RNA was expressed in tissues from treated mice. An increase in

[0268] Example 11 – Systemic Administration of ISIS 396443 sSMA mice and healthy heterozygous control mice were grouped and the effects of ISIS 396443 were studied by bolus ICV injection and / or bolus subcutaneous injection (SC) as follows: did: Group 1 - ICV+SC One ICV injection delivered 20 μg on P1 or P2 (postnatal day 1 or 2); and two subcutaneous injections delivered 50 μg / g between P0 and P3.

[0269] Group 2 - SC+SC Two SC injections delivered 50 μg / g between P0 and P3; and one subcutaneous injection delivered 50 μg / g between P5 and P6; and one subcutaneous injection delivered 50 μg / g between P9 and P10.

[0270] Group 3 - SC Two SC injections delivered 50 μg / g between P0 and P3.

[0271] Group 4 - SMA saline control One ICV injection delivered saline to P1 or P2; and two subcutaneous injections delivered saline. Saline delivered between P0 and P3.

[0272] Group 5 - Heterozygote control One ICV injection delivered 20 μg to P1 or P2; and two subcutaneous injections delivered 50 μg / g. Delivery between P0 and P3 in heterozygous mice.

[0273] Each group contained between 14 and 22 mice. The survival (in days) for individual mice in each group is presented in the table below. Many mice in this study are still alive at the time of preparation for this patent application. Thus, a value marked with ">" indicates that the mouse survived that number of days and is still alive.

[0274] [Table 11]

[0275] Example 12 – Dose-Response of SC Administration The survival of sSMA mice receiving different doses of subcutaneous ISIS 396443 was evaluated according to the following treatment groups:

[0276] Group 1-SC400 (dose range 80 mg / kg to 180 mg / kg) Two SC injections, totaling 400 μg per mouse, were delivered on P0–P3, with the first dose being 150 μg (3 μl volume) on P0 or P1 and the second dose being 250 μg delivered on P2 or P3. (5 μl volume).

[0277] Group 2-SC200 (dose range 40 mg / kg to 90 mg / kg) Two SC injections, totaling 200 μg per mouse, were delivered between P0 and P3, with the first dose being 75 μg (1.5 μl volume) on P0-P1 and the second dose being 125 μg (2.5 μl volume) delivered on P2 or P3.

[0278] Group 3-SC100 (dose range 20 mg / kg to 45 mg / kg) Two SC injections, totaling 100 μg per mouse, were delivered between P0 and P3, with the first dose being 40 μg (2 μl volume) on P0 or P1 and the second dose being 60 μg delivered on P2 or P3. There was (volume 3 μl).

[0279] Group 4 - SMA saline (negative control) Between P0 and P3, two SC injections of saline were administered, the first at P0 or P1 (5 μl volume) and the second at P2 or P3 (5 μl volume).

[0280] Group 5 – heterozygous controls (positive control) Mice without treatment.

[0281] Each group contained between 14 and 26 mice. The survival (in days) for individual mice in each group is presented in the table below. Many mice in this study are still alive at the time of preparation of this patent application. Thus, a value marked with ">" indicates that the mouse survived that number of days and is still alive.

[0282] [Table 12]

[0283] Example 13 – ICV drip vs. ICV bolus Administration by intracerebroventricular bolus injection (ICV bolus) versus continuous intracerebroventricular infusion (ICV infusion) SMA type III transgenic mice were administered ISIS 387954. ICV infusion mice were infused with a total dose of 0 (PBS control), 87.5 μg, 175 μg, 350 μg, or 700 μg over 7 days and then sacrificed 2 days later. ICV bolus mice were infused with ISIS 387954 over 7 days and sacrificed 2 days later. The same total dose of 0 (PBS control), 87.5 μg, 175 μg, 350 μg, or 700 μg was injected in a single ICV injection and then sacrificed 9 days later. There were 5 mice in each group. RNA was administered via the lumbar spinal cord. The pith was harvested and analyzed by real-time PCR. Intron 7 content was normalized to saline-treated controls. The results are summarized in the table below.

[0284] [Table 13]

[0285] In this experiment, the same dose when delivered by ICV bolus injection resulted in a It provided greater activity than when delivered by infusion over a 7-day period.

[0286] Real-time PCR was also performed to measure the expression levels of allograft inflammatory factor 1 (AIF1). Inflammation was assessed by ELISA using a 2-step ELISA. None of the samples from treated mice showed any significant differences compared to control mice.

[0287] Example 14 – ICV bolus dose-response Additional doses of intracerebroventricular bolus administration were tested. Transgenic mice received a single dose of 0, 10.9 μg, 21.9 μg, 43.4 μg, 87.5 μg, or 175 μg of ISIS 387954. The mice were administered by bolus ICV injection and sacrificed 9 days later as described in Example 13. Samples were taken from the brain and lumbar spinal cord. RNA was prepared and analyzed by RT-PCR for changes in intron 7 inclusion and for changes in AIF1. None of the samples showed changes in AIF1 compared to the control. The results from intron 7 inclusion were summarized below. The results are summarized in the table below. The ED50 is approximately 22 μg.

[0288] [Table 14]

Claims

1. A method comprising administering to a subject an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2 pre-mRNA, wherein the antisense compound is administered into cerebrospinal fluid.

2. The method of claim 1 , wherein the administration is performed into the subarachnoid space.

3. The method of claim 1, wherein the administration is into the cerebrospinal fluid in the brain.

4. 4. The method of any one of claims 1 to 3, wherein administering comprises bolus injection.

5. 4. The method of any one of claims 1 to 3, wherein administering comprises infusion using a delivery pump.

6. 6. The method of any one of claims 1 to 5, wherein the antisense compound is administered at a dose of 0.01 to 10 milligrams of antisense compound per kg of subject body weight.

7. 7. The method of claim 6, wherein the dose is 0.01 to 10 milligrams of antisense compound per kg of subject body weight.

8. The dosage is 0.01 to 5 milligrams of antisense compound per kg of subject body weight. The method described in item 6.

9. The dosage is 0.05 to 1 milligram of antisense compound per kg of subject body weight. The method described in item 6.

10. The dosage is 0.01 to 0.5 milligrams of antisense compound per kg of subject body weight. The method according to claim 6.

11. The dose is 0.05 to 0.5 milligrams of antisense compound per kg of subject body weight. The method according to claim 6.

12. 12. The method of any of claims 6 to 11, wherein the dose is administered daily.

13. 12. The method of any of claims 6 to 11, wherein the dose is administered once a week.

14. The antisense compound is administered continuously and the dose is the amount administered per day. The method according to any one of claims 6 to 11.

15. At least one induction dose should be administered during the induction phase and at least one induction dose should be administered during the maintenance phase.

14. The method of any of claims 1 to 13, comprising administering at least one maintenance dose.

16. the induction phase is 0.05 to 5.0 milligrams of antisense compound per kg of subject body weight; 16. The method of claim 15.

17. The maintenance dose is 0.01 to 1.0 milligrams of antisense compound per kg of subject body weight.

17. The method of claim 15 or 16.

18. 18. The method according to any one of claims 15 to 17, wherein the duration of the induction phase is at least one week. 。

19. 19. The method of any one of claims 15 to 18, wherein the duration of the maintenance phase is at least one week. 。

20. 20. The method of claim 15, wherein each induction dose and each maintenance dose comprises one injection. A method described in any of the above.

21. Each induction dose and each maintenance dose may be administered independently as two or more injections.

20. The method of any of claims 15 to 19, comprising:

22. 22. The method of any of claims 1-21, wherein the antisense compound is administered at least twice over a treatment period of at least one week.

23. 23. The method of claim 22, wherein the treatment period is at least one month.

24. 23. The method of claim 22, wherein the treatment period is at least two months.

25. 23. The method of claim 22, wherein the treatment period is at least 4 months.

26. The induction phase is administered by one or more bolus injections, and the maintenance dose is administered by infusion.

26. The method of any one of claims 15 to 25, wherein the administration is by pump.

27. 27. A method according to any preceding claim, comprising assessing the tolerability and / or efficacy of an antisense compound.

28. 28. The method of any one of claims 1 to 27, wherein the dosage or frequency of administration of the antisense compound is reduced according to the indication in which administration of the antisense compound is unacceptable.

29. The method of any one of claims 1 to 28, wherein the dosage or frequency of administration of the antisense compound is maintained or reduced according to the indication for which administration of the antisense compound is effective. Law.

30. 30. The method of any one of claims 1 to 29, wherein the dosage of the antisense compound is increased according to an indication in which administration of the antisense compound is ineffective.

31. 31. The method of any one of claims 1 to 30, wherein the frequency of administration of the antisense compound is reduced according to the indication for which administration of the antisense compound is effective.

32. 32. The method of any one of claims 1 to 31, wherein the frequency of administration of the antisense compound is increased according to an indication for which administration of the antisense compound is not effective.

33. Claims including co-administration of the antisense compound with at least one other treatment. Item 33. The method according to any one of items 1 to 32.

34. 34. The method of claim 33, wherein the antisense compound and at least one other treatment are co-administered simultaneously.

35. The antisense compound is administered prior to the administration of at least one other treatment. Item 35. The method of claim 34.

36. 35. The method of claim 34, wherein the antisense compound is administered after administration of at least one other treatment.

37. At least one other treatment was valproic acid, riluzole, hydroxyurea, and and butyrate. How it is done.

38. Claim 33, wherein the at least one other treatment comprises administering trichostatin-A.

37. A method according to any one of claims 1 to 37.

39. Any of claims 33 to 38, wherein the at least one other treatment comprises administering stem cells. The method described in either

40. 40. The method according to any one of claims 33 to 39, wherein the at least one other treatment is a gene therapy. How it is done.

41. The antisense compound is administered at a concentration of about 0.01 mg / ml, about 0.05 mg / ml, about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 5 mg / ml, about 10 mg / ml, about 50 mg / ml, or about 100 mg / ml; 41. A method according to any one of claims 1 to 40.

42. 1. The method of claim 1, wherein the content of exon 7 of SMN2 mRNA in the motor neurons of the subject is increased.

41. A method according to any one of claims 1 to 41.

43. The content of exon 7 amino acid of SMN2 polypeptide in the motor neurons of the subject is increased.

43. The method of any one of claims 1 to 42, wherein the

44. administering to the subject an antisense oligonucleotide complementary to intron 7 of the nucleic acid encoding human SMN2; A method for increasing the content of exon 7 of SMN2 mRNA in motor neurons of a subject, comprising administering an antisense compound comprising an antisense oligonucleotide, thereby increasing the content of exon 7 of SMN2 mRNA in the motor neurons of the subject.

45. administering to the subject an antisense oligonucleotide complementary to intron 7 of the nucleic acid encoding human SMN2; administering an antisense compound comprising an antisense oligonucleotide, thereby increasing the content of exon 7 amino acids in SMN2 polypeptide in motor neurons of the subject. and detecting exon 7 in the SMN2 polypeptide in the motor neuron of the subject. How to increase amino acid content.

46. 46. ​​The method of any of claims 1 to 45, wherein the subject has SMA.

47. 46. ​​The method of any of claims 1 to 45, wherein the subject has type 1 SMA.

48. 46. ​​The method of any of claims 1 to 45, wherein the subject has SMA type II.

49. 46. ​​The method of any of claims 1 to 45, wherein the subject has SMA type III.

50. 50. The method of any preceding claim, wherein the first dose is administered in uterus.

51. 51. The method of claim 50, wherein the first dose is administered before the formation of the blood-brain barrier is complete. method.

52. 50. The method of claim 1, wherein the first dose is administered within the first week of the subject's life. How to do it.

53. 50. The method of claim 1, wherein the first dose is administered within the first month of the subject's life. How to do it.

54. 50. The method of claim 1, wherein the first dose is administered within the first three months of the subject's life. How to do it.

55. 50. The method of claim 1, wherein the first dose is administered within the first six months of the subject's life. How to do it.

56. 50. The method of any of claims 1-49, wherein the first dose is administered when the subject is 1 to 2 years old.

57. 50. The method of claim 1, wherein the first dose is administered when the subject is 1 to 15 years old. How it is done.

58. 50. The method of any of claims 1-49, wherein the first dose is administered when the subject is 15 years of age or older.

59. 59. The method of any one of claims 1 to 58, wherein the subject is a mammal.

60. 60. The method of claim 59, wherein the subject is a human.

61. 61. The method of any of claims 1 to 60, comprising identifying a subject with SMA.

62. identifying the subject by measuring the electrical activity of one or more of the subject's muscles; 62. The method of claim 61.

63. 62. The method of claim 61, wherein the subject is identified by genetic testing to determine whether the subject has a mutation in the subject's SMN1 gene.

64. 62. The method of claim 61, wherein the subject is identified by muscle biopsy.

65. 43. The method of claim 42, wherein administering the antisense compound results in an increase in the amount of SMN2 mRNA having exon 7 by at least 10%.

66. 66. The method of claim 65, wherein the increase in the amount of SMN2 mRNA having exon 7 is at least 20%.

67. 66. The method of claim 65, wherein the increase in the amount of SMN2 mRNA having exon 7 is at least 50%.

68. 66. The method of claim 65, wherein the increase in the amount of SMN2 mRNA having exon 7 is at least 70%.

69. Administration of antisense compounds resulted in the identification of at least 10% of SMNs containing exon 7 amino acids.

44. The method of claim 43, which results in an increased amount of the two polypeptides.

70. an increase in the amount of SMN2 polypeptide containing amino acids from exon 7 of at least 20% 70. The method of claim 69.

71. 70. The method of claim 69, wherein the increase in the amount of SMN2 polypeptide having amino acids of exon 7 is at least 50%.

72. 70. The method of claim 69, wherein the increase in the amount of SMN2 polypeptide having amino acids of exon 7 is at least 70%.

73. 73. The method of any of claims 1-72, wherein administering an antisense compound ameliorates at least one symptom of SMA in a subject.

74. 74. The method of claim 73, wherein administering the antisense compound results in an improvement in motor function in the subject.

75. 74. The method of claim 73, wherein administering the antisense compound results in slowing or reducing loss of motor function in the subject.

76. 74. The method of claim 73, wherein administering the antisense compound results in improved respiratory function.

77. 74. The method of claim 73, wherein administering the antisense compound results in improved survival.

78. At least one nucleoside of the antisense oligonucleotide contains a modified sugar moiety.

78. The method of any one of claims 1 to 77.

79. 80. The method of claim 78, wherein at least one modified sugar moiety comprises a 2'-methoxyethyl sugar moiety.

80. 80. The method of any of claims 1-79, wherein essentially each nucleoside of the antisense oligonucleotide comprises a modified sugar moiety.

81. 81. The method of claim 80, wherein all of the nucleosides comprising a modified sugar moiety comprise the same sugar modification.

82. 82. The method of claim 81, wherein each modified sugar moiety comprises a 2'-methoxyethyl sugar moiety. Law.

83. 80. The method of any of claims 1-79, wherein each nucleoside of the antisense oligonucleotide comprises a modified sugar moiety.

84. 84. The method of claim 83, wherein all of the nucleosides contain the same sugar modification.

85. 85. The method of claim 84, wherein each modified sugar moiety comprises a 2'-methoxyethyl sugar moiety. Law.

86. At least one internucleoside linkage is a phosphorothioate internucleoside linkage.

86. The method of any one of claims 1 to 85.

87. 87. The method of claim 86, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

88. 88. The method of any one of claims 1 to 87, wherein the antisense oligonucleotide consists of 10 to 25 linked nucleosides.

89. 88. The method of any one of claims 1 to 87, wherein the antisense oligonucleotide consists of 12 to 22 linked nucleosides.

90. 88. The method of any one of claims 1 to 87, wherein the antisense oligonucleotide consists of 15 to 20 linked nucleosides.

91. Claim 1, wherein the antisense oligonucleotide consists of 18 linked nucleosides.

87. A method according to any one of claims 1 to 87.

92. 92. The method of any of claims 1 to 91, wherein the antisense oligonucleotide is at least 90% complementary to a nucleic acid encoding human SMN2.

93. 93. The method of claim 92, wherein the antisense oligonucleotide is fully complementary to a nucleic acid encoding human SMN2.

94. 94. The method of any of claims 1-93, wherein the oligonucleotide has a nucleobase sequence comprising at least 10 consecutive nucleobases of the nucleobase sequence SEQ ID NO:

1.

95. 95. The method of Claim 94, wherein the oligonucleotide has a nucleobase sequence comprising at least 15 consecutive nucleobases of the nucleobase sequence SEQ ID NO:

1.

96. 95. The method of claim 94, wherein the oligonucleotide has a nucleobase sequence comprising the nucleobase sequence SEQ ID NO:

1.

97. 95. The method of claim 94, wherein the oligonucleotide has a nucleobase sequence consisting of the nucleobase sequence SEQ ID NO:

1.

98. 98. The antisense compound according to any one of claims 1 to 97, wherein the antisense compound comprises a conjugate group or a terminal group. How to do it.

99. 98. The method of claim 1, wherein the antisense compound comprises an antisense oligonucleotide. The method described in either

100. The method of any one of claims 1 to 99, wherein the antisense compound is also administered systemically. Law.

101. The method of claim 100, wherein the systemic administration is by intravenous or intraperitoneal injection. Law.

102. 102. The method of claim 100 or 101, wherein the systemic administration and the administration to the central nervous system are performed simultaneously.

103. 102. The method of claim 100 or 101, wherein the systemic administration and the administration to the central nervous system occur at different times.

104. A method according to any one of claims 1 to 103, comprising coding for human SMN2. an antisense oligonucleotide complementary to intron 7 of the nucleic acid to be read; Antisense compounds.

105. For use in treating diseases or conditions associated with survival motor neuron 1 (SMN1).

105. The antisense compound of claim 104.

106. In the manufacture of a medicament for use in the method of any one of claims 1 to 105 , an antisense oligonucleotide complementary to intron 7 of the nucleic acid encoding human SMN2 Use of antisense compounds containing otides.

107. The drug is intended to treat a disease or condition associated with survival motor neuron 1 (SMN1). The use according to claim 106,

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