Combination Therapy for Spinal Muscular Atrophy

Abstract

Aspects of the present application relate to compositions and methods for treating spinal muscular atrophy in a subject. In particular, the present application provides therapeutic combinations of small molecules that promote SMN function, and / or recombinant nucleic acids (e.g., in viral vectors) encoding survival motor neuron 1 (SMN1) protein, and / or antisense oligonucleotides (ASOs) that increase full-length survival motor neuron 2 (SMN2) mRNA (e.g., targeted to a nucleic acid molecule encoding survival motor neuron 2 (SMN2) and promote inclusion of exon 7 in SMN2 mRNA).

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 887,579, filed on Aug. 15, 2019, which is hereby incorporated by reference in its entirety.

[0002] Field This application relates to methods and compositions for treating spinal muscular atrophy (SMA).

Background Art

[0003] Background Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations or deletions in the telomeric SMN1 gene, which encodes a ubiquitously expressed protein (survival motor neuron - SMN) involved in the biogenesis of the spliceosome.

[0004] The SMN gene product is present within cells, and the deficiency of SMN causes selective toxicity to lower motor neurons, leading to progressive neuron loss and muscle weakness. The severity of the disease is modified by the copy number of the centromeric duplication of the homologous gene (SMN2) that results in the production of very small amounts of full - length SMN transcripts. Patients with 1 - 2 copies of SMN2 exhibit a severe form of SMA characterized by onset within the first few months of life and rapid progression to respiratory failure. Patients with 3 copies of SMN2 generally exhibit a milder form of the disease, typically presenting after 6 months of age. Many never achieve the ability to walk, but patients rarely progress to respiratory failure and often live into adulthood. In patients with 4 copies of SMN2, the disease may not manifest until adulthood, and muscle weakness develops gradually.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Several treatments for SMA have been developed, but there remains a need for a treatment that increases intracellular SMN activity in motor neurons involved in spinal muscular atrophy in patients with different levels of disease severity.

Means for Solving the Problems

[0006] Overview In some aspects, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject having SMA, the method comprising administering to the subject a) a small molecule that increases SMN function, and b) a recombinant nucleic acid encoding a survival motor neuron 1 (SMN1) protein.

[0007] In some aspects, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject having SMA, the method comprising administering to the subject a) a small molecule that increases SMN function, and b) an antisense oligonucleotide (ASO) that increases full-length survival motor neuron 2 (SMN2) mRNA.

[0008] In some aspects, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject having SMA, the method comprising administering to the subject a) a small molecule that increases SMN function, b) a recombinant nucleic acid encoding a survival motor neuron 1 (SMN1) protein, and c) an antisense oligonucleotide (ASO) that increases full-length survival motor neuron 2 (SMN2) mRNA.

[0009] In some aspects, the present application relates to a combination therapy for spinal muscular atrophy (SMA) comprising administration (e.g., in combination or sequentially) of a small molecule that increases SMN function and a recombinant nucleic acid encoding survival motor neuron 1 (SMN1) and / or an oligomeric compound that increases full-length survival motor neuron 2 (SMN2) mRNA to a subject having SMA. In some aspects, the small molecule that increases SMN function is a small molecule that increases full-length SMN2 mRNA in the subject. In some aspects, the recombinant nucleic acid encoding SMN1 is provided by a viral vector, e.g., a recombinant adeno-associated virus (rAAV). In some aspects, the oligomeric compound is an antisense oligonucleotide (ASO) that increases full-length SMN2 mRNA in the subject (e.g., by modulating SMN2 pre-mRNA splicing to increase inclusion of exon 7 in SMN2 mRNA).

[0010] In some aspects, the present application relates to a combination therapy for spinal muscular atrophy (SMA) comprising administration (e.g., in combination or sequentially) of a small molecule that increases SMN function and a recombinant nucleic acid encoding survival motor neuron 1 (SMN1) and / or a nucleic acid encoding survival motor neuron 2 (SMN2) mRNA that modulates exon skipping (e.g., promotes exon 7 inclusion) to a subject having SMA. In some aspects, the small molecule that increases SMN function is a small molecule that increases full-length SMN2 mRNA in the subject. In some aspects, the recombinant nucleic acid encoding SMN1 is provided by a viral vector, e.g., a recombinant adeno-associated virus (rAAV). In some aspects, the oligomeric compound that induces exon skipping in the nucleic acid encoding SMN2 is an antisense oligonucleotide (ASO) that modulates exon skipping in SMN2 pre-mRNA (e.g., promotes exon 7 inclusion).

[0011] In some embodiments, the small molecule that increases SMN function is a splice modulator, an HDAC inhibitor, or a molecule that modulates the activity of an mRNA decapping enzyme. In some embodiments, the small molecule is a splice modulator. In some embodiments, the splice modulator is an SMN2 splice modulator. In some embodiments, the splice modulator is a 7-disubstituted phenyltetracycline. In some embodiments, the splice modulator is a substituted isoindolinone. In some embodiments, the splice modulator is a substituted carbazole derivative. In some embodiments, the SMN2 splice modulator is a substituted 1,4-diazepane. In some embodiments, the SMN2 splice modulator is a substituted pyridazine. In some embodiments, the SMN2 splice modulator is risdiplam. In some embodiments, the SMN2 splice modulator is branaplam.

[0012] In some embodiments, small molecules that increase SMN function (e.g., risdiplam or branaplam), recombinant nucleic acids (e.g., in viral vectors such as rAAV), and / or SMN2 ASOs (e.g., nusinersen) are provided as separate compositions, but are administered to a subject together (e.g., simultaneously or contemporaneously, e.g., during the same clinical visit, e.g., within the same time or on the same day). In some embodiments, small molecules that increase SMN function (e.g., risdiplam or branaplam), recombinant nucleic acids (e.g., in viral vectors such as rAAV), and / or SMN2 ASOs (e.g., nusinersen) are provided as separate compositions and are sequentially administered to a subject during separate clinical visits (e.g., at different times, e.g., on different days) during the course of treatment (e.g., during a treatment regimen for 1 week, 2 - 4 weeks, 1 month, 1 - 12 months, 1 year, 2 - 5 years, or longer). In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam) is administered before and / or after a recombinant nucleic acid (e.g., rAAV) and / or SMN2 ASO (e.g., nusinersen). In some embodiments, small molecules that increase SMN function (e.g., risdiplam or branaplam), recombinant nucleic acids (e.g., in viral vectors such as rAAV), and / or SMN2 ASOs (e.g., nusinersen) are administered at different frequencies (e.g., together or sequentially). In some embodiments, a subject is treated with a combination of separate compositions, each comprising a small molecule that increases SMN function (e.g., risdiplam or branaplam), a recombinant nucleic acid (e.g., in viral vectors such as rAAV), or an ASO, where the compositions are administered at different frequencies (e.g., together or sequentially).

[0013] In some embodiments, a small molecule (e.g., risdiplam or branaplam) that increases two or more different SMN functions is administered to a subject. In some embodiments, two or more different recombinant SMN1 nucleic acids (e.g., in rAAV) are administered to a subject. In some embodiments, two or more different SMN2 ASOs are administered to a subject. In some embodiments, different recombinant SMN1 nucleic acids (e.g., in rAAV) and / or different SMN2 ASOs are administered to a subject during different clinic visits.

[0014] Thus, in some embodiments, a method of treating SMA in a subject (e.g., a human subject) having SMA comprises administering to the subject a small molecule (e.g., risdiplam or branaplam) that increases SMN function, a recombinant nucleic acid encoding SMN1 (also referred to as a recombinant SMN1 gene) (e.g., in rAAV), and / or an SMN2 ASO (also referred to as an SMN2 ASO) that increases full-length SMN2 mRNA in the subject. In some embodiments, a method of treating SMA in a subject comprises administering to a subject having SMA an effective amount of a small molecule (e.g., risdiplam or branaplam) that increases SMN function, a recombinant SMN1 gene (e.g., in rAAV), and / or an SMN2 ASO (e.g., nusinersen).

[0015] In some embodiments, a subject having SMA has one or more symptoms of SMA (e.g., atrophy of limb muscles, difficulty walking or inability to walk, breathing difficulty, or other symptoms of SMA). In some embodiments, a subject having SMA has two variant alleles of the genomic SMN1 gene. In some embodiments, the subject has a deletion or mutation (e.g., a loss-of-function point mutation) in each SMN1 allele. In some embodiments, the subject is homozygous for the SMN1 gene mutation. In some embodiments, the subject is heterozygous for two different SMN1 gene mutations.

[0016] In some embodiments, the subject is a human subject. In some embodiments, the subject is selected from the group of pediatric and adult subjects. In some embodiments, the subject is 18 years of age or older (e.g., 18 years of age or older). In some embodiments, the subject is younger than 18 years of age, younger than 10 years of age, or younger than 6 years of age. In some embodiments, the subject is approximately 2 weeks old, 1 month old, 3 months old, 6 months old, 1 year old, 2 years old, 3 years old, 4 years old, or 5 years old.

[0017] In some embodiments, the recombinant SMN1 gene is operably linked to a promoter. In some embodiments, the SMN1 gene is the human SMN1 gene. In some embodiments, the SMN1 gene is codon-optimized (e.g., for expression in humans). In some embodiments, the recombinant nucleic acid encoding the SMN1 gene is a recombinant AAV genome comprising adjacent AAV inverted terminal repeat sequences (ITRs). In some embodiments, the recombinant nucleic acid is administered within an AAV particle. In some embodiments, the AAV particle comprises an AAV capsid protein (e.g., an AAV9, AAVrh10, AAV8 capsid protein). In some embodiments, the AAV particle comprises an AAVhu68 capsid protein. In some embodiments, the AAV particle comprises an AAV9 capsid protein.

[0018] In some embodiments, the SMN2 ASO alters the splicing pattern of the survival motor neuron 2 (SMN2) pre-mRNA. In some embodiments, the SMN2 ASO promotes the inclusion of exon 7 in the survival motor neuron 2 (SMN2) mRNA. In some embodiments, the SMN2 ASO comprises a sequence complementary to intron 6 or intron 7 of a nucleic acid molecule encoding the SMN2 protein (e.g., the SMN2 gene or SMN2 pre-mRNA). In some embodiments, the SMN2 ASO comprises a sequence complementary to intron 6 of a nucleic acid molecule encoding the SMN2 protein (e.g., the SMN2 gene or SMN2 pre-mRNA). In some embodiments, the SMN2 ASO comprises a sequence complementary to intron 7 of a nucleic acid molecule encoding the SMN2 protein (e.g., the SMN2 gene or SMN2 pre-mRNA). In some embodiments, the SMN2 ASO (e.g., nusinersen) comprises the sequence of SEQ ID NO: 1, 25, or 26. In some embodiments, the ASO is nusinersen. In some embodiments, the SMN2 ASO (e.g., nusinersen) comprises one or more nucleic acid base or backbone modifications.

[0019] In some embodiments, the recombinant SMN1 gene (e.g., in a viral vector) is administered (e.g., once or multiple times) to a subject previously treated with a small molecule and / or SMN2 ASO (e.g., nusinersen) therapy that increases SMN function. In some embodiments, the recombinant SMN1 gene (e.g., in a viral vector such as rAAV) is administered (e.g., once or multiple times) to a subject currently being treated with a small molecule (e.g., risdiplam or branaplam) and / or SMN2 ASO (e.g., nusinersen) therapy that increases SMN function. In some embodiments, a treatment is initiated for a subject that includes co-administration or sequential administration of a small molecule (e.g., risdiplam or branaplam) that increases SMN function and a) a recombinant SMN1 gene (e.g., in a viral vector such as rAAV) and / or b) an SMN2 ASO (e.g., nusinersen).

[0020] In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam), and a) rAAV containing a recombinant SMN1 gene (also referred to as SMN1 rAAV) and / or b) an SMN2 ASO (e.g., nusinersen) are administered simultaneously. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV, and / or an SMN2 ASO are administered together. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV, and / or an SMN2 ASO (e.g., nusinersen) are administered separately in different compositions. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV, and / or an SMN2 ASO (e.g., nusinersen) are administered sequentially. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV, and / or an SMN2 ASO (e.g., nusinersen) are administered at different frequencies. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam) is administered 1 to 6 times per year, or more frequently (e.g., weekly, or 2 to 4 times per month). In some embodiments, SMN1 rAAV is administered once. In some embodiments, SMN2 ASO is administered 1 to 6 times per year. In some embodiments, subsequent administrations of a small molecule that increases SMN function (e.g., risdiplam or branaplam) alone and / or together with an SMN2 ASO (e.g., nusinersen) two or more times are performed after the first administration of SMN1 rAAV and SMN2 ASO (e.g., nusinersen). In some embodiments, the subject receives one or more additional administrations of SMN1 rAAV. In some embodiments, the first and second administrations of SMN1 rAAV are provided to the subject more than 6 months apart, or more than 1 year apart. In some embodiments, the first and second SMN1 rAAV compositions contain the same rAAV capsid protein. In some embodiments, the first and second SMN1 rAAV compositions contain different rAAV capsid proteins.

[0021] In some embodiments, the SMN1 rAAV is administered at a dose of 1×10 10 ~5×10 14 GC. In some embodiments, the SMN1 rAAV is administered at a dose of 2×10 10 ~2×10 14 GC. In some embodiments, the SMN1 rAAV is administered at a dose of 3×10 13 ~5×10 14 GC. In some embodiments, the SMN1 rAAV is administered at a dose of 2×10 14 GC.

[0022] In some embodiments, a total of 5 mg to 60 mg of SMN2 ASO per dose is administered to the subject. In some embodiments, a total of 5 mg to 20 mg of SMN2 ASO per dose is administered to the subject. In some embodiments, a total of 12 mg to 50 mg of SMN2 ASO per dose is administered to the subject. In some embodiments, a total of 12 mg to 48 mg of SMN2 ASO per dose is administered to the subject. In some embodiments, a total of 12 mg to 36 mg of SMN2 ASO per dose is administered to the subject. In some embodiments, a total of 28 mg of SMN2 ASO per dose is administered to the subject. In some embodiments, a total of 12 mg of SMN2 ASO per dose is administered to the subject. In some embodiments, the administration volume is 5 mL.

[0023] In some embodiments, the small molecule is administered via a suitable route (e.g., oral), and the rAAV and / or SMN2 ASO are administered independently, e.g., (via injection or infusion), via a route suitable for the treatment(s), e.g., intrathecal, intracisternal space, intravenous or intramuscular administration. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) are administered into the intrathecal space of the subject. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) are administered into the intracisternal space of the subject. In some embodiments, the initial and / or subsequent administrations of a small molecule that increases SMN function (e.g., risdiplam or branaplam), recombinant SMN1 gene (e.g., in rAAV) and / or SMN2 ASO (e.g., nusinersen) are performed intravenously or intramuscularly.

[0024] In some embodiments, the administration of a small molecule that increases SMN function (e.g., risdiplam or branaplam) and SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) increases the intracellular SMN protein level in the subject. In some embodiments, the SMN protein level increases in the cervical, thoracic and lumbar spinal cords of the subject (e.g., in motor neurons in the brain and / or spinal cord of the subject).

[0025] In some embodiments, SMN protein expression in a subject having SMA is increased by administering to the subject a small molecule (e.g., risdiplam or branaplam) that increases an effective amount of SMN function and SMN1 rAAV and / or an SMN2 ASO (e.g., nusinersen) (e.g., together or sequentially). In some embodiments, the subject has been previously treated with a small molecule (e.g., risdiplam or branaplam) that increases SMN function. In some embodiments, the subject has been previously administered SMN1 rAAV. In some embodiments, the subject has been previously treated with an SMN2 ASO (e.g., nusinersen). In some embodiments, SMN protein expression in a subject previously treated with SMN1 rAAV is increased by administering to the subject a small molecule (e.g., risdiplam or branaplam) that increases an effective amount of SMN function and / or an SMN2 ASO (e.g., nusinersen). In some embodiments, SMN protein expression in a subject previously treated with an SMN2 ASO (e.g., nusinersen) is increased by administering to the subject a small molecule (e.g., risdiplam or branaplam) that increases an effective amount of SMN function and / or SMN1 rAAV. In some embodiments, SMN protein expression in a subject previously treated with a small molecule (e.g., risdiplam or branaplam) that increases SMN function is increased by administering to the subject an effective amount of SMN1 rAAV and / or an SMN2 ASO (e.g., nusinersen).

[0026] In some embodiments, the composition comprises a small molecule that increases SMN function (e.g., risdiplam or branaplam). In some embodiments, the composition comprises a recombinant SMN1 gene (e.g., in rAAV). In some embodiments, the composition comprises an SMN2 ASO (e.g., nusinersen). In some embodiments, the pharmaceutical compositions described herein further comprise a pharmaceutically acceptable carrier. In some embodiments, a therapeutically effective amount of the pharmaceutical composition is administered to a subject in need thereof. Any of the compositions described herein can be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprising a small molecule that increases SMN function (e.g., risdiplam or branaplam) is administered to a subject via any known route suitable for administering a small molecule drug (e.g., oral administration). In some embodiments, a pharmaceutical composition comprising a recombinant SMN1 gene is administered to a subject via any known route suitable for administering a recombinant SMN1 gene (e.g., via intravenous injection). In some embodiments, a pharmaceutical composition comprising an SMN2 ASO (e.g., nusinersen) is administered to a subject via any known route suitable for administering an ASO (e.g., intrathecal injection). In some embodiments, one or more of a small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV, and / or SMN2 ASO (e.g., nusinersen) (e.g., as two or three separate compositions) are administered to a subject (e.g., a human subject) via the intrathecal route. In some embodiments, one or more of a small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV, and / or SMN2 ASO are (e.g., as two or three separate compositions) administered to the spinal canal, subarachnoid space, ventricle, or lumbar CSF by suboccipital puncture or by another suitable route (e.g., via injection, infusion, using a pump and catheter, or via another suitable technique).In some embodiments, one or more of a small molecule that increases SMN function (e.g., risdiplam or branaplam), a recombinant SMN1 gene (e.g., in rAAV), and / or an SMN2 ASO are administered (e.g., as two or three separate compositions) to a subject (e.g., a human subject) intracranially, intraventricularly, intracerebrally, parenchymally, intravenously, or via other suitable routes. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam) is administered to the subject via oral administration, while the SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) are administered (e.g., as two or three separate compositions) to the subject (e.g., a human subject) via injection (e.g., intravenous, intrathecal, intramuscular, intracranial, intraventricular, intracerebral, or parenchymal). In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam) is administered to the subject via oral administration, while the SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) are administered (e.g., as two or three separate compositions) to the spinal canal, subarachnoid space, ventricle, or lumbar CSF by suboccipital puncture or by other suitable routes (e.g., via injection, infusion, using a pump and catheter, or via other suitable techniques). Whether administered concomitantly or sequentially, each of the small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV, and SMN2 ASO (e.g., nusinersen) may be administered by any suitable or appropriate means known in the art (e.g., intrathecally, intravenously, etc.), and the small molecule that increases SMN function (e.g., risdiplam or branaplam), SMN1 rAAV, and SMN2 ASO (e.g., nusinersen) may be administered by the same means or by different means (e.g., via the same or different routes of administration).

[0027] In some embodiments, a small molecule (e.g., risdiplam or branaplam) that increases SMN function, SMN1 rAAV, and / or an SMN2 ASO (e.g., nusinersen) are used in the manufacture of a medicament (e.g., as two or three separate medicaments) for treating a disease or condition associated with survival motor neuron protein (SMN), such as spinal muscular atrophy (SMA).

[0028] In some embodiments, the disclosure relates to a method of treating SMA in a subject having spinal muscular atrophy (SMA), the method comprising administering to a subject previously treated with an ASO that increases full-length SMN2 mRNA, in separate compositions, a small molecule (e.g., risdiplam or branaplam) that increases SMN function and / or a recombinant SMN1 gene (e.g., in rAAV). In some embodiments, the ASO treatment is discontinued and the small molecule and / or recombinant SMN1 gene can be provided as a replacement therapy. In some embodiments, the ASO treatment is continued and the small molecule and / or recombinant SMN1 gene can be provided as an additional therapy (e.g., as an adjunctive therapy).

[0029] In some embodiments, the present disclosure is a method of treating spinal muscular atrophy (SMA) in a subject having SMA, the method comprising administering, in separate compositions, a small molecule (e.g., risdiplam or branaplam) that increases SMN function and / or an ASO (e.g., nusinersen) that increases full-length SMN2 mRNA to a subject previously administered a recombinant SMN1 gene (e.g., in rAAV). In some embodiments, the subject does not receive any additional recombinant SMN1 gene after administration of the small molecule and / or ASO has been initiated. In some embodiments, one or more additional administrations of the recombinant SMN1 gene and / or small molecule are administered after administration of the small molecule and / or ASO has been initiated. In some embodiments, the dosing schedule of one or more therapies can be maintained or changed when an adjunct therapy is initiated. In some embodiments, the dosing schedule of the recombinant SMN1 gene can be maintained or changed after administration of a small molecule that increases SMN function and / or an SMN2 ASO has been initiated. In some embodiments, the dosing schedule of the SMN2 ASO is maintained or changed after administration of a small molecule that increases SMN function and / or a recombinant SMN1 gene has been initiated. In some embodiments, the dosing schedule of a small molecule that increases a SMN functional gene is maintained or changed after administration of a recombinant SMN1 gene and / or an SMN2 ASO has been initiated.

[0030] In some aspects, the present disclosure relates to a method of treating spinal muscular atrophy (SMA) in a subject having SMA, the method comprising administering to a subject previously treated with a small molecule (e.g., risdiplam or branaplam) that increases SMN function an effective amount of a recombinant SMN1 gene (e.g., in rAAV) and / or an ASO (e.g., nusinersen) that increases full-length SMN2 mRNA or a separate composition to increase SMN function. In some aspects, the small molecule treatment is discontinued and the ASO and / or recombinant SMN1 gene can be provided as replacement therapy. In some aspects, the small molecule treatment is continued and the ASO and / or recombinant SMN1 gene can be provided as adjunctive therapy (e.g., as supplementary therapy).

[0031] Other aspects of the present disclosure relate to separate compositions comprising a small molecule (e.g., risdiplam or branaplam) that increases SMN function, rAAV encoding SMN1, or an ASO (e.g., nusinersen) that can increase full-length SMN2 mRNA. In some aspects, the rAAV comprises an AAV9 capsid protein. In some aspects, the ASO is nusinersen. In some aspects, the small molecule is risdiplam or branaplam. In some aspects, the composition or separate compositions are pharmaceutical compositions and comprise a pharmaceutically acceptable carrier.

[0032] Other aspects and advantages of the invention will be apparent from the following detailed description of the invention.

[0033] The following drawings form a part of this specification and are included to further demonstrate certain specific aspects of the present application, which can be better understood by reference in combination with the detailed description of the specific aspects shown in one or more of these drawings in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

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[0045] Detailed Description In some embodiments, the present application relates to compositions and methods for treating SMA in a subject, e.g., a human subject having spinal muscular atrophy (SMA).

[0046] In one aspect, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject having SMA, the method comprising administering to the subject a) a small molecule that increases SMN function, and b) a recombinant nucleic acid encoding survival motor neuron 1 (SMN1) protein.

[0047] In one aspect, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject having SMA, the method comprising administering to the subject a) a small molecule that increases SMN function, and b) an antisense oligonucleotide (ASO) that increases full-length survival motor neuron 2 (SMN2) mRNA.

[0048] In one aspect, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject having SMA, the method comprising administering to the subject a) a small molecule that increases SMN function, b) a recombinant nucleic acid encoding survival motor neuron 1 (SMN1) protein, and c) an antisense oligonucleotide (ASO) that increases full-length survival motor neuron 2 (SMN2) mRNA.

[0049] The present application relates to compositions and methods for treating SMA in a subject, such as a human subject having spinal muscular atrophy (SMA), using combination therapy.

[0050] In some embodiments, the combination therapy comprises administering to a subject having SMA a small molecule that increases SMN function in the subject (e.g., risdiplam or branaplam) and a) a recombinant nucleic acid that expresses the SMN1 gene (e.g., in a viral vector such as rAAV encoding SMN1) and / or b) an antisense oligonucleotide (ASO) that increases full-length SMN2 mRNA (e.g., an ASO that promotes inclusion of exon 7 in SMN2 mRNA such as nusinersen), (e.g., together or sequentially). "Combination therapy", "combination treatment", "combination therapy" or "combination treatment" as used herein refers to a method for treating spinal muscular atrophy (SMA) by administering to a subject one or more of the therapies described herein (e.g., recombinant SMN1 gene, SMN2 ASO, small molecule that increases SMN function, or a pharmaceutical composition of any of the foregoing).

[0051] In some embodiments, administration of a small molecule that can increase SMN function (e.g., risdiplam or branaplam), a recombinant nucleic acid that expresses SMN1 (e.g., in rAAV), and / or an SMN2 ASO (e.g., nusinersen) can provide enhanced intracellular SMN protein levels in some motor neurons and an increase in the number of motor neurons in which intracellular survival motor neuron (SMN) protein levels are elevated, compared to treatment with any of the recombinant nucleic acid, SMN2 ASO (e.g., nusinersen), or small molecule that increases SMN function (e.g., risdiplam or branaplam) alone.

[0052] Methods and compositions for administration of a small molecule that can increase SMN function (e.g., risdiplam or branaplam) and / or a recombinant nucleic acid that expresses SMN1 (e.g., in rAAV) and / or an ASO that increases full-length SMN2 mRNA (e.g., an ASO that promotes inclusion of exon 7 in SMN2 mRNA such as nusinersen) may be useful for providing therapeutically effective levels of SMN protein in subjects having SMA and for treating subjects having different levels of disease severity.

[0053] Spinal muscular atrophy or proximal spinal muscular atrophy (SMA) is a hereditary neurodegenerative disorder characterized by the loss of spinal motor neurons. SMA is an early-onset autosomal recessive disease and is currently among the top causes of death in infants. The severity of SMA varies among patients and is therefore classified into different types according to the age of onset and motor development milestones. The designation of SMA0 is proposed to reflect prenatal onset, as well as severe joint contractures, bilateral facial paralysis, and respiratory failure. Three postnatal forms of SMA have been designated. Type I SMA (also called Werdnig-Hoffmann disease) is the most severe form, with onset at birth or within 6 months after birth, and typically leads to death within 2 years. Children with type I SMA cannot sit or walk and have severe respiratory dysfunction. Type II SMA is an intermediate form with onset within the first 2 years. Children with type II SMA can sit but cannot stand or walk. Type III (also called Kugelberg-Welander disease) begins after 18 months of age to 2 years (Lefebvre et al., Hum. Mol. Genet., 1998, 7, 1531-1536) and usually progresses chronically. Children with type III SMA can stand and walk independently, at least during infancy. The adult form (type IV) is the mildest form of SMA with onset after 30 years of age, and only a few cases have been reported. Types III and IV SMA are also known as late-onset SMA.

[0054] The molecular mechanism of SMA results from the loss of both copies of the survival motor neuron gene 1 (SMN1), which may also be known as telomeric SMN, a protein that is part of multiple protein complexes thought to be involved in the biogenesis and recycling of snRNPs. SMN2, an almost identical gene that may also be known as centromeric SMN, is present in a duplicated region on chromosome 5q13 and modulates disease severity. Expression of the normal SMN1 gene alone results in the expression of the survival motor neuron (SMN) protein. SMN1 and SMN2 potentially encode the same protein, but SMN2 contains a translationally silent mutation at position +6 of exon 7, which results in insufficient inclusion of exon 7 in the SMN2 transcript. Thus, the predominant form of SMN2 is a truncated version lacking exon 7, which is unstable and inactive (Cartegni and Krainer, Nat. Genet., 2002, 30, 377-384). Expression of the SMN2 gene results in approximately 10-20% SMN protein and 80-90% unstable / non-functional SMNΔ7 protein. The SMN protein plays a well-established role in spliceosome assembly and may also mediate mRNA transport in neuronal axons and nerve terminals.

[0055] SMA is caused by the loss of homozygosity of both functional copies of the SMN1 gene. However, the SMN2 gene encodes the same protein as SMN1 and thus has the potential to overcome the genetic defect in SMA patients. SMN2 contains a translationally silent mutation (C→T) at position +6 of exon 7, which results in insufficient inclusion of exon 7 in the SMN2 transcript. Thus, the predominant form of SMN2 lacks exon 7 and is unstable and inactive. The full-size protein arising from the SMN2 gene is identical to the protein arising from a similar gene called SMN1. However, only 10-15 percent of all functional SMN protein is produced from the SMN2 gene (the rest is produced from the SMN1 gene). Typically, people have two copies of the SMN1 gene and one or two copies of the SMN2 gene in each cell. However, the number of copies of the SMN2 gene varies, and some people have up to eight copies. The more copies of the SMN2 gene one has, the more SMN protein is produced. Extra copies of the SMN2 gene can modify the severity of SMA. All individuals with spinal muscular atrophy have mutations in both copies of the SMN1 gene, which results in little or no production of SMN protein from SMN1, and the SMN2 gene can help replace some of the lack of SMN protein. In people with spinal muscular atrophy, having multiple copies of the SMN2 gene is usually associated with less severe features of the condition that develop in later life. Affected individuals with one or two functional copies of the SMN2 gene generally have severe muscle weakness beginning at birth or in infancy. Affected individuals with four or more copies of the SMN2 gene typically have mild muscle weakness that may not be noticeable until adulthood. In some embodiments, different doses and / or designs of one or more of the treatments described herein can be administered to different subjects having different numbers of SMN2 genes.

[0056] In some embodiments, intracellular SMN protein levels can be increased by contacting motor neurons with a small molecule (e.g., risdiplam or branaplam) that can increase SMN function and a) a recombinant nucleic acid encoding a recombinant SMN1 gene that promotes intracellular expression of recombinant SMN protein and / or b) an ASO that modulates intracellular SMN2 splicing to increase the percentage of cellular SMN2 transcripts that contain exon 7, thereby resulting in increased expression of full-length SMN protein from the cellular SMN2 transcripts. In some embodiments, the combination therapy comprises administering a small molecule that can increase SMN function, a recombinant nucleic acid encoding the SMN1 gene (also referred to herein as the recombinant SMN1 gene), and an SMN2 ASO that increases full-length SMN2 mRNA (e.g., an ASO that increases the intracellular level of full-length SMN2 mRNA, e.g., by promoting the inclusion of exon 7 in the SMN2 mRNA). In some embodiments, the SMN2 mRNA is nusinersen. In some embodiments, increasing the intracellular level of full-length SMN2 mRNA is useful for targeting multiple aspects of SMA and may be useful for treating a range of subjects with different disease severities, including patients with different types of SMA, including patients with different genomic copy numbers of the SMN2 gene. In some embodiments, the small molecule that increases SMN function and the recombinant SMN1 gene are administered together. In some embodiments, the small molecule that increases SMN function and the SMN2 ASO are administered together. In some embodiments, the small molecule that increases SMN function, the recombinant SMN1 gene, and the SMN2 ASO are administered together. In some embodiments, the small molecule that increases SMN function and the recombinant SMN1 gene are administered sequentially. In some embodiments, the small molecule that increases SMN function and the SMN2 ASO are administered sequentially. In some embodiments, the small molecule that increases SMN function, the recombinant SMN1 gene, and the SMN2 ASO are administered sequentially.

[0057] In some embodiments, the small molecule, recombinant SMN1 gene, or SMN2 ASO that increases SMN function is formulated separately. In some embodiments, the route of administration for each molecule can be different and is affected by the type of molecule being administered to the subject (e.g., known methods suitable for administering a recombinant gene, small molecule, or antisense oligonucleotide).

[0058] In some embodiments, the recombinant SMN1 gene (e.g., in rAAV) is formulated as a pharmaceutical composition suitable for delivering the recombinant gene to a subject. Administration of the recombinant SMN1 gene can be via any known route suitable for administering the recombinant SMN1 gene. In some embodiments, the pharmaceutical composition containing the recombinant SMN1 gene is suitable for rAAV-based delivery (e.g., an injectable solution). In some embodiments, administration of the recombinant SMN1 gene (e.g., in rAAV) for treating SMA is by injection (e.g., intravenous injection, direct injection into the CNS, or via any other suitable route).

[0059] In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branaplam) is formulated as a pharmaceutical composition suitable for delivering the small molecule drug to a subject (e.g., in the form of one or more tablets, pills, capsules, powders, granules, or solutions). Administration of the small molecule that increases SMN function can be via any known route suitable for administering a small molecule drug (e.g., oral administration). In some embodiments, the small molecule that increases SMN function is administered to the subject by oral administration.

[0060] In some embodiments, an SMN2 ASO (e.g., nusinersen) is formulated as a pharmaceutical composition suitable for delivering the oligonucleotide (e.g., as an injectable solution). Administration of the SMN2 ASO (e.g., nusinersen) can be via any known route suitable for administering an ASO. In some embodiments, an SMN2 ASO for treating SMA is administered to a subject by intracerebroventricular (ICV) injection, intravenous (IV) injection, or intrathecal (IT) injection (e.g., via lumbar puncture (LP) and / or intracisternal (ICM) delivery). In some embodiments, an SMN2 ASO for treating SMA is administered to a subject by intrathecal (IT) injection.

[0061] In some embodiments, any of the pharmaceutical compositions described herein further comprises a pharmaceutically acceptable carrier (e.g., an excipient). A pharmaceutically acceptable carrier, as used herein, refers to a carrier that is compatible with the active ingredient and / or gene therapy agent (e.g., rAAV) of the composition (and preferably can stabilize the active ingredient) and is not harmful to the subject to which it is administered. A pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier known in the art, including, but not limited to, excipients, buffers, one or more suitable salts, surfactants, antioxidants, and the like.

[0062] The pharmaceutical composition used in the method can include a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover).

[0063] The pharmaceutical composition used for in vivo administration can be sterile. This can be achieved by any means known in the art, including, but not limited to, filtration through a sterile filtration membrane.

[0064] The pharmaceutical compositions described herein can be suitable unit dosage forms known in the art, including, but not limited to, tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories.

[0065] In some embodiments, the combination treatment comprises administering (e.g., together or sequentially) a first composition comprising a small molecule that increases SMN function and a separate second composition comprising a recombinant SMN1 gene. In some embodiments, the combination treatment comprises administering (e.g., together or sequentially) a first composition comprising a small molecule that increases SMN function and a separate second composition comprising an SMN2 ASO. In some embodiments, the combination treatment comprises administering (e.g., together or sequentially) a first composition comprising a small molecule that increases SMN function, a separate second composition comprising a recombinant SMN1 gene, and a separate third composition comprising an SMN2 ASO. In some embodiments, the first and second compositions are administered together as defined herein. In some embodiments, the first, second, and third compositions are administered together as defined herein. In some embodiments, the first and second compositions are administered sequentially to the subject as defined herein. In some embodiments, the first, second, and third compositions are administered sequentially to the subject as defined herein.

[0066] Concurrent administration, as used herein, refers to the administration of two or more of the treatments described herein for treating SMA (e.g., a recombinant SMN1 gene, an SMN2 ASO, or a small molecule that increases SMN function) to a subject at the same time or at different times during the same visit. For example, during the same visit to a hospital, clinic, or other medical center, the subject is administered two or more of the treatments described herein, but the administrations can be spaced apart as defined by the individual treatments.

[0067] Sequential administration, as used herein, refers to two or more administrations of a treatment described herein (e.g., a recombinant SMN1 gene, an SMN2 ASO, or a small molecule that increases SMN function) for treating SMA under different dosing schedules. For example, the treatment may be administered on different days, weeks, months, or years during different clinic visits. The treatments described herein may be administered to a subject in any order (e.g., as determined in a treatment plan by a physician). In some embodiments, sequential administration includes the administration of each of a recombinant SMN1 gene, an SMN2 ASO, and / or a small molecule that increases SMN function described herein at different frequencies or dosing schedules.

[0068] Accordingly, in some embodiments, the first and second compositions described herein are administered to a subject separately at different times (e.g., at different times of day, on different days of the same week or month, or in different weeks, months, or years). In some embodiments, the first, second, and third compositions described herein are administered to a subject separately at different times (e.g., at different times of day, on different days of the same week, or in different weeks). In some embodiments, the first and second compositions described herein are administered at different frequencies. In some embodiments, the first, second, and third compositions described herein are administered at different frequencies. In some embodiments, a composition comprising a recombinant SMN1 gene (e.g., in rAAV) is administered less frequently than a composition comprising a small molecule that increases SMN function (e.g., risdiplam or branaplam). In some embodiments, a composition comprising a recombinant SMN1 gene (e.g., in rAAV) is administered less frequently than a composition comprising an SMN2 ASO (e.g., nusinersen) or a composition comprising a small molecule that increases SMN function (e.g., risdiplam or branaplam).

[0069] In some embodiments, the recombinant SMN1 gene is administered to a subject before the subject is treated with a small molecule or SMN2 ASO that increases SMN function. However, in other embodiments, the subject has already been treated with a small molecule and / or SMN2 ASO that increases SMN function before being administered the recombinant SMN1 gene. In some embodiments, the recombinant SMN1 gene is administered to a subject who has already received a small molecule and / or SMN2 ASO that increases SMN function.

[0070] In some embodiments, the small molecule that increases SMN function is administered to a subject before the subject is treated with the recombinant SMN1 gene and / or SMN2 ASO. However, in other embodiments, the subject is treated with the recombinant SMN1 gene and / or SMN2 ASO before being administered the small molecule that increases SMN function. In some embodiments, the small molecule that increases SMN function is administered to a subject who has already received the recombinant SMN1 gene and / or SMN2 ASO treatment. In some embodiments, the SMN2 ASO is administered to a subject before the subject is treated with the recombinant SMN1 gene and / or a small molecule that increases SMN function. However, in other embodiments, the subject is treated with the recombinant SMN1 gene and / or a small molecule that increases SMN function before being administered the SMN2 ASO. In some embodiments, the SMN2 ASO is administered to a subject who has already received the recombinant SMN1 gene and / or a small molecule that increases SMN function.

[0071] In some embodiments, the recombinant SMN1 gene (e.g., in rAAV), or the SMN2 ASO alone, or one, two or more subsequent administrations of the recombinant SMN1 gene (e.g., in rAAV) and the SMN2 ASO (e.g., nusinersen) are performed after the first administration of a small molecule that increases SMN function (e.g., risdiplam or branaplam). In some embodiments, the small molecule that increases SMN function (e.g., risdiplam or branaplam), or the SMN2 ASO (e.g., nusinersen) alone, or one, two or more subsequent administrations of the small molecule that increases SMN function (e.g., risdiplam or branaplam) and the SMN2 ASO (e.g., nusinersen) are performed after the first administration of the recombinant SMN1 gene (e.g., in rAAV). In some embodiments, the small molecule that increases SMN function (e.g., risdiplam or branaplam), or the recombinant SMN1 gene, or one, two or more subsequent administrations of the small molecule that increases SMN function (e.g., risdiplam or branaplam) and the recombinant SMN1 gene (e.g., in rAAV) are performed after the first administration of the SMN2 ASO (e.g., nusinersen). In some embodiments, one, two or more subsequent administrations of the small molecule that increases SMN function (e.g., risdiplam or branaplam) are performed after the first administration of the recombinant SMN1 gene (e.g., in rAAV) and the SMN2 ASO (e.g., nusinersen). In some embodiments, the recombinant SMN1 gene (e.g., in rAAV) and the SMN2 ASO (e.g., nusinersen) are administered after the first administration of the small molecule that increases SMN function (e.g., risdiplam or branaplam) alone.

[0072] There are various assays for measuring SMN expression and activity levels in vitro. See, for example, Tanguy et al, 2015, cited above. The methods described herein can also be combined with any other treatment for the treatment of SMA or its symptoms. See also Wang et al, Consensus Statement for Standard of Care in Spinal Muscular Atrophy, which provides a discussion of the current standard of care for SMA, and http: / / www.ncbi.nlm.nih.gov / books / NBK1352 / (Prior TW, Leach ME, Finanger E. Spinal Muscular Atrophy. 2000 Feb 24. GeneReviews). For example, when nutritional intake is a concern in SMA, placement of a gastrostomy tube may be appropriate. As respiratory function deteriorates, tracheostomy or non-invasive respiratory assistance is provided. Sleep disordered breathing can be treated by the nightly use of continuous positive airway pressure. Surgery for scoliosis in individuals with SMA type II and SMA type III can be performed safely if the forced vital capacity exceeds 30% - 40%. Electric wheelchairs and other devices can improve quality of life. See also U.S. Patent No. 8,211,631, which is incorporated herein by reference.

[0073] Small molecules that can increase SMN function In some embodiments, the pharmaceutical composition comprises a small molecule (e.g., risdiplam or branaplam) that increases SMN function and is used in combination with (i) a pharmaceutical composition(s) comprising a recombinant SMN1 gene (e.g., in rAAV) and / or (ii) a pharmaceutical composition(s) comprising an SMN2 ASO (e.g., in parallel or sequential treatment) for treating SMA in a subject.

[0074] In some embodiments, small molecule drugs that increase SMN function can modulate the splicing of the SMN gene (e.g., SMN1 or SMN2), stabilize it, and / or increase its transcription or translation. In some embodiments, small molecule drugs that increase SMN function can improve the activity (e.g., efficacy and / or effectiveness) of other active agents in the composition (e.g., recombinant SMN1 gene (e.g., in rAAV), SMN2 ASO) when administered to a subject in need thereof.

[0075] In some embodiments, the small molecule drug that increases SMN function is a splicing modulator. In some embodiments, the splicing modulator is an SMN2 splicing modulator. In some embodiments, the splicing modulator is a 7-disubstituted phenyltetracycline. Non-limiting examples of 7-substituted phenyltetracycline SMN2 splicing modulators are described in International Publication No. WO 2013 / 181391, the content of which is incorporated herein by reference. In some embodiments, the splicing modulator is a substituted isoindolinone. Non-limiting examples of substituted isoindolinone SMN2 splicing modulators are described in U.S. Patent Application Publication No. US 2009 / 0031435, the content of which is incorporated herein by reference. In some embodiments, the splicing modulator is a substituted carbazole derivative. Non-limiting examples of substituted carbazole derivatives that act as SMN2 splicing modulators are described in International Publication No. WO 2005 / 023255, the content of which is incorporated herein by reference. In some embodiments, the SMN2 splicing modulator is a substituted 1,4-diazepane. Non-limiting examples of substituted 1,4-diazepanes that act as SMN2 splicing modulators are described in International Publication No. WO 2019 / 028440, the content of which is incorporated herein by reference. In some embodiments, the SMN2 splicing modulator is a substituted pyridazine. Non-limiting examples of substituted pyridazines that act as SMN2 splicing modulators are described in International Publication No. WO 2015 / 017589, International Publication No. WO 2014 / 028459, U.S. Patent No. 10,195,196, U.S. Patent No. 9,545,404, U.S. Patent No. 8,729,263, and International Publication No. WO 2015 / 173181, the respective contents of which are incorporated herein by reference.

[0076] In some embodiments, the substituted pyridazine is a compound of formula (I’):

Chemical formula

Chemical formula

Chemical formula

[0077] In some embodiments, the substituted pyridazine is a compound of formula (I):

Chemical formula

Chemical formula

Chemical formula

[0078] In one embodiment, A is C 1 ~C 4 alkyl (wherein two C 1 ~C 4 alkyl groups can combine with the atoms to which they are attached to form a 5- to 6-membered ring and are substituted with 0 or 1 substituent selected from oxo, oxime, and hydroxy), halo C 1 ~C 4 alkyl, dihalo C 1 ~C 4 alkyl, trihalo C 1 ~C 4 alkyl, C 1 ~C 4 alkoxy, C 1 ~C 4 alkoxy-, C 3 ~C 7 cycloalkyl, halo C 1 ~C 4 alkoxy, dihalo C 1 ~C 4 alkoxy, trihalo C 1 ~C 4 alkoxy, hydroxy, cyano, halogen, amino, mono- and di-C 1 ~C 4 alkylamino, heteroaryl, C substituted with hydroxy 1 ~C 4 alkyl, C substituted with aryl 1 ~C 4 alkoxy, amino, -C(O)NH, C 1 ~C 4 alkyl, -heteroaryl, -NHC(O)-, C 1 ~C 4 alkyl-, heteroaryl, C 1 ~C 4 alkyl-C(O)NH-, heteroaryl, C 1 ~C 42-Hydroxyphenyl substituted with 0, 1, 2 or 3 substituents independently selected from alkyl NHC(O)-heteroaryl, 3- to 7-membered cycloalkyl, 5- to 7-membered cycloalkenyl, or 5-, 6- or 9-membered heterocycles containing 1 or 2 heteroatoms independently selected from S, O and N, wherein the heteroaryl has 5, 6 or 9 ring atoms and 1, 2 or 3 ring heteroatoms selected from N, O and S, and is oxo, hydroxy, nitro, halogen, C 1 ~C 4 alkyl, C 1 ~C 4 alkenyl, C 1 ~C 4 alkoxy, C 3 ~C 7 cycloalkyl, C 1 ~C 4 alkyl-OH, trihalo C 1 ~C 4 alkyl, mono- and di-C 1 ~C 4 alkylamino, -C(O)NH 2 、-NH 2 、-NO 2 、hydroxy C 1 ~C 4 alkylamino, hydroxy C 1 ~C 4 alkyl, 4- to 7-membered heterocyclic C 1 ~C 4 alkyl, amino C 1 ~C 4 alkyl and mono- and di-C 1 ~C 4 alkylamino C 1 ~C 4 substituted with 0, 1 or 2 substituents independently selected from alkyl. In some embodiments, A is of the formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0079] In some embodiments, A is optionally substituted at the 3-position with hydroxy and further substituted with 0, 1, or 2 substituents selected from hydroxy, cyano, halogen, C 1 ~C 4 alkyl, C 2 ~C 4 alkenyl, C 1 ~C 5 alkoxy, wherein the alkoxy is unsubstituted or substituted with hydroxy, C 1 ~C 4 alkoxy, amino, N(H)C(O)C 1 ~C 4 alkyl, N(H)C(O) 2 C 1 ~C 4 alkyl, alkylene 4- to 7-membered heterocycle, 4- to 7-membered heterocycle and mono- and di-C 1 ~C 4 alkylamino.

[0080] In some embodiments, A is a 6-membered heteroaryl having 1 to 3 ring nitrogen atoms, and the 6-membered heteroaryl is phenyl, or has 5 or 6 ring atoms and 1 or 2 ring heteroatoms independently selected from N, O, and S, C 1 ~C 4 alkyl, mono- and di-C 1 ~C 4 alkylamino, hydroxy C 1 ~C 4 alkylamino, hydroxy C 1 ~C 4 alkyl, amino C 1 ~C 4 alkyl and mono- and di-C 1 ~C 4 alkylamino C 1 ~C 4 substituted by 0, 1, or 2 substituents independently selected from alkyl.

[0081] In some embodiments, A is a bicyclic heteroaryl having 9 to 10 ring atoms and 1, 2, or 3 ring heteroatoms independently selected from N, O, or S, and the bicyclic heteroaryl is cyano, halogen, hydroxy, C 1 ~C 4 alkyl, C 2 ~C 4 alkenyl, C 2 ~C 4 alkynyl, C 1 ~C 4 alkoxy and hydroxy, C 1 ~C 4 alkoxy, amino, and mono- and di-C 1 ~C 4 substituted by 0, 1, or 2 substituents independently selected from C substituted with alkylamino 1 ~C 4 alkoxy.

[0082] In one aspect, A is a tricyclic heteroaryl having 12 or 13 ring atoms and 1, 2 or 3 ring heteroatoms independently selected from N, O or S, and said tricyclic heteroaryl is cyano, halogen, hydroxy, C 1 ~C 4 alkyl, C 2 ~C 4 alkenyl, C 2 ~C 4 alkynyl, C 1 ~C 4 alkoxy, C substituted with hydroxy 1 ~C 4 alkoxy, C 1 ~C 4 alkoxy, amino, mono- and di-C 1 ~C 4 alkylamino and is substituted with 0, 1 or 2 substituents independently selected from heteroaryl, wherein said heteroaryl has 5, 6 or 9 ring atoms and 1, 2 or 3 ring heteroatoms selected from N, O and S, and is oxo, hydroxy, nitro, halogen, C 1 ~C 4 alkyl, C 1 ~C 4 alkenyl, C 1 ~C 4 alkoxy, C 3 ~C 7 cycloalkyl, C 1 ~C 4 alkyl-OH, trihalo C 1 ~C 4 alkyl, mono- and di-C 1 ~C 4 alkylamino, -C(O)NH 2 ,-NH 2 ,-NO 2 , hydroxy C 1 ~C 4 alkylamino, hydroxy C 1 ~C 4 alkyl, 4- to 7-membered heterocyclic C 1 ~C 4 alkyl, amino C 1 ~C 4 alkyl and mono- and di-C 1 ~C4 Alkylamino C 1 ~C 4 is substituted with 0, 1 or 2 substituents independently selected from alkyl.

[0083] In some embodiments, B is a group of the formula:

Chemical formula

[0084] In some embodiments, B is a group of the formula:

Chemical formula

[0085] In some embodiments, B is

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0086] In some embodiments, the substituted pyridazine of formula (I’) is of formula (II’): [Chemical formula] or a pharmaceutically acceptable salt thereof [wherein, R 16 is a 5-membered heteroaryl having one ring nitrogen atom and 0 or 1 additional ring heteroatom selected from N, O or S, wherein the heteroaryl is optionally substituted with C 1 ~C 4 alkyl].

[0087] In some embodiments, the substituted pyridazine of formula (I) is of formula (II): [Chemical formula] or a pharmaceutically acceptable salt thereof [wherein, R 16 is a 5-membered heteroaryl having one ring nitrogen atom and 0 or 1 additional ring heteroatom selected from N, O or S, wherein the heteroaryl is optionally substituted with C 1 ~C 4 alkyl] In some embodiments, R 16is thiophene, furan, pyrrole, dihydropyrrole, imidazole, pyrazole, pyrazine, isothiazole, isoxazole, triazole, tetrazole, oxazole, isoxazole, thiazole, isothiazole. In some embodiments, R 16 is pyrazole. In some embodiments, R 16 is

Chemical formula

[0088] In some embodiments, the substituted pyridazine of formula (I) is of the formula:

Chemical formula

[0089] In some embodiments, the substituted pyridazine of formula (I) is of the formula:

Chemical formula

[0090] In some embodiments, the substituted pyridazine is a compound of formula (III):

Chemical formula

[0091] In some embodiments, the compound of formula (III) is of the formula:

Chemical formula

[0092] In some embodiments, R 1 is C 1~7 alkyl. In some embodiments, R 1 is methyl.

[0093] In some embodiments, R 2 is hydrogen or C 1~7 alkyl. In some embodiments, R 2 is hydrogen or methyl. In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is methyl.

[0094] In some embodiments, R 3 is hydrogen or C 1~7 alkyl. In some embodiments, R 3 is hydrogen or methyl. In some embodiments, R 3 is hydrogen. In some embodiments, R 3 is methyl.

[0095] In some embodiments, A is N-heterocycloalkyl or NR 12 R 13and wherein N - heterocycloalkyl contains one or two nitrogen ring atoms and is optionally substituted with one, two, three or four substituents selected from R 14 ; R 12 is heterocycloalkyl containing one nitrogen ring atom, wherein the heterocycloalkyl is optionally substituted with one, two, three or four substituents selected from R 14 ; R 13 is hydrogen, C 1~7 alkyl or C 3~8 cycloalkyl; R 14 is hydrogen, C 1~7 alkyl, amino, amino - C 1~7 alkyl, C 3~8 cycloalkyl and heterocycloalkyl independently selected or two R 14 together form C 1~7 alkylene; provided that when A is N - heterocycloalkyl containing only one nitrogen ring atom, at least one R 14 substituent is amino or amino - C 1~7 alkyl.

[0096] In some embodiments, R 12 is piperidinyl optionally substituted with one, two, three or four substituents selected from R 14 .

[0097] In some embodiments, A is of the formula:

Chemical formula

[0098] In some embodiments, A is of the formula:

Chemical formula

[0099] In some embodiments, X is N.

[0100] In some embodiments, n is 1.

[0101] In some embodiments, R 6is hydrogen, methyl or -(CH 2 ) m -NR 9 R 10 . In some embodiments, R 6 is hydrogen or methyl. In some embodiments, R 6 is hydrogen. In some embodiments, R 6 is methyl.

[0102] In some embodiments, R 7 is hydrogen or methyl.

[0103] In some embodiments, m is 0.

[0104] In some embodiments, R 4 and R 5 together form propylene. In some embodiments, R 5 and R 6 together form ethylene. In some embodiments, R 9 and R 10 together form butylene.

[0105] In some embodiments, A is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

[0106] In some embodiments, A is

Chem.

[0107] In some embodiments, the substituted pyridazine of formula (III) is of the formula:

Chem.

[0108] In some embodiments, the SMN2 splice modulator is risdiplam. In some embodiments, the SMN2 splice modulator is branaplam.

[0109] In some embodiments, the small molecule drug that increases SMN function modulates the activity of the mRNA decapping enzyme. In some embodiments, the small molecule drug inhibits the activity of the mRNA decapping enzyme. In some embodiments, the small molecule drug is a DcpS inhibitor. In some embodiments, the DcpS inhibitor is C5-substituted 2,4-diaminoquinazoline (2,4-DAQ). In some embodiments, 2,4-DAQ is RG3039. In some embodiments, the DcpS inhibitor is a 2,4-DAQ derivative. In some embodiments, the 2,4-DAQ derivative is D156844.

[0110] In some embodiments, the small molecule drug that increases the SMN function is an HDAC inhibitor. In some embodiments, the HDAC inhibitor is a cinnamic acid compound and derivatives thereof. Non-limiting examples of cinnamic acid compounds that act as HDAC inhibitors are described in US Patent Application Publication No. 2010 / 0256401 and European Patent Application Publication No. 2236503, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is a hydroxamic acid indane derivative. Non-limiting examples of hydroxamic acid indane derivatives that act as HDAC inhibitors are described in International Publication No. 2017 / 218,950, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is 3-spiro-7-hydroxamic acid tetralin. Non-limiting examples of 3-spiro-7-hydroxamic acid tetralin that act as HDAC inhibitors are described in International Publication No. 2016 / 168660, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is 3-alkyl bicyclic [4,5,0] hydroxamic acid. Non-limiting examples of 3-alkyl bicyclic [4,5,0] hydroxamic acid that act as HDAC inhibitors are described in International Publication No. 2016 / 126722, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is a condensed pyrimidine hydroxamate derivative. Non-limiting examples of condensed pyrimidine hydroxamate derivatives that act as HDAC inhibitors are described in US Patent Application Publication No. 2018 / 0265512, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is a tetrahydroindole and / or tetrahydroindazole derivative. Non-limiting examples of tetrahydroindole and tetrahydroindazoles that act as HDAC inhibitors are described in International Publication No. 2009114470 A2, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is benzimidazole. Non-limiting examples of benzimidazole that act as HDAC inhibitors are described in International Publication No. 2005 / 028447, the contents of which are incorporated herein by reference.In some embodiments, the HDAC inhibitor is a 2-propylpentanoic acid derivative. Non-limiting examples of 2-propylpentanoic acid that act as HDAC inhibitors are described in U.S. Patent Application Publication No. 2012 / 0071554, the content of which is incorporated herein by reference. In some embodiments, the HDAC inhibitor is a pimelic acid derivative. Non-limiting examples of pimelic acid derivatives that act as HDAC inhibitors are described in International Publication No. 2010 / 028193, the content of which is incorporated herein by reference. In some embodiments, the HDAC inhibitor is 6-aminohexanoic acid. Non-limiting examples of 6-aminohexanoic acid that act as HDAC inhibitors are described in U.S. Patent No. 9,796,664, the content of which is incorporated herein by reference. In some embodiments, the HDAC inhibitor is a hydroxamic acid compound. Non-limiting examples of hydroxamic acid compounds that act as HDAC inhibitors are described in International Publication No. 2006 / 101456 and U.S. Patent Application Publication No. 2010 / 0261710, the respective contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is a hydroxamic acid compound. Non-limiting examples of hydroxamic acid compounds that act as HDAC inhibitors are described in U.S. Patent Application Publication No. 2010 / 0105721 and U.S. Patent Application Publication No. 2008 / 0085896, the respective contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is a benzothiophene derivative. Non-limiting examples of benzothiophene derivatives that act as HDAC inhibitors are described in International Publication No. 2006 / 101454, the content of which is incorporated herein by reference. In some embodiments, the HDAC inhibitor is a heteroaryl amide derivative. Non-limiting examples of heteroaryl amide derivatives that act as HDAC inhibitors are described in International Publication No. 2019 / 012172, the content of which is incorporated herein by reference. In some embodiments, the HDAC inhibitor is a substituted bicyclic [4.6.0] hydroxamic acid.Non-limiting examples of substituted bicyclic [4.6.0] hydroxamic acids that act as HDAC inhibitors are described in U.S. Patent Application Publication No. 2016 / 0221997, the content of which is incorporated herein by reference. In some embodiments, the HDAC inhibitor is an aminobenzimidazole derivative. Non-limiting examples of aminobenzimidazole derivatives that act as HDAC inhibitors are described in International Publication No. 2019 / 051125, the content of which is incorporated herein by reference.

[0111] In some embodiments, the HDAC inhibitor is an imidazo[1,2-a]pyridine derivative. Non-limiting examples of imidazo[1,2-a]pyridine derivatives that act as HDAC inhibitors are described in U.S. Patent Application Publication No. 2008 / 0085896, the content of which is incorporated herein by reference.

[0112] In some embodiments, the HDAC inhibitor is a pyrimidine hydroxy compound. Non-limiting examples of pyrimidine hydroxy compounds that act as HDAC inhibitors are described in U.S. Patent Application Publication No. 2017 / 0096403, the content of which is incorporated herein by reference.

[0113] Other non-limiting examples of small molecule drugs that are HDAC inhibitors are described below, the contents of each of which are incorporated herein by reference: WO 2018 / 165520, US 2017 / 0050984, US 2007 / 0219244, US 2017 / 0305900, US 2017 / 0224684 A1, US 2008 / 0312175, WO 2018 / 129533, WO 2018 / 119362, WO 2018 / 017858, WO 2018 / 009531, WO 2017 / 004522, US 2018 / 0057456, WO 2016 / 020369, WO 2014 / 143666, JP 6336562, US 2011 / 0300134, US 2011 / 0218221, US 8,008,344, EP 2045247, JP 2009507829, CN 102271668, US 9,855,267, US 2018 / 0362472, US 2017 / 0349573, JP 5838157, WO 2019 / 007836, TW 200911230, AU 2007 / 21678.

[0114] Exemplary HDAC inhibitors include, but are not limited to, valproic acid, hydroxybutyrate, phenylbutyrate, phenylbutyrate derivatives, trichostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA). An exemplary methylase inhibitor is 5-azacytidine.

[0115] As used herein, the term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or created artificially (e.g., via chemical synthesis). Typically, a small molecule is an organic compound (i.e., it contains carbon). A small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyls, carbonyls, and heterocyclic rings, etc.). In certain embodiments, the molecular weight of a small molecule is at most about 1,000 g / mol, at most about 900 g / mol, at most about 800 g / mol, at most about 700 g / mol, at most about 600 g / mol, at most about 500 g / mol, at most about 400 g / mol, at most about 300 g / mol, at most about 200 g / mol, or at most about 100 g / mol. In certain embodiments, the molecular weight of a small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, or at least about 900 g / mol, or at least about 1,000 g / mol. Combinations of the above ranges (e.g., at least about 200 g / mol and at most about 500 g / mol) are also possible. In certain embodiments, a small molecule is a therapeutic agent such as a drug (e.g., a molecule approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (C.F.R.)). A small molecule may also form a complex with one or more metal atoms and / or metal ions. In this example, the small molecule is also referred to as a "small organometallic molecule". Preferred small molecules are biologically active, and they produce a biological effect in animals, preferably mammals, more preferably humans. In certain embodiments, a small molecule is a drug. Preferably, but not necessarily, a drug has already been considered by an appropriate government or regulatory agency to be safe and effective for use in humans or animals.For example, drugs approved for human use are listed by the FDA under 21 C.F.R. §§ 330.5, 331 - 361 and 440 - 460, which are incorporated herein by reference, and drugs for animal use are listed by the FDA under 21 C.F.R. §§ 500 - 589, which are incorporated herein by reference. All listed drugs are considered acceptable for use according to the present invention.

[0116] The definitions of certain functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the periodic table of the elements on the inside front cover of Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Michael B. Smith, March's Advanced Organic Chemistry, 7 th Edition, John Wiley & Sons, Inc., New York, 2013; Richard C. Larock, Comprehensive Organic Transformations, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0117] The compounds described herein can contain one or more chiral centers and can thus exist in various stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of individual enantiomers, diastereomers or geometric isomers, or in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. The isomers can be isolated from the mixtures by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts, or the preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H., Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present invention further encompasses the compounds as individual isomers substantially free of other isomers, or as mixtures of various isomers.

[0118] The term "tautomer" or "tautomeric" refers to two or more interconvertible compounds that result from the formal migration of at least one hydrogen atom and at least one change in valence (e.g., single bond to double bond, triple bond to single bond, or vice versa). The exact ratio of tautomers depends on several factors including temperature, solvent, and pH. Tautomerization (i.e., the reaction that provides a pair of tautomers) may be catalyzed by an acid or a base. Compounds described herein can include one or more tautomeric forms and thus can exist as tautomers.

[0119] Exemplary tautomerizations include keto-enol, amide-imid, lactam-lactim, enamine-imine, and enamine-(different enamine) tautomerizations. For example, keto-enol tautomerization can include the following.

Chem.

[0120] wherein the bond

Chem.

Chem.

Chem.

[0121] Unless otherwise indicated, the formulae include compounds that do not contain isotopically enriched atoms as well as compounds that contain isotopically enriched atoms. Compounds that contain isotopically enriched atoms can be useful, for example, as analytical tools and / or probes in biological assays.

[0122] When a range of values (a “range”) is listed, it is intended to include each value and sub-ranges within the range. A range includes the two end values of the range unless otherwise indicated. For example, “C 1~6 alkyl” is intended to include C 1 -, C 2 -, C 3 -, C 4 -, C 5 -, C 6 -, C 1~6 -, C 1~5 -, C 1~4 -, C 1~3 -, C 1~2 -, C 2~6 -, C 2~5 -, C 2~4 -, C 2~3 -, C 3~6 -, C 3~5 -, C 3~4 -, C 4~6 -, C 4~5 - and C 5~6 alkyl.

[0123] The term “aliphatic” refers to alkyl, alkenyl, alkynyl and carbocyclic groups. Similarly, the term “heteroaliphatic” refers to heteroalkyl, heteroalkenyl, heteroalkynyl and heterocyclic groups.

[0124] The term “alkyl” refers to a radical of a straight or branched chain saturated hydrocarbon group having from 1 to 20 carbon atoms (“C 1~20 alkyl”). In some embodiments, the alkyl group has from 1 to 12 carbon atoms (“C 1~12 alkyl”). In some embodiments, the alkyl group has from 1 to 10 carbon atoms (“C 1~10 alkyl”). In some embodiments, the alkyl group has from 1 to 9 carbon atoms (“C 1~9 alkyl”). In some embodiments, the alkyl group has from 1 to 8 carbon atoms (“C 1~8 alkyl”). In some embodiments, the alkyl group has from 1 to 7 carbon atoms (“C 1~7 alkyl”). In some embodiments, the alkyl group has from 1 to 6 carbon atoms (“C 1~6"alkyl"). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C 1~5 alkyl"). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C 1~4 alkyl"). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C 1~3 alkyl"). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C 1~2 alkyl"). In some embodiments, the alkyl group has 1 carbon atom ("C 1 alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C 2~6 alkyl"). C 1~6 Examples of alkyl groups include methyl (C 1 ), ethyl (C 2 ), propyl (C 3 ) (e.g., n-propyl, isopropyl), butyl (C 4 ) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C 5 ) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanil, tert-amyl) and hexyl (C 6 ) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C 7 ), n-octyl (C 8 ), n-dodecyl (C 12 ), etc. Unless otherwise specified, each example of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents (e.g., halogen such as F) ("substituted alkyl"). In certain embodiments, the alkyl group is unsubstituted C 1~12 alkyl (unsubstituted C 1~6 alkyl, etc., e.g., -CH 3(Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is substituted C 1~12 alkyl (substituted C 1~6 alkyl such as, for example, -CH 2 F, -CHF 2 , -CF 3 , -CH 2 CH 2 F, -CH 2 CHF 2 , -CH 2 CF 3 or benzyl (Bn)).

[0125] The term "haloalkyl" is a substituted alkyl group in which one or more hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro or iodo. "Perhaloalkyl" is a subset of haloalkyl and refers to an alkyl group in which all hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro or iodo. In some embodiments, the haloalkyl moiety has from 1 to 12 carbon atoms ("C 1~12 haloalkyl"). In some embodiments, the haloalkyl moiety has from 1 to 10 carbon atoms ("C 1~10 haloalkyl"). In some embodiments, the haloalkyl moiety has from 1 to 9 carbon atoms ("C 1~9 haloalkyl"). In some embodiments, the haloalkyl moiety has from 1 to 8 carbon atoms ("C 1~8 haloalkyl"). In some embodiments, the haloalkyl moiety has from 1 to 7 carbon atoms ("C 1~7 haloalkyl"). In some embodiments, the haloalkyl moiety has from 1 to 6 carbon atoms ("C 1~6 haloalkyl"). In some embodiments, the haloalkyl moiety has from 1 to 5 carbon atoms ("C 1~5"Haloalkyl"). In some embodiments, the haloalkyl moiety has from 1 to 4 carbon atoms ("C 1~4 Haloalkyl"). In some embodiments, the haloalkyl moiety has from 1 to 3 carbon atoms ("C 1~3 Haloalkyl"). In some embodiments, the haloalkyl moiety has from 1 to 2 carbon atoms ("C 1~2 Haloalkyl"). In some embodiments, all of the hydrogen atoms of the haloalkyl are independently replaced with fluoro to provide a "perfluoroalkyl" group. In some embodiments, all of the hydrogen atoms of the haloalkyl are independently replaced with chloro to provide a "perchloroalkyl" group. Examples of haloalkyl groups include -CHF 2 , -CH 2 F, -CF 3 , -CH 2 CF 3 , -CF 2 CF 3 , -CF 2 CF 2 CF 3 , -CCl 3 , -CFCl 2 , -CF 2 Cl, and the like.

[0126] The term "heteroalkyl" refers to an alkyl group further comprising at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur, inserted within the parent chain (e.g., between adjacent carbon atoms thereof) and / or disposed at one or more terminal positions of the parent chain. In certain embodiments, the heteroalkyl group refers to a saturated group having from 1 to 12 carbon atoms and one or more heteroatoms within the parent chain ("heteroC 1~12 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having from 1 to 11 carbon atoms and one or more heteroatoms within the parent chain ("heteroC 1~11 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having from 1 to 10 carbon atoms and one or more heteroatoms within the parent chain ("heteroC 1~10"alkyl"). In some embodiments, the heteroalkyl group is a saturated group having 1 to 9 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~9 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having 1 to 8 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~8 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having 1 to 7 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~7 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having 1 to 6 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~6 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having 1 to 5 carbon atoms and one or two heteroatoms in the parent chain ("heteroC 1~5 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having 1 to 4 carbon atoms and one or two heteroatoms in the parent chain ("heteroC 1~4 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having 1 to 3 carbon atoms and one heteroatom in the parent chain ("heteroC 1~3 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having 1 to 2 carbon atoms and one heteroatom in the parent chain ("heteroC 1~2 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having one carbon atom and one heteroatom ("heteroC 1 alkyl"). In some embodiments, the heteroalkyl group is a saturated group having 2 to 6 carbon atoms and one or two heteroatoms in the parent chain ("heteroC 2~6 alkyl"). Unless otherwise specified, each example of a heteroalkyl group is independently unsubstituted ("unsubstituted heteroalkyl") or substituted with one or more substituents ("substituted heteroalkyl"). In certain embodiments, the heteroalkyl group is unsubstituted heteroC 1~12 alkyl. In certain embodiments, the heteroalkyl group is substituted heteroC 1~12It is alkyl.

[0127] The term "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having 1 to 12 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, the alkenyl group has 1 to 12 carbon atoms ("C 1~12 alkenyl"). In some embodiments, the alkenyl group has 1 to 11 carbon atoms ("C 1~11 alkenyl"). In some embodiments, the alkenyl group has 1 to 10 carbon atoms ("C 1~10 alkenyl"). In some embodiments, the alkenyl group has 1 to 9 carbon atoms ("C 1~9 alkenyl"). In some embodiments, the alkenyl group has 1 to 8 carbon atoms ("C 1~8 alkenyl"). In some embodiments, the alkenyl group has 1 to 7 carbon atoms ("C 1~7 alkenyl"). In some embodiments, the alkenyl group has 1 to 6 carbon atoms ("C 1~6 alkenyl"). In some embodiments, the alkenyl group has 1 to 5 carbon atoms ("C 1~5 alkenyl"). In some embodiments, the alkenyl group has 1 to 4 carbon atoms ("C 1~4 alkenyl"). In some embodiments, the alkenyl group has 1 to 3 carbon atoms ("C 1~3 alkenyl"). In some embodiments, the alkenyl group has 1 to 2 carbon atoms ("C 1~2 alkenyl"). In some embodiments, the alkenyl group has 1 carbon atom ("C 1 alkenyl"). One or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). C 1~4 Examples of C 1 alkenyl groups include methylidene (C 2 ), ethenyl (C 3 ), 1-propenyl (C 3 ), 2-propenyl (C 4 ), 1-butenyl (C 4 ), 2-butenyl (C4 ), butadienyl (C 4 ), etc. Examples of C 1~6 alkenyl groups include the aforementioned C 2~4 alkenyl group, and pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ), etc. Additional examples of alkenyl include heptenyl (C 7 ), octenyl (C 8 ), octatriene (C 8 ), etc. Unless otherwise specified, each example of an alkenyl group is independently unsubstituted (``unsubstituted alkenyl'') or substituted with one or more substituents (``substituted alkenyl''). In certain embodiments, the alkenyl group is unsubstituted C 1~12 alkenyl. In certain embodiments, the alkenyl group is substituted C 1~12 alkenyl. In an alkenyl group, a C=C double bond for which the stereochemistry is not specified (e.g., -CH=CHCH 3 or

Chemical formula

[0128] The term ``heteroalkenyl'' refers to an alkenyl group further containing at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur, inserted within the parent chain (e.g., inserted between its adjacent carbon atoms) and / or located at one or more terminal positions of the parent chain. In certain embodiments, the heteroalkenyl group refers to a group having 1 to 12 carbon atoms, at least one double bond, and one or more heteroatoms within the parent chain (``hetero C 1~12 alkenyl''). In certain embodiments, the heteroalkenyl group refers to a group having 1 to 11 carbon atoms, at least one double bond, and one or more heteroatoms within the parent chain (``hetero C 1~11"(alkenyl)". In certain embodiments, a heteroalkenyl group refers to a group having 1 to 10 carbon atoms, at least one double bond and one or more heteroatoms within the parent chain ("hetero C 1~10 "(alkenyl)". In some embodiments, a heteroalkenyl group has 1 to 9 carbon atoms, at least one double bond and one or more heteroatoms within the parent chain ("hetero C 1~9 "(alkenyl)". In some embodiments, a heteroalkenyl group has 1 to 8 carbon atoms, at least one double bond and one or more heteroatoms within the parent chain ("hetero C 1~8 "(alkenyl)". In some embodiments, a heteroalkenyl group has 1 to 7 carbon atoms, at least one double bond and one or more heteroatoms within the parent chain ("hetero C 1~7 "(alkenyl)". In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond and one or more heteroatoms within the parent chain ("hetero C 1~6 "(alkenyl)". In some embodiments, a heteroalkenyl group has 1 to 5 carbon atoms, at least one double bond and one or two heteroatoms within the parent chain ("hetero C 1~5 "(alkenyl)". In some embodiments, a heteroalkenyl group has 1 to 4 carbon atoms, at least one double bond and one or two heteroatoms within the parent chain ("hetero C 1~4 "(alkenyl)". In some embodiments, a heteroalkenyl group has 1 to 3 carbon atoms, at least one double bond and one heteroatom within the parent chain ("hetero C 1~3 "(alkenyl)". In some embodiments, a heteroalkenyl group has 1 to 2 carbon atoms, at least one double bond and one heteroatom within the parent chain ("hetero C 1~2 "(alkenyl)". In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond and one or two heteroatoms within the parent chain ("hetero C 1~6"alkenyl"). Unless otherwise specified, each example of a heteroalkenyl group is independently unsubstituted ("unsubstituted heteroalkenyl") or substituted with one or more substituents ("substituted heteroalkenyl"). In certain embodiments, the heteroalkenyl group is unsubstituted hetero C 1~20 alkenyl. In certain embodiments, the heteroalkenyl group is substituted hetero C 1~20 alkenyl.

[0129] The term "alkynyl" refers to a radical of a straight-chain or branched hydrocarbon group having 1 to 10 carbon atoms ("C 1~10 alkynyl"). In some embodiments, the alkynyl group has 1 to 9 carbon atoms ("C 1~9 alkynyl"). In some embodiments, the alkynyl group has 1 to 8 carbon atoms ("C 1~8 alkynyl"). In some embodiments, the alkynyl group has 1 to 7 carbon atoms ("C 1~7 alkynyl"). In some embodiments, the alkynyl group has 1 to 6 carbon atoms ("C 1~6 alkynyl"). In some embodiments, the alkynyl group has 1 to 5 carbon atoms ("C 1~5 alkynyl"). In some embodiments, the alkynyl group has 1 to 4 carbon atoms ("C 1~4 alkynyl"). In some embodiments, the alkynyl group has 1 to 3 carbon atoms ("C 1~3 alkynyl"). In some embodiments, the alkynyl group has 1 to 2 carbon atoms ("C 1~2 alkynyl"). In some embodiments, the alkynyl group has 1 carbon atom ("C 1 alkynyl"). One or more carbon-carbon triple bonds can be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of C 1 ~4 alkynyl groups include, but are not limited to, methylidinyl (C 1 ), ethynyl (C 2 ), 1-propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C4 ), 2-butynyl (C 4 ), and the like. C 1~6 Examples of the alkynyl group include the aforementioned C 2~4 alkynyl group, and pentynyl (C 5 ), hexynyl (C 6 ), and the like. Additional examples of alkynyl include heptynyl (C 7 ), octynyl (C 8 ), and the like. Unless otherwise specified, each example of the alkynyl group is independently unsubstituted (``unsubstituted alkynyl'') or substituted with one or more substituents (``substituted alkynyl'').

[0130] The term ``heteroalkynyl'' refers to an alkynyl group further comprising at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur, inserted within the parent chain (e.g., inserted between its adjacent carbon atoms) and / or disposed at one or more terminal positions of the parent chain. In certain embodiments, the heteroalkynyl group refers to a group having 1 to 10 carbon atoms, at least one triple bond, and one or more heteroatoms within the parent chain (``hetero-C 1~10 alkynyl''). In some embodiments, the heteroalkynyl group has 1 to 9 carbon atoms, at least one triple bond, and one or more heteroatoms within the parent chain (``hetero-C 1~9 alkynyl''). In some embodiments, the heteroalkynyl group has 1 to 8 carbon atoms, at least one triple bond, and one or more heteroatoms within the parent chain (``hetero-C 1~8 alkynyl''). In some embodiments, the heteroalkynyl group has 1 to 7 carbon atoms, at least one triple bond, and one or more heteroatoms within the parent chain (``hetero-C 1~7 alkynyl''). In some embodiments, the heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and one or more heteroatoms within the parent chain (``hetero-C 1~6"(alkynyl)". In some embodiments, the heteroalkynyl group has 1 to 5 carbon atoms, at least one triple bond, and one or two heteroatoms in the parent chain ("heteroC 1~5 "(alkynyl)". In some embodiments, the heteroalkynyl group has 1 to 4 carbon atoms, at least one triple bond, and one or two heteroatoms in the parent chain ("heteroC 1~4 "(alkynyl)". In some embodiments, the heteroalkynyl group has 1 to 3 carbon atoms, at least one triple bond, and one heteroatom in the parent chain ("heteroC 1~3 "(alkynyl)". In some embodiments, the heteroalkynyl group has 1 to 2 carbon atoms, at least one triple bond, and one heteroatom in the parent chain ("heteroC 1~2 "(alkynyl)". In some embodiments, the heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and one or two heteroatoms in the parent chain ("heteroC 1~6 "(alkynyl)". Unless otherwise specified, each example of a heteroalkynyl group is independently unsubstituted ("unsubstituted heteroalkynyl") or substituted with one or more substituents ("substituted heteroalkynyl").

[0131] The term "carbocyclic" or "carbocyclic ring" refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms ("C 3~10 "carbocyclic") and zero heteroatoms in a non-aromatic ring system. In some embodiments, the carbocyclic group has 3 to 10 ring carbon atoms ("C 3~10 "carbocyclic"). In some embodiments, the carbocyclic group has 3 to 8 ring carbon atoms ("C 3~8 "carbocyclic"). In some embodiments, the carbocyclic group has 3 to 7 ring carbon atoms ("C 3~7 "carbocyclic"). In some embodiments, the carbocyclic group has 3 to 6 ring carbon atoms ("C 3~6 "carbocyclic"). In some embodiments, the carbocyclic group has 4 to 6 ring carbon atoms ("C 4~6"Carbocyclic"). In some embodiments, the carbocyclic group has 5 to 6 ring carbon atoms ("C 5~6 "Carbocyclic"). In some embodiments, the carbocyclic group has 5 to 10 ring carbon atoms ("C 5~10 "Carbocyclic"). Exemplary C 3~6 carbocyclic groups include cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4 ), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ), and the like. Exemplary C 3~8 carbocyclic groups include the aforementioned C 3~6 carbocyclic groups, as well as cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), cyclooctyl (C 8 ), cyclooctenyl (C 8 ), bicyclo[2.2.1]heptanyl (C 7 ), bicyclo[2.2.2]octanyl (C 8 ), and the like. Exemplary C 3~10 carbocyclic groups include the aforementioned C 3~8 carbocyclic groups, as well as cyclononyl (C 9 ), cyclononenyl (C 9 ), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C 9 ), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ), and the like. Exemplary C 3~8 carbocyclic groups include the aforementioned C 3~10Examples include a carbocyclic group and the like. As described in the above examples, in certain embodiments, the carbocyclic group is either monocyclic (“monocyclic carbocyclic”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclic”) or a tricyclic system (“tricyclic carbocyclic”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds. “Carbocyclic” also includes a ring system in which the carbocyclic ring defined above is fused to one or more aryl or heteroaryl groups and the point of attachment is on the carbocyclic ring. In such examples, the number of carbons also represents the number of carbons in the carbocyclic ring system. Unless otherwise specified, each example of a carbocyclic group is independently unsubstituted (“unsubstituted carbocyclic”) or substituted with one or more substituents (“substituted carbocyclic”). In certain embodiments, the carbocyclic group is unsubstituted C 3~10 carbocyclic. In certain embodiments, the carbocyclic group is substituted C 3~10 carbocyclic. In some embodiments, the cycloalkyl group has 3 to 10 ring carbon atoms (“C 3~10 cycloalkyl”). In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms (“C 3~8 cycloalkyl”). In some embodiments, the cycloalkyl group has 3 to 6 ring carbon atoms (“C 3~6 cycloalkyl”). In some embodiments, the cycloalkyl group has 4 to 6 ring carbon atoms (“C 4~6 cycloalkyl”). In some embodiments, the cycloalkyl group has 5 to 6 ring carbon atoms (“C 5~6 cycloalkyl”). In some embodiments, the cycloalkyl group has 5 to 10 ring carbon atoms (“C 5~10 cycloalkyl”). Examples of C 5~6 cycloalkyl groups include cyclopentyl (C 5 ) and cyclohexyl (C 5 ). Examples of C 3~6 cycloalkyl groups include the aforementioned C 5~6Cycloalkyl groups, and cyclopropyl (C 3 ) and cyclobutyl (C 4 ) are included. C 3~8 Examples of cycloalkyl groups include the aforementioned C 3~6 cycloalkyl groups, and cycloheptyl (C 7 ) and cyclooctyl (C 8 ). Unless otherwise specified, each example of a carbocyclic group is independently unsubstituted ( "unsubstituted cycloalkyl") or substituted with one or more substituents ( "substituted cycloalkyl"). In certain embodiments, the cycloalkyl group is unsubstituted C 3~14 cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C 3~14 cycloalkyl. In certain embodiments, the carbocyclic ring contains 0, 1, or 2 C = C double bonds in the carbocyclic ring system as long as the valence allows.

[0132] The term "heterocyclyl" or "heterocyclic" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("3- to 14-membered heterocyclyl"). In a heterocyclyl group containing one or more nitrogen atoms, the point of attachment can be a carbon atom or a nitrogen atom, as valence permits. The heterocyclyl group can be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a fused, bridged, or spiro ring system such as a bicyclic system ("bicyclic heterocyclyl") or a tricyclic system ("tricyclic heterocyclyl")), and can be saturated or can contain one or more carbon-carbon double or triple bonds. The heterocyclyl polycyclic ring system can contain one or more heteroatoms in one or both rings. "Heterocyclyl" includes a ring system in which the heterocyclyl ring as defined above is fused to one or more carbocyclic groups and the point of attachment is on either the carbocyclic ring or the heterocyclyl ring, or a ring system in which the heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups and the point of attachment is on the heterocyclyl ring. In such examples, the number of ring members also represents the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each example of a heterocyclyl group is independently unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted hetero 3- to 14-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted hetero 3- to 14-membered heterocyclyl. In certain embodiments, heterocyclyl is a substituted or unsubstituted 3- to 7-membered monocyclic heterocyclyl, and 1, 2, or 3 atoms in the heterocyclic ring system are independently oxygen, nitrogen, or sulfur, as valence permits.

[0133] In some embodiments, the heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 10-membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 8-membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 6-membered heterocyclyl"). In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0134] Exemplary 3-membered heterocyclyl groups containing one heteroatom include azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrol-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing three heteroatoms include triazinyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include azocanyl, oxecanyl, and thiocanyl.Exemplary bicyclic heterocyclyl groups include indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

[0135] The term "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system having 6 to 14 ring carbon atoms and 0 heteroatoms provided in an aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) ("C 6~14 aryl"). In some embodiments, an aryl group has 6 ring carbon atoms ("C 6 aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 aryl"; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14"Aryl"; for example, anthracyl). "Aryl" includes ring systems in which the aryl ring defined above is condensed with one or more carbocyclic or heterocyclic groups and the radical or point of attachment is on the aryl ring. Even in such examples, the number of carbon atoms also represents the number of carbons in the aryl ring system. Unless otherwise specified, each example of an aryl group is independently unsubstituted ( "unsubstituted aryl") or substituted with one or more substituents ( "substituted aryl"). In certain embodiments, the aryl group is unsubstituted C 6~14 aryl. In certain embodiments, the aryl group is substituted C 6~14 aryl.

[0136] "Aralkyl" is a subset of "alkyl" and refers to an alkyl group substituted by an aryl group, with the point of attachment on the alkyl moiety.

[0137] The term "heteroaryl" refers to a radical of a 5- to 14-membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1 to 4 heteroatoms provided in an aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 14-membered heteroaryl"). In a heteroaryl group containing one or more nitrogen atoms, the point of attachment can be a carbon atom or a nitrogen atom as valence permits. The heteroaryl polycyclic ring system can contain one or more heteroatoms in one or both rings. "Heteroaryl" includes a ring system in which the heteroaryl ring defined above is fused to one or more carbocyclic groups or heterocyclic groups and the point of attachment is on the heteroaryl ring, and in such examples, the number of ring members also represents the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes a ring system in which the heteroaryl ring defined above is fused to one or more aryl groups and the point of attachment is on either the aryl ring or the heteroaryl ring, and in such examples, the number of ring members represents the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. The point of attachment of a polycyclic heteroaryl group in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.) can be on any ring, e.g., on the ring having a heteroatom (e.g., 2-indolyl) or on the ring not containing a heteroatom (e.g., 5-indolyl). In certain embodiments, heteroaryl is a substituted or unsubstituted 5- or 6-membered monocyclic heteroaryl, and 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur. In certain embodiments, heteroaryl is a substituted or unsubstituted 9- or 10-membered bicyclic heteroaryl, and 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur.

[0138] In some embodiments, a heteroaryl group is a 5- to 10-membered aromatic ring system having ring carbon atoms provided in the aromatic ring system and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 10-membered heteroaryl"). In some embodiments, a heteroaryl group is a 5- to 8-membered aromatic ring system having ring carbon atoms provided in the aromatic ring system and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 8-membered heteroaryl"). In some embodiments, a heteroaryl group is a 5- to 6-membered aromatic ring system having ring carbon atoms provided in the aromatic ring system and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 6-membered heteroaryl"). In some embodiments, the 5- to 6-membered heteroaryl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each example of a heteroaryl group is independently unsubstituted (an "unsubstituted heteroaryl") or substituted with one or more substituents (a "substituted heteroaryl"). In certain embodiments, the heteroaryl group is an unsubstituted 5- to 14-membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5- to 14-membered heteroaryl.

[0139] Exemplary 5-membered heteroaryl groups containing one heteroatom include pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0140] "Heteroaralkyl" is a subset of "alkyl" and refers to an alkyl group substituted by a heteroaryl group, with the point of attachment being on the alkyl moiety.

[0141] The term "unsaturated bond" refers to a double bond or a triple bond.

[0142] The term "unsaturated" or "partially unsaturated" refers to a moiety containing at least one double bond or triple bond.

[0143] The term "saturated" or "fully saturated" refers to a moiety containing neither a double bond nor a triple bond, for example, a moiety containing only single bonds.

[0144] Attaching the suffix "-ene" indicates that the group is a divalent moiety. For example, alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.

[0145] Unless otherwise explicitly indicated, the group is optionally substituted. The term "optionally substituted" refers to being either substituted or unsubstituted. In certain embodiments, an alkyl group, alkenyl group, alkynyl group, heteroalkyl group, heteroalkenyl group, heteroalkynyl group, carbocyclic group, heterocyclic group, aryl group, and heteroaryl group are optionally substituted. "Optionally substituted" refers to a group that can be either substituted or unsubstituted (e.g., a "substituted" or "unsubstituted" alkyl group, "substituted" or "unsubstituted" alkenyl group, "substituted" or "unsubstituted" alkynyl group, "substituted" or "unsubstituted" heteroalkyl group, "substituted" or "unsubstituted" heteroalkenyl group, "substituted" or "unsubstituted" heteroalkynyl group, "substituted" or "unsubstituted" carbocyclic group, "substituted" or "unsubstituted" heterocyclic group, "substituted" or "unsubstituted" aryl group, or "substituted" or "unsubstituted" heteroaryl group). Generally, the term "substituted" means that at least one hydrogen present in the group is replaced by an acceptable substituent, i.e., a substituent that, upon substitution, results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as rearrangement, cyclization, elimination, or other reactions. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and if two or more positions in any given structure are substituted, the substituents can be either the same or different at each position. The term "substituted" is intended to include substitution by all acceptable substituents of organic compounds and includes any of the substituents described herein that result in the formation of a stable compound. The present invention contemplates any such combinations to arrive at a stable compound. For the purposes of the present invention, a heteroatom such as nitrogen may have any suitable substituent described herein that satisfies the valence of the hydrogen substituent and / or the heteroatom and results in the formation of a stable moiety. The present invention is not intended to be limited in any way by the exemplary substituents described herein.

[0146] Exemplary substituents for the carbon atom include halogen, -CN, -NO 2 、-N 3 、-SO 2 H、-SO 3 H、-OH、-OR aa 、-ON(R bb ) 2 、-N(R bb ) 2 、-N(R bb ) 3 + X - 、-N(OR cc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO 2 H、-CHO、-C(OR cc ) 2 、-CO 2 R aa 、-OC(=O)R aa 、-OCO 2 R aa 、-C(=O)N(R bb ) 2 、-OC(=O)N(R bb ) 2 、-NR bb C(=O)R aa 、-NR bb CO 2 R aa 、-NR bb C(=O)N(R bb ) 2 、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb ) 2 、-OC(=NR bb )N(R bb ) 2 、-NR bb C(=NR bb )N(R bb ) 2, -C(=O)NR bb SO 2 R aa , -NR bb SO 2 R aa , -SO 2 N(R bb ) 2 , -SO 2 R aa , -SO 2 OR aa , -OSO 2 R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa ) 3 , -OSi(R aa ) 3 -C(=S)N(R bb ) 2 , -C(=O)SR aa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)(R aa ) 2 , -P(=O)(OR cc ) 2 , -OP(=O)(R aa ) 2 , -OP(=O)(OR cc ) 2 , -P(=O)(N(R bb ) 2 ) 2 , -OP(=O)(N(R bb ) 2 ) 2 , -NR bb P(=O)(R aa ) 2 , -NR bb P(=O)(OR cc ) 2 , -NR bb P(=O)(N(R bb ) 2 ) 2 , -P(R cc ) 2, -P(OR cc ) 2 , -P(R cc ) 3 + X - , -P(OR cc ) 3 + X - , -P(R cc ) 4 , -P(OR cc ) 4 , -OP(R cc ) 2 , -OP(R cc ) 3 + X - , -OP(OR cc ) 2 , -OP(OR cc ) 3 + X - , -OP(R cc ) 4 , -OP(OR cc ) 4 , -B(R aa ) 2 , -B(OR cc ) 2 , -BR aa (OR cc ), C 1~20 alkyl, C 1~20 perhaloalkyl, C 1~20 alkenyl, C 1~20 alkynyl, heteroC 1~20 alkyl, heteroC 1~20 alkenyl, heteroC 1~20 alkynyl, C 3~10 carbocyclic, 3 - 14 membered heterocyclic, C 6~14 aryl and 5 - 14 membered heteroaryl are included, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic, heterocyclic, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups; wherein X - is a counterion; alternatively, two geminal hydrogens on a carbon atom are a group =O, =S, =NN(Rbb ) 2 、 =NNR bb C(=O)R aa 、 =NNR bb C(=O)OR aa 、 =NNR bb S(=O) 2 R aa 、 =NR bb or =NOR cc is replaced by; each R aa examples are, independently, C 1~20 alkyl, C 1~20 perhaloalkyl, C 1~20 alkenyl, C 1~20 alkynyl, heteroC 1~20 alkyl, heteroC 1~20 alkenyl, heteroC 1~20 alkynyl, C 3~10 carbocyclic, 3- to 14-membered heterocyclic, C 6~14 aryl and 5- to 14-membered heteroaryl, or two R aa groups are joined to form a 3- to 14-membered heterocyclic ring or a 5- to 14-membered heteroaryl ring, where each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic, heterocyclic, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups; each R bb examples are, independently, hydrogen, -OH, -OR aa 、 -N(R cc ) 2 、 -CN, -C(=O)R aa 、 -C(=O)N(R cc ) 2 、 -CO 2 R aa 、 -SO 2 R aa 、 -C(=NR cc )OR aa 、 -C(=NR cc )N(R cc ) 2 、 -SO 2 N(R cc )2 、 -SO 2 R cc 、 -SO 2 OR cc 、 -SOR aa 、 -C(=S)N(R cc ) 2 、 -C(=O)SR cc 、 -C(=S)SR cc 、 -P(=O)(R aa ) 2 、 -P(=O)(OR cc ) 2 、 -P(=O)(N(R cc ) 2 ) 2 、 C 1~20 alkyl, C 1~20 perhaloalkyl, C 1~20 alkenyl, C 1~20 alkynyl, hetero C 1~20 alkyl, hetero C 1~20 alkenyl, hetero C 1~20 alkynyl, C 3~10 carbocyclic, 3 - to 14 - membered heterocyclic, C 6~14 aryl and 5 - to 14 - membered heteroaryl, or two R bb groups are joined to form a 3 - to 14 - membered heterocyclic ring or a 5 - to 14 - membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups; each R cc is independently selected from hydrogen, C 1~20 alkyl, C 1~20 perhaloalkyl, C 1~20 alkenyl, C 1~20 alkynyl, hetero C 1~20 alkyl, hetero C 1~20 alkenyl, hetero C 1~20 alkynyl, C 3~10 carbocyclic, 3 - to 14 - membered heterocyclic, C 6~14 aryl and 5 - to 14 - membered heteroaryl, or two Rcc The groups combine to form a 3- to 14-membered heterocyclic ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic, heterocyclic, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups; Examples of each R dd are independently halogen, -CN, -NO 2 , -N 3 , -SO 2 H, -SO 3 H, -OH, -OR ee , -ON(R ff ) 2 , -N(R ff ) 2 , -N(R ff ) 3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO 2 H, -CO 2 R ee , -OC(=O)R ee , -OCO 2 R ee , -C(=O)N(R ff ) 2 , -OC(=O)N(R ff ) 2 , -NR ff C(=O)R ee , -NR ff CO 2 R ee , -NR ff C(=O)N(R ff ) 2 , -C(=NR ff )OR ee , -OC(=NR ff )R ee , -OC(=NR ff )OR ee , -C(=NR ff )N(R ff )2 ,-OC(=NR ff )N(R ff ) 2 ,-NR ff C(=NR ff )N(R ff ) 2 ,-NR ff SO 2 R ee ,-SO 2 N(R ff ) 2 ,-SO 2 R ee ,-SO 2 OR ee ,-OSO 2 R ee ,-S(=O)R ee ,-Si(R ee ) 3 ,-OSi(R ee ) 3 ,-C(=S)N(R ff ) 2 ,-C(=O)SR ee ,-C(=S)SR ee ,-SC(=S)SR ee ,-P(=O)(OR ee ) 2 ,-P(=O)(R ee ) 2 ,-OP(=O)(R ee ) 2 ,-OP(=O)(OR ee ) 2 ,C 1~10 alkyl,C 1~10 perhaloalkyl,C 1~10 alkenyl,C 1~10 alkynyl,heteroC 1~10 alkyl,heteroC 1~10 alkenyl,heteroC 1~10 alkynyl,C 3~10 carbocyclic,3- to 10-membered heterocyclic,C 6~10 aryl,5- to 10-membered heteroaryl,selected from, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic, heterocyclic, aryl and heteroaryl is independently 0, 1, 2, 3, 4 or 5 R ggsubstituted with a radical or two geminal R dd The substituents can combine to form =O or =S; wherein X - is a counterion; each R ee is independently selected from C 1~10 alkyl, C 1~10 perhaloalkyl, C 1~10 alkenyl, C 1~10 alkynyl, hetero-C 1~10 alkyl, hetero-C 1~10 alkenyl, hetero-C 1~10 alkynyl, C 3~10 carbocyclic, C 6~10 aryl, 3- to 10-membered heterocyclyl and 3- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R gg groups; each R ff is independently selected from hydrogen, C 1~10 alkyl, C 1~10 perhaloalkyl, C 1~10 alkenyl, C 1~10 alkynyl, hetero-C 1~10 alkyl, hetero-C 1~10 alkenyl, hetero-C 1~10 alkynyl, C 3~10 carbocyclic, 3- to 10-membered heterocyclyl, C 6~10 aryl and 5- to 10-membered heteroaryl, or two R ff groups combine to form a 3- to 10-membered heterocyclyl ring or a 5- to 10-membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R gg groups; each R gg is independently selected from halogen, -CN, -NO2 、 -N 3 、 -SO 2 H、 -SO 3 H、 -OH、 -OC 1~6 アルキル、 -ON(C 1~6 アルキル) 2 、 -N(C 1~6 アルキル) 2 、 -N(C 1~6 アルキル) 3 + X - 、 -NH(C 1~6 アルキル) 2 + X - 、 -NH 2 (C 1~6 アルキル) + X - 、 -NH 3 + X - 、 -N(OC 1~6 アルキル)(C 1~6 アルキル)、 -N(OH)(C 1~6 アルキル)、 -NH(OH)、 -SH、 -SC 1~6 アルキル、 -SS(C 1~6 アルキル)、 -C(=O)(C 1~6 アルキル)、 -CO 2 H、 -CO 2 (C 1~6 アルキル)、 -OC(=O)(C 1~6 アルキル)、 -OCO 2 (C 1~6 アルキル)、 -C(=O)NH 2 、 -C(=O)N(C 1~6 アルキル) 2 、 -OC(=O)NH(C 1~6 アルキル)、 -NHC(=O)(C 1~6 アルキル)、 -N(C 1~6 アルキル)C(=O)(C 1~6 アルキル)、 -NHCO 2 (C 1~6 アルキル)、 -NHC(=O)N(C 1~6 アルキル) 2 、 -NHC(=O)NH(C 1~6 アルキル)、 -NHC(=O)NH 2 、 -C(=NH)O(C 1~6(alkyl), -OC(=NH)(C 1~6 (alkyl), -OC(=NH)OC 1~6 (alkyl, -C(=NH)N(C 1~6 (alkyl) 2 , -C(=NH)NH(C 1~6 (alkyl), -C(=NH)NH 2 , -OC(=NH)N(C 1~6 (alkyl) 2 , -OC(NH)NH(C 1~6 (alkyl), -OC(NH)NH 2 , -NHC(NH)N(C 1~6 (alkyl) 2 , -NHC(=NH)NH 2 , -NHSO 2 (C 1~6 (alkyl), -SO 2 N(C 1~6 (alkyl) 2 , -SO 2 NH(C 1~6 (alkyl), -SO 2 NH 2 , -SO 2 C 1~6 (alkyl, -SO 2 OC 1~6 (alkyl, -OSO 2 C 1~6 (alkyl, -SOC 1~6 (alkyl, -Si(C 1~6 (alkyl) 3 , -OSi(C 1~6 (alkyl) 3 -C(=S)N(C 1~6 (alkyl) 2 , C(=S)NH(C 1~6 (alkyl), C(=S)NH 2 , -C(=O)S(C 1~6 , -C(=S)SC 1~6 (alkyl, -SC(=S)SC 1~6 (alkyl, -P(=O)(OC 1~6 (alkyl) 2 , -P(=O)(C 1~6 (alkyl) 2 , -OP(=O)(C 1~6 (alkyl) 2 , -OP(=O)(OC 1~6(Alkyl) 2 、 C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 1~10 Alkenyl, C 1~10 Alkynyl, hetero C 1~10 Alkyl, hetero C 1~10 Alkenyl, hetero C 1~10 Alkynyl, C 3~10 Carbocyclic, C 6~10 Is it aryl, 3- to 10-membered heterocyclyl or 5- to 10-membered heteroaryl; or two geminal R gg Substituents can combine to form =O or =S; and Each X - Is a counterion.

[0147] In certain embodiments, the substituents on the carbon atoms are independently halogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~6 Alkyl, -OR aa , -SR aa , -N(R bb ) 2 , -CN, -SCN, -NO 2 , -C(=O)R aa , -CO 2 R aa , -C(=O)N(R bb ) 2 , -OC(=O)R aa , -OCO 2 R aa , -OC(=O)N(R bb ) 2 , -NR bb C(=O)R aa , -NR bb CO 2 R aa Or -NR bb C(=O)N(R bb ) 2 In certain embodiments, the substituents on the carbon atoms are independently halogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 Alkyl, -OR aa , -SR aa, -N(R bb ) 2 , -CN, -SCN, -NO 2 , -C(=O)R aa , -CO 2 R aa , -C(=O)N(R bb ) 2 , -OC(=O)R aa , -OCO 2 R aa , -OC(=O)N(R bb ) 2 , -NR bb C(=O)R aa , -NR bb CO2R aa or -NR bb C(=O)N(R bb ) 2 , wherein R aa is hydrogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 alkyl, an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl or benzoyl) when bonded to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridinesulfenyl, 2-pyridinesulfenyl or triphenylmethyl) when bonded to a sulfur atom; each R bb is independently hydrogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 alkyl or a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In certain embodiments, the substituents on the carbon atom are independently halogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~6 alkyl, -OR aa , -SR aa , -N(R bb ) 2 , -CN, -SCN or -NO 2It is. In certain embodiments, the substituent of the carbon atom is independently halogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 alkyl, -OR aa , -SR aa , -N(R bb ), 2 , -CN, -SCN or -NO 2 , wherein R aa is hydrogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 alkyl, an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl or benzoyl) when bonded to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridinesulfenyl, 2-pyridinesulfenyl or triphenylmethyl) when bonded to a sulfur atom; each Rbb is independently hydrogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 alkyl or a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts).

[0148] In certain embodiments, the molecular weight of the substituent of the carbon atom is less than 250 g / mol, less than 200 g / mol, less than 150 g / mol, less than 100 g / mol, or less than 50 g / mol. In certain embodiments, the substituent of the carbon atom consists of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, nitrogen and / or silicon atoms. In certain embodiments, the substituent of the carbon atom consists of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur and / or nitrogen atoms. In certain embodiments, the substituent of the carbon atom consists of carbon, hydrogen, fluorine, chlorine, bromine and / or iodine atoms. In certain embodiments, the substituent of the carbon atom consists of carbon, hydrogen, fluorine and / or chlorine atoms.

[0149] The term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br) or iodine (iodo, -I).

[0150] The term "hydroxyl" or "hydroxy" refers to the -OH group. The term "substituted hydroxyl" or "substituted hydroxy" in turn refers to a hydroxyl group in which the oxygen atom directly bonded to the parent molecule is substituted with a group other than hydrogen, -OR aa 、-ON(R bb ) 2 、-OC(=O)SR aa 、-OC(=O)R aa 、-OCO 2 R aa 、-OC(=O)N(R bb ) 2 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-OC(=NR bb )N(R bb ) 2 、-OS(=O)R aa 、-OSO 2 R aa 、-OSi(R aa ) 3 、-OP(R cc ) 2 、-OP(R cc ) 3 + X - 、-OP(OR cc ) 2 、-OP(OR cc ) 3 + X - 、-OP(=O)(R aa ) 2 、-OP(=O)(OR cc ) 2 and -OP(=O)(N(R bb )) 2 and includes a group selected from, where X - 、R aa 、R bb and R ccis as defined herein.

[0151] The terms "thiol" or "thio" refer to an -SH group. The term "substituted thiol" or "substituted thio" thus refers to a thiol group in which the sulfur atom directly bonded to the parent molecule is substituted with a group other than hydrogen, -SR aa , -S=SR cc , -SC(=S)SR aa , -SC(=S)OR aa , -SC(=S)N(R bb ) 2 , -SC(=O)SR aa , -SC(=O)OR aa , -SC(=O)N(R bb ) 2 and -SC(=O)R aa and includes a group selected from, wherein R aa and R cc are as defined herein.

[0152] The term "amino" refers to an -NH 2 group. The term "substituted amino" thus refers to a mono-substituted amino, di-substituted amino or tri-substituted amino. In certain embodiments, "substituted amino" is a mono-substituted amino group or a di-substituted amino group.

[0153] The term "mono-substituted amino" refers to an amino group in which the nitrogen atom directly bonded to the parent molecule is substituted with one hydrogen and one group other than hydrogen, -NH(R bb ), -NHC(=O)R aa , -NHCO 2 R aa , -NHC(=O)N(R bb ) 2 , -NHC(=NR bb )N(R bb ) 2 , -NHSO 2 R aa , -NHP(=O)(OR cc ) 2 and -NHP(=O)(N(R bb ) 2 )2 comprises a group selected from, wherein R aa , R bb and R cc are as defined herein, and the R bb in the -NH(R bb ) group is not hydrogen.

[0154] The term "disubstituted amino" refers to an amino group in which the nitrogen atom directly bonded to the parent molecule is substituted with two groups other than hydrogen, -N(R bb ), 2 , -NR bb C(=O)R aa , -NR bb CO 2 R aa , -NR bb C(=O)N(R bb ) 2 , -NR bb C(=NR bb )N(R bb ) 2 , -NR bb SO 2 R aa , -NR bb P(=O)(OR cc ) 2 and -NR bb P(=O)(N(R bb ) 2 ) 2 comprises a group selected from, wherein R aa , R bb and R cc are as defined herein, provided that the nitrogen atom directly bonded to the parent molecule is not substituted with hydrogen.

[0155] The term "trisubstituted amino" refers to an amino group in which the nitrogen atom directly bonded to the parent molecule is substituted with three groups, -N(R bb ) 3 and -N(R bb ) 3 + X - comprises a group selected from, wherein R bb and X - are as defined herein.

[0156] The term "sulfonyl" refers to a group selected from -SO 2 N(R bb ) 2 -, -SO 2 R aa and -SO 2 OR aa , wherein R aa and R bb are as defined herein.

[0157] The term "sulfinyl" refers to the -S(=O)R aa group, wherein R aa is as defined herein.

[0158] The term "acyl" refers to the general formula -C(=O)R X1 , -C(=O)OR X1 , -C(=O)-O-C(=O)R X1 , -C(=O)SR X1 , -C(=O)N(R X1 ) 2 , -C(=S)R X1 , -C(=S)N(R X1 ) 2 and -C(=S)S(R X1 ), -C(=NR X1 )R X1 , -C(=NR X1 )OR X1 , -C(=NR X1 )SR X1 , as well as -C(=NR X1 )N(R X1 ) 2 having groups, wherein R X1is hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphatic oxy, heteroaliphatic oxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphatic thioxy, heteroaliphatic thioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or dialiphatic amino, mono- or diheteroaliphatic amino, mono- or dialkylamino, mono- or diheteroalkylamino, mono- or diarylamino or mono- or diheteroarylamino; or two R X1 groups together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehyde (-CHO), carboxylic acid (-CO 2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Examples of acyl substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thioxo, cyano, isocyano, amino, azide, nitro, hydroxyl, thiol, halo, aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphatic thioxy, heteroaliphatic thioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, etc., each of which may be further substituted or may not be further substituted).

[0159] The term "carbonyl" refers to a group in which the carbon directly attached to the parent molecule is sp 2 hybridized and is substituted with an oxygen, nitrogen, or sulfur atom, e.g., a ketone (-C(=O)R aa ), a carboxylic acid (-CO 2 H), an aldehyde (-CHO), an ester (-CO 2 R aa , -C(=O)SR aa , -C(=S)SR aa ), an amide (-C(=O)N(R bb ) 2 , -C(=O)NR bb SO 2 R aa , -C(=S)N(R bb ) 2 ) and an imine (-C(=NR bb )R aa , -C(=NR bb )OR aa ), -C(=NR bb )N(R bb ) 2 ), where Raa and R bb are as defined herein.

[0160] As used herein, the term "salt" or "salts" refers to acid addition salts or base addition salts of the compounds of the invention. "Salts" include, in particular, "pharmaceutically acceptable salts".

[0161] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of the invention and that are typically neither biologically nor otherwise undesirable. In many cases, the compounds of the invention can form salts with acids and / or bases due to the presence of amino and / or carboxyl groups, or groups similar thereto.

[0162] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, for example, acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methyl sulfate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate and trifluoroacetate.

[0163] Examples of inorganic acids from which salts can be derived include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.

[0164] Examples of organic acids from which the salt may be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonate, toluenesulfonic acid, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.

[0165] Examples of inorganic bases from which the salt may be derived include, for example, ammonium salts and metals from Groups I - XII of the periodic table. In certain embodiments, the salt is derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper, and particularly preferred salts include salts of ammonium, potassium, sodium, calcium, and magnesium.

[0166] Examples of organic bases from which the salt may be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain specific organic amines include isopropylamine, benzathine, cholineate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0167] The pharmaceutically acceptable salts of the present disclosure can be synthesized from the basic or acidic moieties of the parent compound by conventional chemical methods. In general, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of an appropriate base (e.g., hydroxides, carbonates, bicarbonates of Na, Ca, Mg or K, etc.) or by reacting the free base form of these compounds with a stoichiometric amount of an appropriate acid. Such reactions are typically carried out in water, in an organic solvent, or in a mixture of the two. In general, the use of a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is desirable when available. A further list of suitable salts can be found, for example, in "Remington's Pharmaceutical Sciences", 20th ed., Mack Publishing Company, Easton, Pa., (1985); and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH. Weinheim, Germany, 2002).

[0168] A recombinant nucleic acid encoding SMN1 In some embodiments, a combination therapy for treating SMA includes administration (e.g., together or sequentially) of a recombinant nucleic acid encoding SMN1 (e.g., administered with a viral vector such as rAAV), in addition to other therapies described herein (e.g., SMN2 ASO or small molecules that increase SMN function). In some embodiments, the recombinant nucleic acid encoding SMN1 (also referred to herein as the recombinant SMN1 gene) includes the SMN1 gene operably linked to a promoter (e.g., a promoter active in motor neuron cells). In some embodiments, the recombinant nucleic acid encoding SMN1 is provided in a non-viral vector (e.g., a non-viral plasmid). However, in some embodiments, the recombinant nucleic acid encoding SMN1 is provided in a recombinant viral vector (e.g., in a recombinant viral genome packaged within a viral capsid). In some embodiments, the recombinant SMN1 gene is provided in a recombinant adeno-associated virus (rAAV) genome and packaged within AAV capsid particles.

[0169] In some embodiments, the recombinant SMN1 gene is administered to a subject with a viral vector. In some embodiments, the recombinant SMN1 gene is administered with a recombinant AAV genome that includes adjacent AAV inverted terminal repeat sequences (ITRs). Thus, in some embodiments, recombinant viral particles (e.g., rAAV particles) that include a gene encoding SMN1 are administered to a subject together with an SMN2 ASO.

[0170] Figure 2 provides a non-limiting example of a recombinant viral genome comprising the SMN1 gene operably linked to a promoter. Figure 2 illustrates the SMN1 gene flanked by AAV ITRs. The SMN1 gene contains a human SMN1 codon-optimized SMN1 open reading frame and is operably linked to the CB7 promoter (a chicken beta-actin promoter with a cytomegalovirus (CMV) enhancer). The recombinant AAV genome also contains a chicken beta-actin intron and a rabbit beta-globin polyA signal. The rAAV genome illustrated in Figure 2 is non-limiting, and alternative SMN1 coding sequences, promoters, and other regulatory elements can be used.

[0171] In some embodiments, the rAAV genome is packaged into a viral capsid. In some embodiments, the capsid protein is a hu68 serotype capsid protein. However, other capsid proteins of other serotypes can be used.

[0172] These and other embodiments of the recombinant SMN1 gene are described in more detail in the following paragraphs.

[0173] SMN1 coding sequence: In some embodiments, a coding sequence encoding a wild-type human SMN protein (e.g., an SMN1 cDNA sequence) is provided. Nucleic acid sequences encoding human SMN1 are known in the art. For example, for non-limiting examples of nucleic acid sequences of human SMN1, see GenBank accession numbers NM_001297715.1; NM_000344.3; NM_022874.2, DQ894095, NM_000344, NM_022874, and BC062723. Non-limiting examples of amino acid sequences for wild-type human SMN protein are provided in UniProtKB / Swiss-Prot: Q16637.1. Other publications in which the SMN1 coding sequence is described include, for example, International Publication No. WO 2010 / 129021 A1 and International Publication No. WO 2009 / 151546 A2, the entire contents of which are incorporated herein by reference.

[0174] In some embodiments, a coding sequence encoding a functional SMN protein is provided. In some embodiments, the amino acid sequence of functional SMN1 is that of human SMN1 protein or a sequence sharing 95% identity therewith.

[0175] In some embodiments, a modified hSMN1 coding sequence is provided. In some embodiments, the modified hSMN1 coding sequence has less than about 80% identity, preferably about 75% or less identity, with the full-length native hSMN1 coding sequence. In some embodiments, the modified hSMN1 coding sequence is characterized by an improved translation rate compared to native hSMN1 after AAV-mediated delivery (e.g., using rAAV particles). In some embodiments, the modified hSMN1 coding sequence shares less than about 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or less identity with the full-length native hSMN1 coding sequence.

[0176] The terms "percent identity (%)", "sequence identity", "percent sequence identity" or "percent identical" in the context of nucleic acid sequences refer to residues in two sequences that are the same when aligned for comparison. The length of the sequence identity comparison may span the entire length of the genome, the entire length of the gene coding sequence, or preferably at least a fragment of about 500 - 5000 nucleotides. However, identity between smaller fragments, for example, at least about 9 nucleotides, usually at least about 20 - 24 nucleotides, at least about 28 - 32 nucleotides, at least about 36 or more nucleotides may also be desirable.

[0177] An "aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that, when compared to a reference sequence, often contain corrections for deletions or additions of bases or amino acids.

[0178] Alignment can be performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, for example, the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match - Box" programs are available. Generally, any of these programs are used with default settings, but those skilled in the art can change these settings if necessary. Alternatively, those skilled in the art can utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the reference algorithm and reference program. See, for example, J. D. Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682 - 2690 (1999).

[0179] Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal W", "CAP Sequence Assembly", "BLAST", "MAP" and "MEME", which are accessible through web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility is also used. There are also many algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG version 6.1. Fasta™ provides an alignment of the optimal overlapping regions and the percent sequence identity between the query sequence and the search sequence. For example, the percent sequence identity between nucleic acid sequences can be determined using Fasta™ as provided in GCG version 6.1, which is incorporated herein by reference, and its default parameters (word size 6, and NOP AM coefficient for the scoring matrix).

[0180] In some embodiments, the modified hSMN1 coding sequence is a codon-optimized sequence optimized for expression in the subject species. As used herein, "subject" is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate such as a monkey, chimpanzee, baboon or gorilla. In some embodiments, the subject is a human. Thus, in some embodiments, the SMN1 coding sequence is codon-optimized for expression in humans.

[0181] The codon-optimized coding region can be designed in a variety of different ways. This optimization can be performed using methods available online (e.g., GeneArt), published methods, or companies that provide codon optimization services, such as DNA2.0 (Menlo Park, CA). One codon optimization method is described, for example, in U.S. International Publication No. 2015 / 012924, which is hereby incorporated by reference in its entirety. See also, for example, U.S. Patent Application Publication No. 2014 / 0032186 and U.S. Patent Application Publication No. 2006 / 0136184.

[0182] In some embodiments, the entire length of the open reading frame (ORF) is modified. However, in some embodiments, only a fragment of the ORF is changed. By using one of these methods, a frequency can be applied to any given polypeptide sequence to generate a nucleic acid fragment of a codon-optimized coding region that encodes the polypeptide. Thus, in some embodiments, a codon-optimized SMN1 coding sequence is used (e.g., a codon-optimized hSMN1 ORF). In some embodiments, one or more portions of the SMN1 coding sequence (e.g., up to the entire ORF) are codon-optimized for expression in humans.

[0183] A number of options are available for making the actual changes to the codons or for synthesizing the codon-optimized coding regions designed as described herein. Such modifications or syntheses can be carried out using standard and routine molecular biology manipulations well known to those of skill in the art. In one approach, a series of complementary oligonucleotide pairs corresponding to the desired sequence length, each 80 - 90 nucleotides in length, are synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing they form 80 - 90 base pair double-stranded fragments containing sticky ends. For example, each oligonucleotide in a pair is synthesized such that it extends 3, 4, 5, 6, 7, 8, 9, 10 or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides are designed to anneal with the single-stranded ends of another pair of oligonucleotides. The oligonucleotide pairs are capable of annealing, and then approximately 5 - 6 of these double-stranded fragments can anneal together via the sticky single-stranded ends and then they are ligated together and cloned into a standard bacterial cloning vector, e.g., the TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Some of these constructs consisting of 5 - 6 fragments of 80 - 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared such that the entire desired sequence is represented by a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector and sequenced. Additional or alternative methods can also be used (e.g., including commercially available gene synthesis services).

[0184] In some embodiments, the SMN1 cDNA sequence can be synthetically made in vitro using techniques known in the art. For example, as described in Xiong et al, PCR-based accurate synthesis of long DNA sequences, Nature Protocols 1, 791 - 797 (2006), a PCR-based accurate synthesis (PAS) method for long DNA sequences may be utilized. Methods combining the double asymmetric PCR method and the overlap extension PCR method are described by Young and Dong, Two-step total gene synthesis method, Nucleic Acids Res. 2004; 32(7): e59. See also Gordeeva et al, J Microbiol Methods. Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification. 2010 May;81(2): 147-52. Epub 2010 Mar 10; also see Gene Seq. 2012 Apr;6(l): 10-21; U.S. Patent No. 8008005 and U.S. Patent No. 7985565 for oligonucleotide synthesis and gene synthesis. Each of these documents is incorporated herein by reference. In addition, kits and protocols for making DNA via PCR are commercially available. These include, but are not limited to, the use of polymerases including Taq polymerase; OneTaq® (New England Biolabs); Q5® High-Fidelity DNA Polymerase (New England Biolabs); and GoTaq® G2 Polymerase (Promega). DNA may also be made from cells transfected with a plasmid containing the hSMN sequence described herein.Kits and protocols are known and commercially available and include, but are not limited to, QIAGEN plasmid kits; Chargeswitch® Pro Filter Plasmid Kits (Invitrogen); and GenElute™ Plasmid Kits (Sigma Aldrich). Other techniques useful herein include sequence-specific isothermal amplification methods, excluding the need for thermal cycling. Instead of heat, these methods typically use strand-displacing DNA polymerases such as Bst DNA Polymerase, Large Fragment (New England Biolabs) to separate double-stranded DNA. DNA may also be made from RNA molecules by amplification through the use of reverse transcriptase (RT), an RNA-dependent DNA polymerase. RT polymerizes a strand of DNA that is complementary to the original RNA template and is referred to as cDNA. This cDNA can then be further amplified by the PCR or isothermal methods outlined above. Custom DNA can also be made commercially from companies including, but not limited to, GenScript; GENEWIZ®; GeneArt® (Life Technologies); and Integrated DNA Technologies.

[0185] "Functional SMN1" refers to the gene encoding the native SMN protein, or another gene encoding an SMN protein that provides a biological activity level of native survival motor neuron protein that is at least about 50%, at least about 75%, at least about 80%, at least about 90%, or substantially the same, or higher than 100%, or those natural variants or polymorphisms not associated with disease. In addition, SMN2, an SMN1 homolog, also encodes the SMN protein, but processing of the functional protein is less efficient. Based on the copy number of SMN2, subjects lacking the functional hSMN1 gene demonstrate varying degrees of SMA. Thus, for some subjects, it may be desirable for the SMN protein to provide less than 100% of the biological activity of the native SMN protein.

[0186] In some embodiments, such a functional SMN has a sequence having at least about 95% or higher identity to the native protein, or at the amino acid level, at least about 97% or higher identity, or about 99% identity. Such a functional SMN protein may also include natural polymorphisms. Identity can be determined by preparing an alignment of the sequences and using various algorithms and / or computer programs known or commercially available in the art (e.g., BLAST, ExPASy; ClustalO; FASTA; e.g., using the Needleman-Wunsch algorithm, the Smith-Waterman algorithm).

[0187] The percent identity can be readily determined for the full length of the protein, the polypeptide, the amino acid sequence over about 32 amino acids, about 330 amino acids or a peptide fragment thereof, or the corresponding nucleic acid sequence encoding the sequence. Suitable amino acid fragments can be at least about 8 amino acids in length and up to about 700 amino acids. Generally, when referring to "identity", "homology" or "similarity" between two different sequences, "identity", "homology" or "similarity" is determined with respect to the "aligned" sequences.

[0188] In some embodiments, the modified SMN1 (e.g., hSMN1) gene described herein is engineered into a suitable genetic element (e.g., a vector), such as naked DNA, phage, transposon, cosmid, episome, etc., that is useful for creating a viral vector and / or delivering it to a host cell, and that transfers the SMN1 sequence carried therein. The vector selected can be delivered by any suitable method including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection, and protoplast fusion. The methods used to create such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic, recombinant, and synthetic techniques. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0189] In some embodiments, an expression cassette comprising an SMN1 (e.g., hSMN1) nucleic acid sequence(s) is provided. As used herein, an “expression cassette” refers to a nucleic acid molecule that comprises an SMN1 sequence operably linked to a promoter and may include other regulatory sequences. In some embodiments, the expression cassette is packaged into the capsid (e.g., viral particle) of a viral vector. Typically, such an expression cassette for creating a viral vector contains the SMN1 (e.g., hSMN1) sequence described herein flanked by a packaging signal of the viral genome and other expression control sequences such as those described herein. For example, for an AAV viral vector, the packaging signals are the 5′ inverted terminal repeat (ITR) and the 3′ ITR. When packaged into an AAV capsid, the ITRs together with the expression cassette are herein referred to as the “recombinant AAV (rAAV) genome” or “vector genome” within the rAAV particle or capsid.

[0190] The term "expression" is used herein in its broadest sense and includes the production of RNA or the production of RNA and protein. With respect to RNA, the terms "expression" or "translation" specifically relate to the production of peptides or proteins. Expression may be transient or stable.

[0191] The term "translation" in the context of the present invention relates to the process at the ribosome by which an mRNA strand controls the assembly of an amino acid sequence to produce a protein or peptide.

[0192] Promoters and regulatory elements: In some embodiments, the expression construct includes one or more regions that promote the expression of the coding sequence of the SMN1 gene, e.g., expression control sequences operably linked to the coding sequence. Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, start sites, termination signals, and poly(A) tails. Any combination of such control sequences is contemplated herein (e.g., a promoter and an enhancer).

[0193] In one aspect, the expression cassette contains a promoter sequence as part of the expression control sequence and is located, for example, between the 5’ ITR sequence and the SMN1 coding sequence. The exemplary plasmids and vectors described herein use the ubiquitous chicken β-actin promoter (CB) with the CMV immediate early enhancer (CMV IE). Alternatively, other neuron-specific promoters may be used (see, for example, the Lockery Lab Neuron-Specific Promoter Database accessible at http: / / chinook.uoregon.edu / promoters.html). Such neuron-specific promoters include, but are not limited to, synapsin I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase, and platelet-derived growth factor beta chain promoter. See Hioki et al, Gene Therapy, June 2007, 14(11):872-82, which is incorporated herein by reference. Other neuron-specific promoters include the promoters of 67 kDa glutamate decarboxylase (GAD67), homeobox Dlx5 / 6, glutamate receptor 1 (GluR1), preprotachykinin 1 (Tac1), neuron-specific enolase (NSE), and dopamine receptor 1 (Drd1a). See, for example, Delzor et al, Human Gene Therapy Methods. August 2012, 23(4): 242-254. In another aspect, the promoter is the GUSb promoter of http: / / www.jci.Org / articles / view / 41615#B30.

[0194] Other promoters such as constitutive promoters, inducible promoters (see, for example, WO 2011 / 126808 and WO 2013 / 04943), or promoters responsive to physiological cues may be used. The promoter(s) can be from different sources, such as the human cytomegalovirus (CMV) immediate early enhancer / promoter, SV40 early enhancer / promoter, JC polyomavirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latency-associated promoter (LAP), Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, chicken beta-actin (CBA) promoter, and matrix metalloprotein (MPP) promoter.

[0195] In addition to the promoter, the expression cassette and / or vector may contain one or more other suitable transcription initiation sequences, termination sequences, enhancer sequences, effective RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA, such as WPRE; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance the secretion of the encoded product. Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. An example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include those suitable for the symptoms of the CNS. In some embodiments, the expression cassette includes one or more expression enhancers. In some embodiments, the expression cassette contains two or more expression enhancers. These enhancers may be the same as each other or different from each other. For example, the enhancer may include the CMV immediate early enhancer. This enhancer may be present in two copies located adjacent to each other. Alternatively, the double copy of the enhancer may be separated by one or more sequences. In yet another embodiment, the expression cassette further contains an intron, such as the chicken beta-actin intron. Other suitable introns include those known in the art, for example, as described in WO 2011 / 126808. In some embodiments, the intron is incorporated upstream of the coding sequence to improve 5'-capping and stability of the mRNA. Optionally, one or more other sequences may be selected such that the mRNA is stabilized. An example of such a sequence is a modified WPRE sequence, which can be engineered at positions upstream of the polyA sequence and downstream of the coding sequence (see, for example, MA Zanta-Boussif, et al, Gene Therapy (2009) 16: 605-619).

[0196] In some embodiments, these control sequences are "operably linked" to the SMN1 gene sequence. As used herein, the term "operably linked" refers to both an expression control sequence adjacent to the gene of interest and an expression control sequence that acts in trans, either adjacent to or separate from, to control the gene of interest.

[0197] Recombinant viral vector: In some embodiments, an adeno-associated virus vector comprising an AAV capsid and at least one expression cassette is provided. In some embodiments, the at least one expression cassette comprises a nucleic acid sequence encoding SMN1 and an expression control sequence that directs the expression of the SMN1 sequence in a host cell. The rAAV vector genome can also include AAV ITR sequences. In some embodiments, the ITR is derived from an AAV serotype different from the serotype of the capsid protein used to package the rAAV genome. In some embodiments, the ITR sequence is derived from AAV2 or a deleted version thereof (AITR), which can be used for convenience and to expedite regulatory approval. However, ITRs from other AAV origins may be selected. When the origin of the ITR is from AAV2 and the AAV capsid is from another AAV origin, the resulting vector may be referred to as pseudotyped. Typically, the rAAV vector genome includes an AAV 5' ITR, the SMN1 coding sequence and any regulatory sequences, and an AAV 3' ITR. However, other arrangements of these elements may also be suitable. A shortened version of the 5' ITR designated AITR has been described, in which the D-sequence and the terminal resolution site (trs) are deleted. In other embodiments, the full-length AAV 5' ITR and AAV 3' ITR are used.

[0198] The ITR sequences of the nucleic acids or nucleic acid vectors described herein can be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), or can be derived from two or more serotypes. In some embodiments, the ITR sequences and plasmids containing the ITR sequences are known in the art and commercially available (e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca and Addgene, Cambridge, MA; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14082-7; and Curtis A. Machida. Methods in Molecular Medicine (trademark). Viral Vectors for Gene Therapy Methods and Protocols. 10.1385 / 1-59259-304-6:201 (C) Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; see U.S. Patent Nos. 5,139,941 and 5,962,313, all of which are incorporated herein by reference).

[0199] In some embodiments, the rAAV nucleic acid or genome can be single-stranded (ss). However, in some embodiments, the rAAV nucleic acid or genome can be a self-complementary (sc) AAV nucleic acid vector. In some embodiments, the recombinant AAV particles comprise a nucleic acid vector such as a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector. In some embodiments, the nucleic acid vector contains one or more regions comprising the SMN1 gene and inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) adjacent to the expression construct. In some embodiments, the nucleic acid is encapsidated by a viral capsid.

[0200] Accordingly, in some embodiments, the AAV particles comprise a viral capsid and a nucleic acid vector described herein that is encapsidated by the viral capsid. In some embodiments, the viral capsid comprises 60 capsid protein subunits including VP1, VP2, and VP3. In some embodiments, the VP1, VP2, and VP3 subunits are present in the capsid in a ratio of approximately 1:1:10, respectively.

[0201] In some embodiments, recombinant adeno-associated virus (rAAV) is an AAV DNase-resistant particle having an AAV protein capsid in which a nucleic acid sequence is packaged for delivery to a target cell. In some embodiments, the AAV capsid is composed of VP1, VP2, and VP3 of 60 capsid (cap) protein subunits arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. The AAV capsid may be selected from those known to those of skill in the art, including its variants. In some embodiments, the AAV capsid is selected from those that efficiently transduce neuronal cells. In some embodiments, the AAV capsid is selected from AAV1, AAV2, AAV7, AAV8, AAV9, AAVrh10, AAV5, AAVhu11, AAV8DJ, AAVhu32, AAVhu37, AAVpi2, AAVrh8, AAVhu48R3, AAVhu68, and their variants. See International Publication No. WO 2018 / 160585 A2, International Publication No. WO 2018 / 160582 A1, Royo, et al, Brain Res, 2008 Jan, 1190: 15-22; Petrosyan et al, Gene Therapy, 2014 Dec, 21(12):991-1000; Holehonnur et al, BMC Neuroscience, 2014, 15:28; and Cearley et al, Mol Ther. 2008 Oct; 16(10): 1710-1718, each of which is incorporated herein by reference. Other AAV capsids useful herein include AAVrh39, AAVrh20, AAVrh25, AAV10, AAVbb1, and AAVbb2, and their variants. For example, other AAV serotypes, including variants of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAVrh64Rl, AAVrh64R2, AAVrh8, and any AAV known or mentioned or not yet discovered, may be selected as the origin for the capsid of the AAV viral vector (DNase-resistant viral particle).See, for example, U.S. Patent Application Publication No. 2007-0036760 A1; U.S. Patent Application Publication No. 2009-0197338 A1; European Patent Application Publication No. 1310571. See also International Publication No. 2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent No. 7,790,449 and U.S. Patent No. 7,282,199 (AAV8), International Publication No. 2005 / 033321 and U.S. Patent No. 7,906,111 (AAV9), as well as International Publication No. 2006 / 110689, and International Publication No. 2003 / 042397 (rh10). Alternatively, recombinant AAVs based on any of the AAVs listed may be used as the origin for the AAV capsid. These documents also describe other AAVs that may be selected for making AAVs and are incorporated herein by reference. In some embodiments, the AAV cap for use in a viral vector can be made by mutagenesis of one of the aforementioned AAV Caps or its coding nucleic acid (e.g., by insertion, deletion, or substitution). In some embodiments, the AAV capsid is a chimera that includes domains derived from two or three or four or more of the aforementioned AAV capsid proteins. In some embodiments, the AAV capsid is a mosaic of Vp1, Vp2, and Vp3 monomers derived from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition includes two or more of the aforementioned Caps. As used herein, with respect to AAV, the term variant means any AAV sequence derived from a known AAV sequence and includes those that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or higher sequence identity across the amino acid or nucleic acid sequence. In another embodiment, AAV capsids include variants that may contain up to about 10% variation from any of the described or known AAV capsid sequences. That is, the AAV capsid shares about 90% to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with the AAV capsids provided herein and / or known in the art.In some embodiments, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, the comparison can be made across any of the variable proteins (e.g., vp1, vp2, or vp3). In some embodiments, the AAV capsid shares at least 95% identity with AAV8 vp3.

[0202] In some embodiments, self-complementary AAV is provided. The abbreviation "sc" in this context refers to self-complementary. "Self-complementary AAV" refers to constructs in which the coding regions carried by the recombinant AAV nucleic acid sequences are designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cellular-mediated synthesis of the second strand, the two complementary halves of scAAV associate to form one double-stranded DNA (dsDNA) unit that is immediately ready for replication and transcription. See, for example, D M McCarty et al, "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAV is described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0203] Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, U.S. Patent Application Publication No. 2007 / 0036760 (Feb. 15, 2007), U.S. Patent No. 7,790,449; U.S. Patent No. 7,282,199; International Publication No. 2003 / 042397; International Publication No. 2005 / 033321, International Publication No. 2006 / 110689; and U.S. Patent No. 7,588,772 B2. In one system, the production cell line is transiently transfected with a construct encoding a transgene flanked by ITRs, as well as a construct (s) encoding rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, and separation of rAAV from contaminating viruses is required. Systems that do not require infection with a helper virus to recover AAV have also been developed, and the necessary helper functions (e.g., adenovirus E1, E2a, VA and E4, or herpesvirus UL5, UL8, UL52 and UL29, as well as herpesvirus polymerase) are also supplied in trans by the system. In these systems, the helper functions can be supplied by transient transfection of cells with a construct encoding the necessary helper functions, or the cells can be engineered to stably contain a gene encoding the helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In yet another system, the transgene flanked by ITRs and the rep / cap genes are introduced into insect cells by infection with a baculovirus-based vector.For a general review of these production systems, see, for example, Zhang et al, 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the entire contents of which are incorporated herein by reference. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the entire contents of each of which are incorporated herein by reference: U.S. Patent Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0204] Optionally, the SMN1 gene described herein can be used to generate viral vectors other than rAAV, which can also be used in combination therapy with SMN2 ASO. Such other viral vectors can include any virus suitable for use in gene therapy, including, but not limited to, adenovirus; herpes virus; lentivirus; retrovirus, etc. Preferably, when one of these other vectors is generated, it is produced as a replication-deficient viral vector.

[0205] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged into a viral capsid or envelope, where any viral genomic sequences packaged within the viral capsid or envelope are also replication-defective, i.e., they are unable to generate progeny virions but retain the ability to infect target cells. In some embodiments, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "attenuated" and contains only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), and these genes can be supplied during production. Thus, replication and infection by progeny virions can occur only in the presence of viral enzymes required for replication, and are therefore considered safe for use in gene therapy. Such replication-defective viruses can be adeno-associated virus (AAV), adenovirus, lentivirus (integrating or non-integrating), or another suitable viral origin.

[0206] Also provided are host cells comprising at least one of the disclosed AAV particles, expression constructs or nucleic acid vectors. Such host cells include mammalian host cells, such as human host cells, and can be isolated either in cell culture or tissue culture. In the case of a genetically modified animal model (e.g., a mouse), the host cells to be transformed may be contained within the body of the non-human animal itself.

[0207] Oligomeric compounds that increase the production of full-length SMN2 mRNA In some embodiments, a combination therapy for treating SMA includes administering, in addition to other therapies described herein (e.g., recombinant SMN1 gene and / or small molecules that increase SMN function), an ASO complementary to the pre-mRNA encoding SMN2 (also referred to herein as SMN2 ASO) (e.g., in combination or sequentially). In some embodiments, the ASO increases full-length SMN2 mRNA. In some embodiments, the ASO changes the splicing of SMN2 pre-mRNA. In some embodiments, the ASO promotes exon 7 inclusion in SMN2 mRNA. Some sequences and regions useful for changing the splicing of SMN2 can be found in PCT / US06 / 024469 (published as WO 2007 / 002390) and WO 2018 / 014041 A2, which are hereby incorporated by reference in their entirety for any purpose.

[0208] In some embodiments, the SMN2 ASO efficiently modulates the splicing of SMN2, resulting in an increase in exon 7 inclusion in SMN2 mRNA and ultimately an increase in SMN2 protein containing the amino acids corresponding to exon 7. Such alternative SMN2 proteins are 100% identical to the wild-type SMN protein.

[0209] An ASO that efficiently modulates the expression of SMN2 mRNA to produce a functional SMN protein is considered an active ASO. Modulation of the expression of SMN2 can be measured in a body fluid, which may or may not contain animal cells, tissues or organs. Methods for obtaining samples for analysis, e.g., body fluids (e.g., sputum, serum, CSF), tissues (e.g., biopsies) or organs, and methods for preparing the samples to enable analysis are well known to those of skill in the art. The effect of treatment can be evaluated by measuring biomarkers associated with target gene expression in one or more biological fluids, tissues or organs collected from an animal that has been contacted with one or more of the compositions described in this application.

[0210] In some embodiments, an increase in full-length SMN2 mRNA means that the intracellular level of full-length SMN2 mRNA is higher than the level of full-length SMN2 mRNA in a reference level, e.g., a control (e.g., a subject not administered with an SMN2 ASO). The increase in intracellular full-length SMN2 mRNA can be measured as an increase in the level of full-length protein and / or mRNA produced from the SMN2 gene. In some embodiments, the increase in full-length SMN2 mRNA can be determined by investigation of the external characteristics of the cell or organism (e.g., as described in the examples below), or by assay techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, monitoring gene expression by microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), nucleic acid sequencing, Western blot, radioimmunoassay (RIA), other immunoassays, fluorescence-activated cell analysis (FACS), or any other technique or combination of techniques capable of detecting the presence of full-length SMN2 mRNA or protein (e.g., in a subject or a sample obtained from a subject).

[0211] In some embodiments, the degree to which an SMN2 ASO increases full-length SMN2 mRNA can be determined by comparing the level of full-length SMN2 mRNA in a sample obtained from a subject treated with an SMN2 ASO with the level of full-length SMN2 mRNA in a subject not treated with an SMN2 ASO. In some embodiments, the reference level of full-length SMN2 mRNA is obtained from the same subject prior to receiving the SMN2 ASO. In some embodiments, the reference level of full-length SMN2 mRNA is within the range determined by a population of subjects not receiving an SMN2 ASO.

[0212] In some embodiments, the increased level of full-length SMN2 mRNA is, for example, higher than 1-fold, 1.5 - 5-fold, 5 - 10-fold, 10 - 50-fold, 50 - 100-fold, about 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or higher than the reference value.

[0213] In some embodiments, it is possible to determine whether an SMN2 ASO has resulted in an increase in full-length SMN2 mRNA by comparing the ratio of the shorter SMN2 mRNA (e.g., SMN2 mRNA without exon 7) to the full-length SMN2 mRNA in a subject receiving SMN2 ASO administration with a reference ratio. In some embodiments, the reference ratio is the ratio of the shorter SMN2 mRNA (e.g., SMN2 mRNA without exon 7) to the full-length SMN2 mRNA before administration of the SMN2 ASO. In some embodiments, the ratio of the shorter SMN2 mRNA (e.g., SMN2 mRNA without exon 7) to the full-length SMN2 mRNA in a subject receiving SMN2 ASO is, for example, higher than 1-fold, 1.5 - 5-fold, 5 - 10-fold, 10 - 50-fold, 50 - 100-fold, about 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or higher than the reference ratio.

[0214] In some embodiments, an increase in full-length SMN2 mRNA in a subject can be indicated by an increase in full-length SMN protein as compared to a reference level. In some embodiments, the reference level of full-length SMN protein is the level of full-length SMN protein obtained from a subject having SMA prior to treatment or a subject at risk of having SMA. In some embodiments, the production of exon 7-containing SMN protein increases in a subject receiving SMN2 ASO administration with an enhancement of the exon 7-containing SMN protein level by at least about, for example, more than 1-fold, 1.5 to 5-fold, 5 to 10-fold, 10 to 50-fold, 50 to 100-fold, about 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or higher than the reference value. Also contemplated is a method by contacting a body fluid, organ or tissue with an effective amount of one or more of the compositions described in this application. The body fluid, organ or tissue can be contacted with one or more of the compositions, resulting in modulation of SMN1 expression and SMN2 expression in the cells of the body fluid, organ or tissue. The effective amount of the composition can be determined by monitoring the effect on the expression of functional SMN protein of the recombinant SMN1 gene and SMN2 ASO administered to the subject or contacted with the cells.

[0215] 1. Antisense oligonucleotide (ASO) In some embodiments, an ASO comprising a sequence complementary to a nucleic acid encoding human SMN2 is provided for use in the treatment of diseases or conditions associated with survival motor neuron protein (SMN), such as spinal muscular atrophy (SMA) (e.g., by a recombinant SMN1 gene and / or a small molecule that increases SMN function). In some embodiments, an ASO comprising a sequence complementary to a nucleic acid encoding human SMN2 is provided for use in the treatment of diseases or conditions associated with survival motor neuron protein (SMN) (e.g., by a recombinant SMN1 gene and / or a small molecule that increases SMN function) by direct administration of the ASO to the central nervous system (CNS) or cerebrospinal fluid (CSF).

[0216] As used herein, the term "oligomeric compound" refers to a compound that includes an oligonucleotide. In some embodiments, the oligomeric compound consists of an oligonucleotide. As used herein, the term "oligonucleotide" refers to a compound that includes a phosphoric acid linking group, a heterocyclic base moiety, and a sugar moiety. In some embodiments, the oligomeric compound further includes one or more conjugate groups and / or terminal groups. In some embodiments, the oligomeric compound is an antisense oligonucleotide (ASO). As used herein, the term "antisense oligonucleotide" or "ASO" refers to an oligomeric compound, at least a portion of which is at least partially complementary to a target nucleic acid to which it hybridizes, wherein such hybridization results in at least one antisense activity.

[0217] In some examples, an antisense oligonucleotide (ASO) increases full-length SMN protein in a subject. In some examples, the ASO increases full-length SMN2 mRNA in a subject. In some embodiments, the ASO that increases full-length SMN2 mRNA is an antisense oligonucleotide that is complementary to a nucleic acid encoding SMN2. In some embodiments, the ASO increases full-length SMN2 mRNA by altering the splicing pattern of SMN2 pre-mRNA. In some embodiments, the ASO promotes exon skipping during splicing of SMN2 pre-mRNA. In some embodiments, the ASO promotes inclusion of exon 7 in SMN2 mRNA. In some embodiments, the ASO is designed to target the boundary between intron 6, intron 7, or exon 7 and the adjacent intron of SMN2 pre-mRNA to promote inclusion of exon 7 in SMN2 mRNA. In some embodiments, the ASO comprises a nucleobase sequence complementary to intron 6 of SMN2 pre-mRNA. In some embodiments, the ASO comprises a nucleobase sequence complementary to exon 6 of SMN2 pre-mRNA. In some embodiments, the ASO comprises a nucleobase sequence complementary to intron 7 of SMN2 pre-mRNA. In some embodiments, the ASO targeting intron 7 of SMN2 pre-mRNA comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the ASO targeting intron 7 of SMN2 pre-mRNA is nusinersen. In some embodiments, one or more of the ASOs described herein can be administered to a subject to increase the levels of full-length SMN protein and / or full-length SMN2 mRNA. Non-limiting examples of sequences and regions useful for altering the splicing of SMN2 can be found in PCT / US06 / 024469, which is hereby incorporated by reference in its entirety for any purpose. In some embodiments, the antisense oligonucleotide has a nucleobase sequence that is complementary to intron 7 of SMN2. Non-limiting examples of such nucleobase sequences are exemplified in the table below.

[0218]

Table 1-1

Table 1-2

[0219] In some embodiments, the ASO targets intron 7 of the SMN2 pre-mRNA. In some embodiments, the ASO comprises a nucleobase sequence comprising at least 10 nucleobases of the sequence: TCACTTTCATAATGCTGG sequence (SEQ ID NO: 1). In some embodiments, the ASO has a nucleobase sequence comprising at least 11 nucleobases of SEQ ID NO: 1. In some embodiments, the ASO has a nucleobase sequence comprising at least 12 nucleobases of SEQ ID NO: 1. In some embodiments, the ASO has a nucleobase sequence comprising at least 13 nucleobases of SEQ ID NO: 1. In some embodiments, the ASO has a nucleobase sequence comprising at least 14 nucleobases of SEQ ID NO: 1. In some embodiments, the ASO has a nucleobase sequence comprising at least 15 nucleobases of SEQ ID NO: 1. In some embodiments, the ASO has a nucleobase sequence comprising at least 16 nucleobases of SEQ ID NO: 1. In some embodiments, the ASO has a nucleobase sequence comprising at least 17 nucleobases of SEQ ID NO: 1. In some embodiments, the ASO has a nucleobase sequence comprising the nucleobases of SEQ ID NO: 1. In some embodiments, the ASO has a nucleobase sequence consisting of the nucleobases of SEQ ID NO: 1. In some embodiments, the ASO consists of 10-18 linked nucleosides and has a nucleobase sequence that is 100% identical to a portion of the same length of the sequence: TCACTTTCATAATGCTGG (SEQ ID NO: 1).

[0220] In some embodiments, the SMN2 ASO is complementary to a nucleic acid molecule encoding the SMN2 protein. In some embodiments, the ASO is complementary to intron 6, exon 7 (or the exon 7 and adjacent intron boundary) or intron 7 of a nucleic acid molecule encoding the SMN2 protein. In some embodiments, the ASO targets intron 7 of the SMN2 pre-mRNA. In some embodiments, the SMN2 ASO that targets intron 7 of the SMN2 pre-mRNA is nusinersen. An exemplary nucleotide sequence for nusinersen is 5’-UCACUUUCAUAAUGCUGG-3’ (SEQ ID NO: 26). The nusinersen (also referred to as ISIS396443) of the active substance has the sequence: 5’- Me U Me CA Me C Me U Me U Me U Me CA Me UAA Me UG Me C Me UGG-3’ (SEQ ID NO: 25) and is a uniformly modified 2’-O-(2-methoxyethyl) phosphorothioate antisense oligonucleotide consisting of 18 nucleotide residues. In some embodiments, the SMN2 ASO comprises a nucleobase sequence comprising the nucleobases of SEQ ID NO: 25 or 26.

[0221] The chemical name of nusinersen sodium is 2’-O-(2-methoxyethyl)-5-methyl-P-thiouridyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiocytidyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-P-thioadenyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiocytidyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiouridyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiouridyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiouridyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiocytidyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-P-thioadenyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiouridyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-P-thioadenyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-P-thioadenyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiouridyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-P-thioguanidyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiocytidyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-5-methyl-P-thiouridyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl)-P-thioguanidyl-(3’-O→5’-O)-2’-O-(2-methoxyethyl) guanosine, which corresponds to the molecular formula C234H323N61O128P17S17Na17, has a relative molecular mass of 7501.0 g / mol and the structure shown in Figure 3.

[0222] Antisense is an effective means for modulating the expression of one or more specific gene products and is uniquely useful in many therapeutic, diagnostic, and research applications. Antisense compounds useful for modulating gene expression via an antisense mechanism of action that includes an antisense mechanism based on target occupancy are provided herein. In one aspect, the antisense compounds provided herein modulate the splicing of a target gene. Such modulation includes promoting or inhibiting the inclusion of an exon. Antisense compounds that target cis-splicing regulatory elements present in a pre-mRNA molecule, including exon splicing enhancers, exon splicing silencers, intron splicing enhancers, and intron splicing silencers, are further provided herein. Disruption of cis-splicing regulatory elements is thought to alter splice site selection, which can result in a change in the composition of splice products.

[0223] The processing of eukaryotic pre-mRNAs is a complex process that requires numerous signals and protein factors to achieve proper mRNA splicing. The definition of exons by the spliceosome requires more than the standard splicing signals that define intron-exon boundaries. One such additional signal is provided by cis-acting regulatory enhancer and silencer sequences. Exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs), and intronic splicing silencers (ISSs) have been identified as either suppressing or enhancing the use of splice donor or splice acceptor sites, depending on their location and mechanism of action (Yeo et al. 2004, Proc. Natl. Acad. Sci. U.S.A. 101(44): 15700-15705). Binding of proteins (trans-factors) specific to these regulatory sequences directs the splicing process, promotes or inhibits the use of specific splice sites, and thus modulates the ratio of splicing 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).

[0224] In some embodiments, an antisense oligonucleotide comprises one or more modifications compared to oligonucleotides of naturally occurring oligomers such as DNA or RNA. Such modified antisense oligonucleotides may have one or more desirable properties. In some embodiments, the modifications alter the antisense activity of the antisense oligonucleotide, for example, by increasing the affinity of the antisense oligonucleotide for its target nucleic acid, increasing its resistance to one or more nucleases, and / or altering the pharmacokinetics or tissue distribution of the oligonucleotide. In some embodiments, the modified antisense oligonucleotide comprises one or more modified nucleosides and / or one or more modified nucleoside linkages and / or one or more conjugate groups.

[0225] a. Modified nucleosides In some embodiments, an antisense oligonucleotide comprises one or more modified nucleosides. Such modified nucleosides can include a modified sugar and / or a modified nucleobase. In some embodiments, incorporation of such modified nucleosides into an oligonucleotide results in an increase in affinity for a target nucleic acid and / or an increase in stability, including, but not limited to, an increase in resistance to degradation by nucleases, and / or an improvement in the toxicity and / or uptake properties of the modified oligonucleotide.

[0226] i. Nucleobases The base moieties of naturally occurring nucleosides are heterocyclic bases and are typically purines and pyrimidines. In addition to the "unmodified" or "natural" nucleobases such as adenine (A) and guanine (G) of purine nucleobases and thymine (T), cytosine (C), and uracil (U) of pyrimidine nucleobases, many modified nucleobases or nucleobase mimics known to those skilled in the art are suitable for incorporation into the compounds described herein. In some embodiments, the modified nucleobases are nucleobases that have a structure fairly similar to the parent nucleobase, such as, for example, 7-deazapurine, 5-methylcytosine, or G-clamp. In some embodiments, the nucleobase mimics include more complex structures such as, for example, tricyclic phenoxazine nucleobase mimics. Methods for preparing modified nucleobases are well known to those skilled in the art.

[0227] ii. Modified sugars and sugar substitutes The antisense oligonucleotides of the present application can, if desired, contain one or more nucleosides in which the sugar moiety is modified compared to the natural sugar. Oligonucleotides containing sugar-modified nucleosides can have enhanced nuclease stability, increased binding affinity, or some other advantageous biological property. Such modifications include, but are not limited to, for example, 2'-F-5'-methyl-substituted nucleosides (see PCT International Application Publication No. WO 2008 / 101157, published August 21, 2008, for other disclosed 5',2'-bis-substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see U.S. Patent Application Publication No. US 20050130923, published June 16, 2005) or 5'-substitution of alternative BNA (see PCT International Application Publication No. WO 2007 / 134181, published November 22, 2007, where LNA is substituted, for example, with a 5'-methyl group or a 5'-vinyl group), addition of substituents, crosslinking of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R) or C(R 1 )(R) 2 (R = H, C 1 ~C 12Replacement with an alkyl or a protecting group, and combinations thereof are included.

[0228] Examples of nucleosides having a modified sugar moiety include, but are not limited to, 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, and 2'-O(CH 2 ) 2 OCH 3 Nucleosides containing substituents are included. Substituents at the 2'-position may also be allyl, amino, azido, thio, O-allyl, O-C 1 ~C 10 alkyl, OCF 3 , O(CH 2 )SCH 3 , O(CH 2 ) 2 -O-N(R m )(R n ) and O-CH 2 -C(=O)-N(R m )(R n )(wherein each R m and R n is independently selected from H or substituted or unsubstituted C 1 ~C 10 alkyl).

[0229] Examples of bicyclic nucleic acids (BNA) include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the antisense compounds provided herein include one or more BNA nucleosides in which the bridge comprises one of the following formulas: 4'-beta-D-(CH 2 )-O-2'(beta-D-LNA); 4'-(CH 2 )-S-2:4'-alpha-L-(CH 2 )-O-2'(alpha-L-LNA); 4'-(CH 2 ) 2 -O-2'(ENA); 4'-C(CH 3 ) 2 -O-2'(see PCT / US2008 / 068922); 4'-CH(CH 3 )-O-2' and 4'-C-H(CH2 OCH 3 )-O-2’(see U.S. Patent No. 7,399,845, issued July 15, 2008); 4’-CH 2 -N(OCH 3 )-2’(see PCT / US2008 / 064591); 4’-CH 2 -O-N(CH 3 )-2’(see U.S. Patent Application Publication No. 2004-0171570, published September 2, 2004); 4’-CH 2 -N(R)-O-2’(see U.S. Patent No. 7,427,672, issued September 23, 2008); 4’-CH 2 -C(CH 3 )-2’ and 4’-CH 2 -C(=CH 2 )-2’(see PCT / US2008 / 066154); where R is independently H, C 1 ~C 12 alkyl, or a protecting group.

[0230] In some embodiments, the modified nucleoside containing a modified sugar moiety is not a bicyclic sugar moiety. In some embodiments, the sugar ring of the nucleoside may be modified at any position. Examples of useful sugar modifications include, but are not limited to, compounds containing a sugar substituent selected from OH, F, O-alkyl, S-alkyl, N-alkyl or O-alkyl-O-alkyl, where alkyl, alkenyl and alkynyl are substituted or unsubstituted C 1 ~C 10 alkyl or C 2 ~C 10 alkenyl and C 2 ~C 10 alkynyl. In some embodiments, such substituents are at the 2’ position of the sugar.

[0231] In some embodiments, the modified nucleoside contains a substituent at the 2’ position of the sugar. In some embodiments, such substituents are halides (including, but not limited to, F), allyl, amino, azide, thio, O-allyl, O-C 1 ~C10 Alkyl, -OCF 3 , O-(CH 2 ) 2 -O-CH 3 , 2’-O(CH 2 ) 2 SCH 3 , O-(CH 2 ) 2 -O-N(R m )(R n ) or O-CH 2 -C(=O)-N(R m )(R n (wherein each R m and R n is independently selected from H or substituted or unsubstituted C 1 ~C 10 alkyl).

[0232] In some embodiments, modified nucleosides suitable for use in the present invention are 2-methoxyethoxy, 2’-O methyl (2’-O CH 3 ), 2’-fluoro (2’-F).

[0233] In some embodiments, O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) 2 CH 3 , O(CH 2 ) n ONH 2 , OCH 2 C(=O)N(H)CH 3 and O(CH 2 ) n ON[(CH 2 ) n CH 3 ) 2 (wherein n and m are from 1 to about 10), modified nucleosides having a substituent at the 2’ position. Other 2’-sugar substituents include C 1 ~C 10Alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF 3 、OCF 3 、SOCH 3 、SO 2 CH 3 、ONO 2 、NO 2 、N 3 、NH 2 、heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, a group for improving the pharmacokinetic properties of an oligomeric compound or a group for improving the pharmacodynamic properties of an oligomeric compound, and other substituents having similar properties.

[0234] In some embodiments, the modified nucleoside comprises a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitutions have been described as having improved binding affinity compared to unmodified nucleosides, as well as other modified nucleosides such as 2'-O-methyl, O-propyl and O-aminopropyl. Oligonucleotides having a 2'-MOE substituent have also been shown to be antisense inhibitors of gene expression with promising features for in vivo use (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 Nucleotides, 1997, 16, 917-926).

[0235] In some embodiments, the 2'-sugar substituent is in either the arabino (upward) or ribo (downward) position. In some embodiments, the 2'-arabino modification is 2'-F arabino (FANA). Similar modifications can also be made at other positions of the sugar, particularly at the 3'-position of the sugar of the 3'-terminal nucleoside or 2'-5'-linked oligonucleotide, and at the 5'-position of the 5'-terminal nucleotide.

[0236] In some embodiments, suitable nucleosides have a sugar substitute such as cyclobutyl instead of the ribofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 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 hereby incorporated by reference in its entirety.

[0237] In some embodiments, the nucleoside contains a modification at the 2'-position of the sugar. In some embodiments, the nucleoside contains a modification at the 5'-position of the sugar. In some embodiments, the nucleoside contains modifications at the 2'- and 5'-positions of the sugar. In some embodiments, the modified nucleoside may be useful for incorporation into an oligonucleotide. In some embodiments, the modified nucleoside is incorporated into an oligonucleoside at the 5'-terminus of the oligonucleotide.

[0238] b. Inter-nucleoside linkages Antisense oligonucleotides can optionally contain one or more modified internucleoside linkages. Two main classes of linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (P=O), phosphorotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylene methylimino (-CH 2 -N(CH 3 )-O-CH2), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-), siloxane (-O-Si(H) 2 -O-), and N,N'-dimethylhydrazine (-CH 2 -N(CH 3 )-N(CH 3 )-). Oligonucleotides having non-phosphorus linking groups are referred to as oligonucleosides. Modified linkages can be used, typically to increase, the nuclease resistance of oligonucleotides compared to the native phosphodiester bond. In some embodiments, linkages having chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for the preparation of phosphorus-containing and non-phosphorus-containing linkages are well known to those of skill in the art.

[0239] The antisense oligonucleotides described herein can contain one or more chiral centers and thus can give rise to enantiomers, diastereomers, and other stereoisomeric arrangements that can be defined as (R) or (S) for sugar anomers, etc., or as (D) or (L) for amino acids, etc., with respect to absolute stereochemistry. The antisense compounds provided herein can include all such possible isomers as well as their racemic and optically pure forms.

[0240] In some embodiments, the antisense oligonucleotide has at least one modified internucleoside linkage. In some embodiments, the antisense oligonucleotide has at least two modified internucleoside linkages. In some embodiments, the antisense oligonucleotide has at least three modified internucleoside linkages. In some embodiments, the antisense oligonucleotide has at least ten modified internucleoside linkages. In some embodiments, each internucleoside linkage of the antisense oligonucleotide is a modified internucleoside linkage. In some embodiments, such modified internucleoside linkages are phosphorothioate linkages.

[0241] c. Length In some embodiments, the present invention provides antisense oligonucleotides of any length within various ranges. In some embodiments, the antisense compound or antisense oligonucleotide comprises or consists of a linked nucleoside of X-Y, 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 some embodiments, the antisense compound or antisense oligonucleotide 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,comprising or consisting of linked nucleosides of 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 29~30.

[0242] In some embodiments, the antisense compound or antisense oligonucleotide is 15 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 16 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 17 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 18 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 19 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 20 nucleosides in length.

[0243] d. Oligonucleotide Motif In some embodiments, the antisense oligonucleotide has chemically modified subunits arranged in a specific orientation along their length. In some embodiments, the antisense oligonucleotide is fully modified. In some embodiments, the antisense oligonucleotide is uniformly modified. In some embodiments, the antisense oligonucleotide is uniformly modified and each nucleoside contains a 2'-MOE sugar moiety. In some embodiments, the antisense oligonucleotide is uniformly modified and each nucleoside contains a 2'-OMe sugar moiety. In some embodiments, the antisense oligonucleotide is uniformly modified and each nucleoside contains a morpholino sugar moiety.

[0244] In some embodiments, the oligonucleotide contains an alternating motif. In some embodiments, the alternating modification type is selected from 2'-MOE, 2'-F, bicyclic sugar-modified nucleosides, and DNA (unmodified 2'-deoxy). In some embodiments, each alternating region contains a single nucleoside.

[0245] In some embodiments, the oligonucleotide contains one or more blocks of a first type of nucleoside and one or more blocks of a second type of nucleoside.

[0246] In some embodiments, one or more alternating regions in the alternating motif contain more than a certain type of single nucleoside. For example, the oligomeric compound has the following nucleoside motif: [Table 2] and may contain one or more regions of any of the above, where Nu1 is a first type of nucleoside and Nu2 is a second type of nucleoside. In some embodiments, one of Nu1 and Nu2 is a 2'-MOE nucleoside, and the other of Nu1 and Nu2 is selected from a 2'-OMe modified nucleoside, a BNA, and an unmodified DNA or RNA nucleoside.

[0247] 2. Oligomeric Compound In some embodiments, the oligomeric compound is composed of only oligonucleotides. In some embodiments, the oligomeric compound includes oligonucleotides and one or more conjugate groups and / or terminal groups. Such conjugate groups and / or terminal groups can be attached to oligonucleotides having any of the chemical motifs described in this application. Thus, for example, an oligomeric compound containing an oligonucleotide having one or more regions of alternating nucleosides can include a terminal group.

[0248] a. Conjugate Group In some embodiments, the oligonucleotide is modified by the attachment of one or more conjugate groups. Generally, conjugate groups modify one or more properties of the conjugated oligomeric compound, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cell distribution, cellular uptake, charge, and clearance. Conjugate groups are routinely used in the art of chemistry and are directly attached to the parent compound, such as an oligomeric compound like an oligonucleotide, or are attached via a conjugate linking moiety or conjugate linking group as needed. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholanic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. Certain conjugate groups have been previously described, e.g.: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholanic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. 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, e.g., dodecane-diol residues 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 chains 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), palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine moieties or hexylamino-carbonyl-oxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0249] In some embodiments, the conjugate group includes an active pharmaceutical substance such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folic acid, benzothiadiazide, chlorothiazide, diazepine, indomethacin, barbiturate, cephalosporin, sulfa drug, antidiabetic drug, antibacterial drug or antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. Patent Application No. 09 / 334,130.

[0250] Representative U.S. patents that teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S.: 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; 5,414,077; 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; 5,451,463; 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. The conjugate group can be attached to either or both ends (terminal conjugate groups) and / or at any internal portion of the oligonucleotide.

[0251] b. Terminal group In some embodiments, the oligomeric compound includes terminal groups at one or both ends. In some embodiments, the terminal group may include any of the conjugate groups described in this application. In some embodiments, the terminal group may include additional nucleosides and / or abasic nucleosides. In some embodiments, the terminal group is a stabilizing group.

[0252] In some embodiments, the oligomeric compound comprises one or more terminal stabilizing groups that enhance properties such as, for example, nuclease stability. The stabilizing groups include a cap structure. The term “cap structure” or “terminal cap moiety” as used herein refers to a chemical modification that can be attached to one or both of the termini of an oligomeric compound. Certain terminal modifications can protect an oligomeric compound having a terminal nucleic acid moiety from degradation by exonucleases and can assist in delivery into and / or localization within a cell. The cap can be present at the 5′ terminus (5′-cap) or the 3′ terminus (3′-cap), or at both termini (for more non-limiting details, see International PCT Publication No. WO 97 / 26270 by Wincott et al.; Beaucage and Tyer, 1993, Tetrahedron 49, 1925; U.S. Patent Application Publication No. 2005 / 0020525; and International Publication No. WO 03 / 004602).

[0253] In some embodiments, one or more additional nucleosides are added to one or both termini of the oligonucleotide of the oligomeric compound. Such additional terminal nucleosides are referred to herein as terminal group nucleosides. In a double-stranded compound, such terminal group nucleosides are terminal (3′ and / or 5′) overhangs. In the context of a double-stranded antisense compound, such terminal group nucleosides may or may not be complementary to the target nucleic acid. In some embodiments, the terminal group is a non-nucleoside terminal group. Such non-terminal groups can be any terminal group other than a nucleoside.

[0254] c. Oligomeric Compound Motif In some embodiments, the oligomeric compound has the motif: T-(Nu 1 ) n1 ,-(Nu 2 ) n2 -(Nu 1 ) n3 -(Nu 2 ) n4 -(Nu 1 ) n5 -T2 [wherein, Nu 1 is a first type of nucleoside, Nu 2 is a second type of nucleoside, each of n1 and n5 is independently 0 to 3, the sum of n2 and n4 is between 10 and 25, n3 is 0 to 5, each T 1 and T 2 is independently H, a hydroxyl protecting group, an optionally linked conjugate group or a capping group] is included.

[0255] In some embodiments, the sum of n2 and n4 is 13 or 14, n1 is 2, n3 is 2 or 3, and n5 is 2. In some embodiments, the oligomeric compound comprises a motif selected from Table A.

[0256]

Table A

[0257] 3. Antisense In some embodiments, the oligomeric compound is an antisense compound. Thus, in some embodiments, the oligomeric compound hybridizes to a target nucleic acid (e.g., a target pre-mRNA or a target mRNA) and provides antisense activity.

[0258] a. Hybridization In some embodiments, the antisense compound specifically hybridizes to the target nucleic acid if there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired (e.g., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays).

[0259] Accordingly, "stringent hybridization conditions" or "stringent conditions" means conditions under which an antisense compound hybridizes to a target sequence while minimizing the number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. The "stringent conditions" under which an antisense oligonucleotide hybridizes to a target sequence are determined by the nature and composition of the antisense oligonucleotide, as well as the assay in which they are being investigated.

[0260] In the art, it is understood that the incorporation of nucleotide affinity modifications can allow for a greater number of mismatches compared to unmodified compounds. Similarly, certain nucleobase sequences may be more tolerant of mismatches than other nucleobase sequences. One of ordinary skill 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 known to those of ordinary skill in the art. For example, the techniques described by Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) enable one of ordinary skill in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA duplexes.

[0261] b. Pre-mRNA processing In some embodiments, the antisense compounds provided herein are complementary to pre-mRNA. In some embodiments, such antisense compounds alter the splicing of pre-mRNA. In some embodiments, the ratio of one variant of the mature mRNA corresponding to the target pre-mRNA to another variant of that mature mRNA is altered. In some embodiments, the ratio of one variant of the protein expressed from the target pre-mRNA to another variant of that protein is altered. Certain oligomeric compounds and nucleobase sequences that can be used to alter pre-mRNA splicing can be found, for example, in U.S. Patent No. 6,210,892; U.S. Patent No. 5,627,274; U.S. Patent No. 5,665,593; U.S. Patent No. 5,916,808; U.S. Patent No. 5,976,879; U.S. Patent Application Publication No. 2006 / 0172962; U.S. Patent Application Publication No. 2007 / 002390; U.S. Patent Application Publication No. 2005 / 0074801; U.S. Patent Application Publication No. 2007 / 0105807; U.S. Patent Application Publication No. 2005 / 0054836; International Publication No. 2007 / 090073; International Publication No. 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 J. of Human Genetics (April 2008) 82, 1-15, each of which is incorporated herein by reference in its entirety for any purpose. In some embodiments, the antisense sequences that alter splicing are modified according to the motifs described in this application.

[0262] In some embodiments, the ASO or oligomeric compound may include one or more modifications described in International Publication No. 2018 / 014043 (PCT / US2017 / 042465), International Publication No. 2018 / 014042 (PCT / US2017 / 042464), International Publication No. 2018 / 014041 (PCT / US2017 / 042463), the contents of which are incorporated herein in their entirety.

[0263] Administration and treatment In some embodiments, a small molecule (e.g., risdiplam or branaplam) that can increase the amount of SMN function that is “therapeutically effective,” a recombinant SMN1 gene (e.g., in a viral vector, e.g., in rAAV), and / or an SMN2 ASO (e.g., nusinersen) are delivered to a subject described herein (e.g., via parallel or sequential administration) to achieve a desired result, e.g., treatment of SMA or one or more of its symptoms. In some embodiments, SMA is evaluated by clinical symptoms such as weight loss, decreased muscle strength, decreased muscle tone, scoliosis, the presence of tremors or contractures, and / or decreased respiratory health. In some embodiments, SMA is evaluated by a scale of motor function appropriate for age and ability, as well as electrophysiological measurements of the health of motor units. In some embodiments, the motor neuron function of a subject can be tested by the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) (e.g., Glanzman AM, et al. The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND): test development and reliability. Neuromuscul Disord. 2010;20(3):155-161; Glanzman AM, Validation of the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND). Pediatr Phys Ther. 2011;23(4):322-326, the content regarding CHOP INTEND is incorporated herein by reference).In some embodiments, the motor neuron function of a subject with late-onset SMA is evaluated by the Expanded Hammersmith Functional Motor Scale (HFMSE) (e.g., Glanzman AM et al; the Pediatric Neuromuscular Clinical Research Network for Spinal Muscular Atrophy (PNCR), and the Muscle Study Group (MSG). Validation of the Expanded Hammersmith Functional Motor Scale in spinal muscular atrophy type II and III. J Child Neurol. 2011;26(12):1499-1507;The Pediatric Neuromuscular Clinical Research Network for SMA. Expanded Hammersmith Functional Motor Scale for SMA (HFMSE). March 7, 2009, the content of the HFMSE is incorporated herein by reference). In some embodiments, compound muscle action potential (CMAP) and / or motor unit number estimation (MUNE) are used to evaluate the electrophysiological function of motor neurons. The CAMP response is a measure of the electrophysiological output from a specific muscle or muscle group after stimulation of the innervating nerve, which is described in Arnold WD, Sheth KA, et al. Electrophysiological motor unit number estimation (MUNE) measuring compound muscle action potential (CMAP) in mouse hindlimb muscles. J Vis Exp. 2015;103:1-8), the content of which is incorporated herein by reference. In some embodiments, the CMAP value decreases in subjects with SMA. In some embodiments, CMAP decreases before physical symptoms appear.Motor unit number estimation (MUNE) is an electrophysiological method for estimating the number of lower motor neurons that innervate a group of muscles supplied by a nerve, and is well-suited for assessing motor neuron loss in the SMA, as described in Bromberg MB, Swoboda KJ. Motor unit number estimation in infants and children with spinal muscular atrophy. Muscle Nerve. 2002;25(3):445-447, the content of which is incorporated herein by reference. The MUNE value is calculated from the ratio of the mean single motor unit potential (SMUP) to the maximal compound muscle action potential (CMAP).

[0264] In some aspects, desired results include reducing muscle weakness, increasing muscle strength and tone, preventing or reducing scoliosis, or maintaining or increasing respiratory health, or reducing tremors or contractions. Other desired endpoints may be determined by a physician.

[0265] In some embodiments, small molecules (e.g., risdiplam or branaplam) and recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function are administered to a subject (e.g., together and sequentially) to increase body weight. In some embodiments, small molecules (e.g., risdiplam or branaplam) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to increase body weight. In some embodiments, small molecules (e.g., risdiplam or branaplam), recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO are administered to a subject (e.g., together and sequentially) to increase body weight. In some embodiments, small molecules (e.g., risdiplam or branaplam) and recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce muscle strength decline. In some embodiments, small molecules (e.g., risdiplam or branaplam) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce muscle strength decline. In some embodiments, small molecules (e.g., risdiplam or branaplam), recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce muscle strength decline. In some embodiments, small molecules (e.g., risdiplam or branaplam) and recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function are administered to a subject (e.g., together and sequentially) to increase muscle strength. In some embodiments, small molecules and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to increase muscle strength. In some embodiments, small molecules (e.g., risdiplam or branaplam), recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to increase muscle strength.In some embodiments, small molecules (e.g., risdiplam or branaplam) and recombinant SMN1 gene for increasing SMN function are administered to a subject (e.g., together and sequentially) to increase muscle tone. In some embodiments, small molecules (e.g., risdiplam or branaplam) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to increase muscle tone. In some embodiments, small molecules (e.g., risdiplam or branaplam) for increasing SMN function, recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to increase muscle tone. In some embodiments, small molecules and recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce scoliosis. In some embodiments, small molecules (e.g., risdiplam or branaplam) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce scoliosis. In some embodiments, small molecules, recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce scoliosis. In some embodiments, small molecules (e.g., risdiplam or branaplam) and recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function are administered to a subject (e.g., together and sequentially) to reduce tremors or contractions. In some embodiments, small molecules (e.g., risdiplam or branaplam) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to reduce tremors or contractions. In some embodiments, small molecules (e.g., risdiplam or branaplam) for increasing SMN function, recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to reduce tremors or contractions.In some embodiments, small molecules (e.g., risdiplam or branaplam) and recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function are administered to a subject (e.g., together and sequentially) to maintain or increase respiratory health. In some embodiments, small molecules (e.g., risdiplam or branaplam) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to maintain or increase respiratory health. In some embodiments, small molecules (e.g., risdiplam or branaplam), recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to maintain or increase respiratory health. In some embodiments, small molecules (e.g., risdiplam or branaplam) and recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce neuronal loss. In some embodiments, small molecules (e.g., risdiplam or branaplam) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce neuronal loss. In some embodiments, small molecules (e.g., risdiplam or branaplam), recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO (e.g., nusinersen) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce neuronal loss. In some embodiments, small molecules (e.g., risdiplam or branaplam) and recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce motor neuron loss. In some embodiments, small molecules and SMN2 ASO for increasing SMN function are administered to a subject (e.g., together and sequentially) to prevent or reduce motor neuron loss.In some embodiments, small molecules (e.g., risdiplam or branaplam) for increasing SMN function, recombinant SMN1 genes (e.g., in rAAV), and SMN2 ASOs (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to prevent or reduce motor neuron loss. In some embodiments, small molecules (e.g., risdiplam or branaplam) for increasing SMN function and recombinant SMN1 genes (e.g., in rAAV) are administered to a subject (e.g., together and sequentially) to improve the score of either a motor neuron function test and / or an electrophysiological test. In some embodiments, small molecules for increasing SMN function and SMN2 ASOs (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to improve the score of either a motor neuron function test and / or an electrophysiological test. In some embodiments, small molecules (e.g., risdiplam or branaplam) for increasing SMN function, recombinant SMN1 genes (e.g., in rAAV), and SMN2 ASOs (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to improve the score of either a motor neuron function test and / or an electrophysiological test.

[0266] In some embodiments, administration of a small molecule (e.g., risdiplam or branaplam) and a recombinant SMN1 gene (e.g., in rAAV) to increase SMN function, or a small molecule and an SMN2 ASO (e.g., nusinersen) to increase SMN function, or a small molecule (e.g., risdiplam or branaplam), a recombinant SMN1 gene (e.g., in rAAV) and an SMN2 ASO (e.g., nusinersen) produces a synergistic effect as measured by any of the assays described herein. In some embodiments, the methods described herein enhance the effect of a small molecule (e.g., risdiplam or branaplam) that increases SMN function and allow for a lower dose of the small molecule that increases SMN function administered to a subject. In some embodiments, the methods described herein enhance the effect of a recombinant SMN1 gene (e.g., in rAAV) and allow for a lower dose (e.g., a lower dose of rAAV encoding the recombinant SMN1 gene) delivered to a subject. In some embodiments, the methods described herein enhance the effect of an SMN2 ASO (e.g., nusinersen) and allow for a lower dose of the ASO (e.g., nusinersen) administered to a subject. In some embodiments, the lower dose of rAAV encoding the recombinant SMN1 gene is less than 1×10 10 GC. In some embodiments, the lower dose of rAAV encoding the recombinant SMN1 gene is from 1.0×10 8 to 1.0×10 10 GC. In some embodiments, the lower dose of rAAV encoding the recombinant SMN1 gene is from 1.0×10 9 to 1.0×10 10 GC. In some embodiments, the lower dose of rAAV encoding the recombinant SMN1 gene is from 1.0×10 10 to 1.0×10 13 GC. In some embodiments, the lower dose of rAAV encoding the recombinant SMN1 gene administered to a human subject is 3×10 13 GC. In some embodiments, the lower dose of rAAV encoding the recombinant SMN1 gene administered to a human subject is 1×1014 less than 1×10 13 ~1×10 14 GC, 1×10 12 ~1×10 13 GC, 1×10 11 ~1×10 12 GC, 1×10 10 ~1×10 11 GC or 1×10 9 ~1×10 10 GC, or less. In some embodiments, a lower dose of SMN2 ASO (e.g., nusinersen) is 12 mg. A total of 5 mg to 60 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to the subject. In some embodiments, a total of 12 mg to 48 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to the subject. In some embodiments, a total of 12 mg to 36 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to the subject. In some embodiments, a total of 12 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to the subject.

[0267] In some examples, SMA is detected in the fetus around 30 to 36 weeks of gestation. In this situation, it may be desirable to treat the neonate as soon as possible after delivery. It may also be desirable to treat the fetus in utero. Accordingly, a method for rescuing and / or treating a neonate subject having SMA, comprising administering a small molecule (e.g., risdiplam or branaplam) and a recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function, or a small molecule (e.g., risdiplam or branaplam) and an SMN2 ASO (e.g., nusinersen) for increasing SMN function, or a small molecule (e.g., risdiplam or branaplam), a recombinant SMN1 gene (e.g., in rAAV) and an SMN2 ASO (e.g., nusinersen) to neuronal cells of a fetal and / or neonatal subject (e.g., a human fetus and / or neonate), (e.g., together or sequentially) is provided. In some embodiments, a method for rescuing and / or treating a fetus having SMA, comprising administering a small molecule (e.g., risdiplam or branaplam) and a recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function, or a small molecule and an SMN2 ASO (e.g., nusinersen) for increasing SMN function, or a small molecule (e.g., risdiplam or branaplam), a recombinant SMN1 gene and an SMN2 ASO (e.g., nusinersen) to neuronal cells of a fetus in utero, (e.g., together or sequentially) is provided. In some embodiments, the method comprises administering (e.g., together or sequentially) one or more of the compositions described herein via intrathecal injection.In some embodiments, the in utero treatment is defined as administering (e.g., in combination and sequentially) a small molecule (e.g., risdiplam or branaplam) and a recombinant SMN1 gene (e.g., in rAAV) to increase the SMN function described herein after detection of SMA in the fetus, or a small molecule (e.g., risdiplam or branaplam) and an SMN2 ASO (e.g., nusinersen) to increase the SMN function, or a small molecule (e.g., risdiplam or branaplam) to increase the SMN2 function, a recombinant SMN1 gene (e.g., in rAAV) and an SMN2 ASO (e.g., nusinersen). See, for example, David et al, Recombinant adeno-associated virus-mediated in utero gene transfer gives therapeutic transgene expression in the sheep, Hum Gene Ther. 2011 Apr;22(4):419-26. doi: 10.1089 / hum.2010.007. Epub 2011 Feb 2, which is incorporated herein by reference.

[0268] In some embodiments, the neonatal treatment comprises delivering at least one dose of a combination of a small molecule (e.g., risdiplam or branaplam) and a recombinant SMN1 gene (e.g., in rAAV) to increase the SMN function, or a small molecule (e.g., risdiplam or branaplam) and an SMN2 ASO (e.g., nusinersen) to increase the SMN2 function, or a small molecule (e.g., risdiplam or branaplam) to increase the SMN function, a recombinant SMN1 gene (e.g., in rAAV) and an SMN2 ASO (e.g., nusinersen) within 8 hours, within the first 12 hours, within the first 24 hours or within the first 48 hours of delivery. In another embodiment, particularly for primates (human or non-human), neonatal delivery is within a period of about 12 hours to about 1 week, 2 weeks, 3 weeks or about 1 month, or about 24 hours to about 48 hours later.

[0269] In some embodiments, for late-onset SMA, small molecules (e.g., risdiplam or branaplam) and recombinant SMN1 gene (e.g., in rAAV) to increase SMN function, or a combination of small molecules (e.g., risdiplam or branaplam) and SMN2 ASO (e.g., nusinersen) to increase SMN function, or a combination of small molecules (e.g., risdiplam or branaplam), recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO (e.g., nusinersen) to increase SMN function are administered after the onset of symptoms. In some embodiments, treatment of the patient (e.g., the first injection) is initiated before the patient reaches 1 year of age. In another embodiment, treatment is initiated after 1 year of age, or after 2 - 3 years of age, 5 years of age, 11 years of age, or at an older age.

[0270] In some embodiments, small molecules and recombinant SMN1 gene (e.g., in rAAV) to increase SMN2 function, or small molecules (e.g., risdiplam or branaplam) and SMN2 ASO (e.g., nusinersen) to increase SMN function, or small molecules (e.g., risdiplam or branaplam), recombinant SMN1 gene (e.g., in rAAV) and SMN2 ASO (e.g., nusinersen) to increase SMN function are readministered at a later date.

[0271] In some embodiments, two or more re - administrations are provided. Such re - administrations can include re - administering the recombinant SMN1 gene with the same type of viral vector, with a different viral vector (e.g., using AAV capsid proteins of different serotypes), or via non - viral delivery. For example, in the event that a patient is treated with a first rAAV (e.g., rAAV9) encoding SMN1 and requires a second treatment with the recombinant SMN1 gene (in addition to receiving, e.g., a small molecule (e.g., risdiplam or branaplam) or a small molecule and SMN2 ASO to increase SMN function), a second different rAAV (e.g., rAAVhu68) encoding the recombinant SMN1 gene can be administered subsequently, and vice versa. Also, if a patient has neutralizing antibodies against the first rAAV serotype, as a result, a second different rAAV serotype can be used to deliver a second dose of the recombinant SMN1 gene to the subject.

[0272] In some embodiments, treatment of SMA patients with a small molecule (e.g., risdiplam or branaplam) to increase SMN function and a recombinant SMN1 gene (e.g., in rAAV), or a small molecule (e.g., risdiplam or branaplam) to increase SMN function and an SMN2 ASO (e.g., nusinersen), or a small molecule (e.g., risdiplam or branaplam) to increase SMN function, a recombinant SMN1 gene (e.g., in rAAV) and an SMN2 ASO may require additional treatments such as transient co - treatment with an immunosuppressive agent before, during, and / or after treatment with the compositions described in this application. Immunosuppressive agents for such co - treatment include, but are not limited to, steroids, antimetabolites, T - cell inhibitors, and alkylating agents, or procedures to remove circulating antibodies such as plasmapheresis. For example, such a transient treatment can include a steroid (e.g., prednisone or prednisolone) administered once daily for 7 days at a decreasing dose starting at about 60 mg and decreasing by 10 mg / day (no administration on day 7). Other doses and immunosuppressive agents may be selected.

[0273] In some embodiments, the subject has one or more indicators of SMA. In some embodiments, the subject has reduced electrical activity of one or more muscles. In some embodiments, the subject has a mutant SMN1 gene (e.g., two mutant alleles of the SMN1 gene). In some embodiments, the subject's SMN1 gene (e.g., both alleles of the SMN1 gene) is absent or unable to produce a functional SMN protein. In some embodiments, the subject has a deletion or loss-of-function point mutation in each SMN1 allele. In some embodiments, the subject is homozygous for the SMN1 gene mutation. In some embodiments, the subject is diagnosed by genetic testing. In some embodiments, the subject is identified by muscle biopsy. In some embodiments, the subject is unable to sit with the back extended. In some embodiments, the subject is unable to stand or walk. In some embodiments, the subject requires assistance with breathing and / or feeding. In some embodiments, the subject is identified by electrophysiological measurement of muscle and / or muscle biopsy.

[0274] In some embodiments, the subject has type I SMA. In some embodiments, the subject has type II SMA. In some embodiments, the subject has type III SMA. In some embodiments, the subject is diagnosed with SMA in utero. In some embodiments, the subject is diagnosed with SMA within one week after birth. In some embodiments, the subject is diagnosed with SMA within one month after birth. In some embodiments, the subject is diagnosed with SMA by three months of age. In some embodiments, the subject is diagnosed with SMA by six months of age. In some embodiments, the subject is diagnosed with SMA by one year of age. In some embodiments, the subject is diagnosed with SMA between one and two years of age. In some embodiments, the subject is diagnosed with SMA between one and fifteen years of age. In some embodiments, the subject is diagnosed with SMA when the subject is older than fifteen years.

[0275] In some embodiments, the first dose of a pharmaceutical composition (e.g., a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, a recombinant SMN1 gene (e.g., in rAAV), an SMN2 ASO (e.g., nusinersen), or both) is administered in utero. In some such embodiments, the first dose is administered prior to the complete development of the blood-brain barrier. In some embodiments, the first dose is administered systemically to the subject in utero. In some embodiments, the first dose is administered in utero after the formation of the blood-brain barrier. In some embodiments, the first dose is administered to the CSF.

[0276] In some embodiments, the first dose of a pharmaceutical composition (e.g., a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, a recombinant SMN1 gene (e.g., in rAAV), an SMN2 ASO (e.g., nusinersen), or both) is administered when the subject is less than 1 week old. In some embodiments, the first dose is administered when the subject is less than 1 month old. In some embodiments, the first dose is administered when the subject is less than 3 months old. In some embodiments, the first dose is administered when the subject is less than 6 months old. In some embodiments, the first dose is administered when the subject is less than 1 year old. In some embodiments, the first dose is administered when the subject is less than 2 years old. In some embodiments, the first dose is administered when the subject is less than 15 years old. In some embodiments, the first dose is administered when the subject is 15 years or older.

[0277] In some embodiments, a small molecule (e.g., risdiplam or branaplam) and / or an SMN2 ASO (e.g., nusinersen) for increasing SMN function is administered 1 to 6 times per year, and a recombinant SMN1 gene (e.g., in rAAV) is administered initially once. In some embodiments, two or more subsequent administrations of a small molecule (e.g., risdiplam or branaplam) and / or an SMN2 ASO (e.g., nusinersen) for increasing SMN function are performed after the first administration of the small molecule (e.g., risdiplam or branaplam), SMN2 ASO (e.g., nusinersen), and recombinant SMN1 gene (e.g., in rAAV) for increasing SMN function. In some embodiments, an SMN2 ASO (e.g., nusinersen) is administered twice monthly. In some embodiments, such administrations are performed monthly. In some embodiments, an SMN2 ASO (e.g., nusinersen) is administered every two months. In some embodiments, an SMN2 ASO (e.g., nusinersen) is administered every six months. In some embodiments, a recombinant SMN1 gene (e.g., in rAAV) is readministered, for example, 1 year or more after the first administration (e.g., 2 - 5 years, 5 - 10 years, 10 - 15 years, 15 - 20 years, or more thereafter).

[0278] In some embodiments, administration of at least one pharmaceutical composition (e.g., a small molecule (e.g., risdiplam or branaplam) to increase SMN function, a recombinant SMN1 gene (e.g., in rAAV) and / or an SMN2 ASO (e.g., nusinersen)) results in a phenotypic change in a subject. In some embodiments, such phenotypic changes include, but are not limited to, an increase in the absolute amount of recombinant SMN mRNA and / or cellular SMN mRNA containing exon 7; an increase in the ratio of SMN mRNA containing exon 7 to SMN mRNA lacking exon 7; an increase in the absolute amount of SMN protein containing exon 7; an increase in the ratio of SMN protein containing exon 7 to SMN protein lacking exon 7; improvement in muscle strength; improvement in electrical activity in at least one muscle; improvement in respiration; weight gain; and survival. In some embodiments, at least one phenotypic change is detected in the motor neurons of the subject. In some embodiments, administration of at least one pharmaceutical composition described in the present application enables the subject to lift the upper body, stand and / or walk. In some embodiments, administration of at least one pharmaceutical composition enables the subject to eat, drink and / or breathe without assistance. In some embodiments, the effectiveness of the treatment is evaluated by electrophysiological assessment of the muscle. In some embodiments, administration of the pharmaceutical composition improves at least one symptom of SMA with little or no inflammatory effect. In some embodiments, the absence of an inflammatory effect is determined by the absence of a significant increase in Aif1 levels during treatment.

[0279] In some embodiments, administration of at least one pharmaceutical composition delays the onset of at least one symptom of SMA. In some embodiments, administration of at least one pharmaceutical composition slows the progression of at least one symptom of SMA. In some embodiments, administration of at least one pharmaceutical composition reduces the severity of at least one symptom of SMA. In some embodiments, administration of at least one pharmaceutical composition results in undesirable side effects. In some embodiments, a treatment regimen is identified that provides a desirable improvement in symptoms while avoiding undesirable side effects.

[0280] Dosage and formulation Thus, in some embodiments, a therapeutically effective amount of an SMN2 ASO (e.g., nusinersen) is administered to a subject having SMA. In some embodiments, the SMN2 ASO (e.g., nusinersen) is administered to the subject alone. In some embodiments, the SMN2 ASO (e.g., nusinersen) is administered to the subject together with other compounds and / or pharmaceutical compositions. In some embodiments, an SMN2 ASO (e.g., nusinersen) and a recombinant nucleic acid (e.g., in rAAV), or an SMN2 ASO (e.g., nusinersen) and a small molecule for increasing SMN function (e.g., risdiplam or branaplam) are administered to the subject. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branaplam), an SMN2 ASO (e.g., nusinersen) and / or a recombinant nucleic acid encoding SMN1 (e.g., in rAAV) are administered to the subject together (e.g., simultaneously, or during the same clinic visit), or sequentially (e.g., during different clinic visits). In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branaplam), an SMN2 ASO (e.g., nusinersen) and a recombinant nucleic acid are administered to the subject separately.

[0281] In some embodiments, a small molecule (e.g., risdiplam or branaplam) for increasing SMN function and a recombinant nucleic acid encoding SMN1 (e.g., in rAAV) are administered to a subject together (e.g., simultaneously, or at different times during a visit to a hospital, clinic, or other medical center, e.g., at different times during the same day of an outpatient visit). In some embodiments, a small molecule (e.g., risdiplam or branaplam) for increasing SMN function and an SMN2 ASO (e.g., nusinersen) are administered to a subject together. In some embodiments, a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, a recombinant nucleic acid encoding SMN1 (e.g., in rAAV), and an SMN2 ASO (e.g., nusinersen) are administered to a subject together. Thus, in some embodiments, co-administering a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, an SMN2 ASO (e.g., nusinersen), and a recombinant nucleic acid encoding SMN1 means administering them during the same outpatient visit (e.g., during the same clinic day). In some embodiments, co-administering a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, an SMN2 ASO (e.g., nusinersen), and a recombinant nucleic acid encoding SMN1 means administering them at different times during the same visit (e.g., during the same clinic day). In some embodiments, co-administration of a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, an SMN1 gene (e.g., in rAAV), and an SMN2 ASO (e.g., nusinersen) is the initiation of a new treatment. In other embodiments, co-administration of a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, an SMN1 gene (e.g., in rAAV), and an SMN2 ASO (e.g., nusinersen) is an adjunctive therapy for a subject currently being treated with a different composition.

[0282] In some embodiments, a small molecule (e.g., risdiplam or branaplam) for increasing SMN function and a recombinant nucleic acid encoding SMN1 (e.g., in rAAV) are sequentially administered to a subject at different visits (e.g., different clinic days). In some embodiments, a small molecule (e.g., risdiplam or branaplam) for increasing SMN function and an SMN2 ASO (e.g., nusinersen) are sequentially administered to a subject at different visits (e.g., different clinic days). In some embodiments, a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, a recombinant nucleic acid encoding SMN1 and an SMN2 gene (e.g., in rAAV) are sequentially administered to a subject at different visits (e.g., different clinic days). In some embodiments, sequentially administering a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, an SMN2 ASO (e.g., nusinersen) and a recombinant nucleic acid encoding SMN1 means administering the recombinant nucleic acid encoding SMN1 (e.g., in rAAV) during a first visit, followed by administering the small molecule and / or SMN2 ASO (e.g., nusinersen) at different visits (e.g., different clinic days). In some embodiments, sequentially administering a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, an SMN2 ASO (e.g., nusinersen) and a recombinant nucleic acid encoding SMN1 means administering the SMN2 ASO (e.g., nusinersen) during a first visit, followed by administering the small molecule for increasing SMN function and / or the recombinant nucleic acid encoding SMN1 (e.g., in rAAV) at different visits (e.g., different clinic days). In some embodiments, sequentially administering a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, an SMN2 ASO (e.g., nusinersen) and a recombinant nucleic acid encoding SMN1 means administering the small molecule for increasing SMN function (e.g., risdiplam or branaplam) during a first visit, followed by administering the recombinant nucleic acid encoding SMN1 and / or the SMN2 ASO (e.g., nusinersen) at different visits (e.g., different clinic days).In some embodiments, a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, a recombinant nucleic acid encoding SMN1, and an SMN2 ASO (e.g., nusinersen) are administered at different frequencies. As used herein, sequential administration may include an administration protocol in which the administration of a first treatment (e.g., a small molecule for increasing SMN2 function such as risdiplam or branaplam) during a clinic visit is followed by, or precedes, one or more administrations of a second treatment (e.g., an SMN2 ASO (e.g., nusinersen) and / or a recombinant nucleic acid encoding SMN1 (e.g., in rAAV), or a combination thereof) during one or more different clinic visits.

[0283] In some embodiments, small molecules (e.g., risdiplam or branaplam) for increasing SMN2 function, SMN2 ASOs (e.g., nusinersen), and recombinant SMN1 genes (e.g., in rAAV) are administered at different frequencies. In some embodiments, an SMN2 ASO (e.g., nusinersen) or a small molecule for increasing SMN2 function (e.g., risdiplam or branaplam) is administered to a subject 1 to 6 times per year. In some embodiments, a recombinant SMN1 gene (e.g., in rAAV) is administered once. In some embodiments, two or more subsequent administrations of a small molecule for increasing SMN2 function (e.g., risdiplam or branaplam) and / or an SMN2 ASO (e.g., nusinersen) are performed after the first administration of the SMN2 ASO (e.g., nusinersen) and the recombinant SMN1 gene. In some embodiments, an SMN2 ASO (e.g., nusinersen) is administered to a subject prior to administration of a small molecule for increasing SMN2 function (e.g., risdiplam or branaplam), an SMN2 ASO, and / or a recombinant SMN1 gene (e.g., in rAAV). In some embodiments, an SMN2 ASO (e.g., nusinersen) is administered to a subject at a dose of 0.01 to 25 milligrams per kilogram of the subject's body weight (e.g., 0.01 to 10 milligrams, 0.05 to 5 milligrams, 0.1 to 2 milligrams, or 0.5 to 1 milligram), and a recombinant SMN1 gene (e.g., in rAAV) is administered at a dose of 2×10 10 ~2×10 14 GC (e.g., 1.0×10 13 ~1.0×10 14 GC, or for example, for IT administration, about 1.0×10 13 ~5.0×10 14 GC) by rAAV. In some embodiments, an SMN2 ASO is administered to a subject at a dose of 0.001 to 25 milligrams per kilogram of the subject's body weight (e.g., 0.001 to 10 milligrams, 0.005 to 5 milligrams, 0.01 to 2 milligrams, or 0.05 to 1 milligram), and a recombinant SMN1 gene (e.g., in rAAV) is administered at a dose of 1×10 10 ~2×10 14 GC (e.g., 1.0×1013 ~1.0×10 14 For GC, or for example, for IT administration, about 1.0×10 13 ~5.0×10 14 (For GC), or for example, for IV administration, about 3×10 13 ~5×10 14 It is administered with rAAV at a dose of GC. In some embodiments, the SMN2 ASO (e.g., nusinersen) is administered at a dose of 0.01 to 10 milligrams per kilogram of the subject's body weight. In some embodiments, the SMN2 ASO (e.g., nusinersen) is administered at a dose of 0.001 to 10 milligrams per kilogram of the subject's body weight. In some embodiments, the SMN2 ASO (e.g., nusinersen) is administered at a dose of less than 0.001 milligrams per kilogram of the subject's body weight.

[0284] In some embodiments, a total of 5 mg to 60 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to a subject. In some embodiments, a total of 5 mg to 20 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to a subject. In some embodiments, a total of 12 mg to 48 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to a subject. In some embodiments, a total of 12 mg to 36 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to a subject. In some embodiments, a total of 28 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to a subject. In some embodiments, a total of 12 mg of SMN2 ASO (e.g., nusinersen) per dose is administered to a subject. In some embodiments, the SMN2 ASO (e.g., nusinersen) and / or recombinant SMN1 gene is administered to the subject intravenously or intramuscularly. In some embodiments, the SMN2 ASO (e.g., nusinersen) and / or recombinant SMN1 gene is administered into the intrathecal space of the subject. In some embodiments, the SMN2 ASO (e.g., nusinersen) and / or recombinant SMN1 gene is administered into the intracisternal space of the subject. In some embodiments, administration of the SMN2 ASO (e.g., nusinersen) and recombinant nucleic acid increases the intracellular SMN protein level in the subject. In some embodiments, administration of the SMN2 ASO (e.g., nusinersen) and recombinant nucleic acid increases the intracellular SMN protein level in the cervical, thoracic, and lumbar spinal cord motor neurons in the subject.

[0285] In some embodiments, the dosages of a small molecule (e.g., risdiplam or branaplam) to increase SMN2 function, a recombinant SMN1 gene (e.g., in rAAV), and an SMN2 ASO (e.g., nusinersen) are administered by bolus injection into the CSF. In some embodiments, the dosages are administered by LP and / or ICM bolus injection. In some embodiments, the dosages are administered by bolus systemic injection (e.g., subcutaneous, intramuscular, or intravenous injection). In some embodiments, the subject receives a bolus injection into the CSF and a bolus systemic injection. In some embodiments, the dosages of the CSF bolus and the systemic bolus may be the same as or different from each other. In some embodiments, the CSF and systemic dosages are administered at different frequencies.

[0286] In some embodiments, a pharmaceutical composition is provided that comprises a small molecule (e.g., risdiplam or branaplam) to increase SMN2 function, a recombinant SMN1 gene (e.g., in rAAV), and / or an SMN2 ASO (e.g., nusinersen). The pharmaceutical composition can be designed for delivery to a subject in need thereof by any suitable route (e.g., by different routes suitable for each treatment). For example, one or more compositions can be administered to a human subject using routes including intracerebroventricular (ICV), intravenous (IV), and intrathecal (IT) (e.g., via lumbar puncture (LP) and / or intracisternal (ICM) delivery).

[0287] In some embodiments, direct delivery to the CNS is desirable and can be accomplished via intrathecal injection. The term "intrathecal administration" refers to delivery targeting cerebrospinal fluid (CSF). This can be done by direct injection into the ventricular or lumbar CSF, by suboccipital puncture, or by other suitable means. Meyer et al, Molecular Therapy (31 October 2014) demonstrated the efficacy of direct CSF injection, which results in widespread transgene expression throughout the spinal cord of mice and non-human primates when using a dose 10-fold lower compared to IV application. This document is incorporated herein by reference. In some embodiments, the recombinant SMN1 gene is delivered via intracerebroventricular viral injection (see, e.g., Kim et al, J Vis Exp. 2014 Sep 15;(91):51863, which is incorporated herein by reference). See also Passini et al, Hum Gene Ther. 2014 Jul;25(7):619-30, which is incorporated herein by reference. In some embodiments, the composition is delivered via lumbar injection.

[0288] In some embodiments, the delivery means and formulation are designed to avoid direct systemic delivery of a suspension containing the AAV composition(s) described in this application. Preferably, this can have the advantage of reducing systemic exposure, reducing toxicity, and / or reducing an unwanted immune response to AAV and / or the transgene product compared to systemic administration.

[0289] Compositions comprising a small molecule (e.g., risdiplam or branaplam) to increase SMN2 function, a recombinant SMN1 gene (e.g., in rAAV), and / or an SMN2 ASO (e.g., nusinersen) can be formulated for any suitable route of administration (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, and other parenteral routes).

[0290] In some embodiments, the recombinant SMN1 gene delivery constructs described in the present application can be delivered in a single composition or multiple compositions. In some embodiments, two or more different AAVs can be delivered (see, e.g., WO 2011 / 126808 and WO 2013 / 049493). In some embodiments, such multiple viruses can contain different replication-deficient viruses (e.g., AAV, adenovirus, and / or lentivirus). Alternatively, delivery can be mediated, for example, by non-viral constructs, such as "naked DNA", "naked plasmid DNA", RNA, and mRNA, in combination with various delivery compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, poly-glycan compositions, and other polymers, lipids, and / or cholesterol-based nucleic acid conjugates; see, e.g., X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: March 21, 2011; WO 2013 / 182683, WO 2010 / 053572, and WO 2012 / 170930, each of which is incorporated herein by reference. Non-viral SMN1 delivery constructs can also be formulated for any suitable route of administration.

[0291] Viral vectors or non-viral DNA or RNA transfer moieties can be formulated using physiologically acceptable carriers for use in gene delivery and gene therapy applications. A number of suitable purification methods can be selected. Examples of suitable purification methods for separating empty capsids from vector particles are described, for example, in International Patent Application No. PCT / US16 / 65976, filed December 9, 2016, entitled "Scalable Purification Method for AAV8", and its priority documents, U.S. Patent Application No. 62 / 322,098, filed April 13, 2016, and U.S. Patent Application No. 62 / 266,341, filed December 11, 2015, which are incorporated herein by reference. Also see the purification methods described in International Patent Application No. PCT / US16 / 65974, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,083, filed April 13, 2016, and U.S. Patent Application No. 62 / 266,351, filed December 11, 2015 (AAV1); International Patent Application No. PCT / US16 / 66013, filed December 9, 2016, and its priority documents, U.S. Provisional Application No. 62 / 322,055, filed April 13, 2016, and U.S. Patent Application No. 62 / 266,347, filed December 11, 2015 (AAVrh10); and International Patent Application No. PCT / US16 / 65970, filed December 9, 2016, and its priority applications, U.S. Provisional Application No. 62 / 266,357 and U.S. Provisional Application No. 62 / 266,357 (AAV9), which are incorporated herein by reference. Briefly, a two-step purification scheme for selectively capturing and isolating genome-containing rAAV vector particles from the clarified and concentrated supernatant of rAAV producer cell cultures has been described. The process utilizes an affinity capture method performed at high salt concentration, followed by an anion exchange resin method performed at high pH, to provide rAAV vector particles substantially free of rAAV intermediates.

[0292] In the case of an AAV viral vector, quantification of genomic copies ("GC") can be used as a measure of the dose contained in the formulation. The number of genomic copies (GC) of the replication-deficient viral composition of the present invention can be determined using any method known in the art. One method for titrating the AAV GC number is as follows. The purified AAV vector sample is first treated with DNase to eliminate contaminating host DNA from the production process. The DNase-resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genome is then quantified by real-time PCR using a primer / probe set that targets a specific region of the viral genome (e.g., the polyA signal). Another suitable method for determining genomic copies is quantitative PCR (qPCR), particularly optimized qPCR or digital droplet PCR (Lock Martin, et al, Human Gene Therapy Methods. April 2014, 25(2): 115-125. doi: 10.1089 / hgtb.2013.131, published online December 13, 2013 prior to editing).

[0293] In some embodiments, the replication-deficient viral composition is formulated in a dosage unit to contain an amount of from about 1.0×10 9 GC to about 1.0×10 15 GC (e.g., for treating an average subject weighing 70 kg), preferably for human patients, from 1.0×10 12 GC to 1.0×10 14 GC of replication-deficient virus. The total dose administered to a subject can depend on the route of administration. In some embodiments, the composition contains at least 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9It is formulated to contain GC. In another aspect, the composition contains at least 1×10 per dose, including all integer or fractional amounts within the range. 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 or 9×10 10 It is formulated to contain GC. In another aspect, the composition contains at least 1×10 per dose, including all integer or fractional amounts within the range. 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 or 9×10 11 It is formulated to contain GC. In another aspect, the composition contains at least 1×10 per dose, including all integer or fractional amounts within the range. 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 or 9×10 12 It is formulated to contain GC. In another aspect, the composition contains at least 1×10 per dose, including all integer or fractional amounts within the range. 13 、2×10 13 、3×10 13 、4×10 13 、5×10 13 、6×10 13 、7×10 13 、8×10 13 or 9×10 13 It is formulated to contain GC. In another aspect, the composition contains at least 1×10 per dose, including all integer or fractional amounts within the range. 14 、2×10 14 、3×1014 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 or 9×10 14 is formulated to contain GC. In another aspect, the composition contains at least 1×10 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 or 9×10 15 is formulated to contain GC. In some aspects, for human applications, the dose of the virus (e.g., rAAV) can range from 1×10 10 to about 1×10 12 GC per dose, including all integer or fractional amounts within the range.

[0294] These above dosages can be administered in various volumes of pharmaceutically acceptable carriers, excipients or buffer formulations ranging from about 25 microliters to about 1,000 microliters, or up to about 10 milliliters, or up to 20 milliliters at most, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration and the desired effect of the method. In some embodiments, the volume of the pharmaceutically acceptable carrier, excipient or buffer is at least about 25 μl. In some embodiments, the volume is about 50 μl. In another embodiment, the volume is about 75 μl. In another embodiment, the volume is about 100 μl. In another embodiment, the volume is about 125 μl. In another embodiment, the volume is about 150 μl. In another embodiment, the volume is about 175 μl. In yet another embodiment, the volume is about 200 μl. In another embodiment, the volume is about 225 μl. In yet another embodiment, the volume is about 250 μl. In yet another embodiment, the volume is about 275 μl. In yet another embodiment, the volume is about 300 μl. In yet another embodiment, the volume is about 325 μl. In another embodiment, the volume is about 350 μl. In another embodiment, the volume is about 375 μl. In another embodiment, the volume is about 400 μl. In another embodiment, the volume is about 450 μl. In another embodiment, the volume is about 500 μl. In another embodiment, the volume is about 550 μl. In another embodiment, the volume is about 600 μl. In another embodiment, the volume is about 650 μl. In another embodiment, the volume is about 700 μl. In another embodiment, the volume is about 700 - 1000 μl.

[0295] In other embodiments, a volume of from about 1 μl to 150 mL can be selected, with more volume being selected for adults. Typically, for neonates, a suitable volume is from about 0.5 mL to about 10 mL. For older infants, from about 0.5 mL to about 15 mL can be selected. For toddlers, a volume of from about 0.5 mL to about 20 mL can be selected. For children, a volume of up to about 30 mL can be selected. For pre-adolescents and adolescents, a volume of up to about 50 mL can be selected. In yet other embodiments, a patient can receive intrathecal administration at a volume of from about 5 mL to about 15 mL, or at a volume of from about 7.5 mL to about 10 mL. Other suitable volumes and dosages may be determined. The dosage is adjusted to balance the therapeutic benefit against any side effects, and such dosage can vary depending on the therapeutic use for which the recombinant vector is employed.

[0296] The recombinant SMN1 gene, such as that in a viral vector (e.g., packaged in rAAV), can be delivered to a host cell using a suitable method. Preferably, rAAV suspended in a physiologically compatible carrier (e.g., a pharmaceutically acceptable carrier) can be administered to a human or non-human mammalian patient. In some embodiments, the composition includes a pharmaceutically acceptable carrier, diluent, excipient, and / or adjuvant. A suitable carrier can be selected depending on the route of administration. For example, one suitable carrier is saline, which can be formulated using various buffer solutions (e.g., phosphate buffered saline). Other exemplary pharmaceutically acceptable carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.

[0297] In some embodiments, the composition may contain, in addition to SMN1 rAAV, a small molecule (e.g., risdiplam or branaplam) and / or an ASO (e.g., nusinersen) for increasing SMN function, and a pharmaceutically acceptable carrier(s), other conventional pharmaceutical ingredients such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0298] In some embodiments, a composition comprising a small molecule (e.g., risdiplam or branaplam) for increasing SMN function, SMN1 rAAV, and / or an SMN2 ASO (e.g., nusinersen) may include a pharmaceutically acceptable carrier and / or may be mixed with suitable excipients designed for delivery to a subject via injection, osmotic pump, intrathecal catheter, or for delivery by another device or route. In one example, the composition is formulated for intrathecal delivery. In some embodiments, intrathecal delivery includes injection into the spinal canal, e.g., the subarachnoid space.

[0299] The viral vectors described in this application are used to deliver SMN1 to a subject (e.g., a human patient) in need thereof to provide functional SMN to the subject and / or to prepare a medicament (e.g., administered concomitantly or sequentially) for treating spinal muscular atrophy in combination therapy with one or more SMN2 ASOs.

[0300] In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable carrier (e.g., a buffer, a salt, and / or other components of a pharmaceutical formulation) containing rAAV is selected to include one or more components that prevent the attachment of rAAV to an infusion tube but do not interfere with the binding activity of rAAV in vivo.

[0301] In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated for delivery (e.g., for systemic administration) with an amount of ASO in the range of 5 mg to 60 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated for delivery (e.g., for systemic administration) with an amount of ASO in the range of 5 mg to 20 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated for delivery (e.g., for systemic administration) with an amount of ASO in the range of 12 mg to 50 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated for delivery (e.g., for systemic administration) with an amount of ASO in the range of 12 mg to 48 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated for delivery (e.g., for systemic administration) with an amount of ASO in the range of 12 mg to 36 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated for delivery (e.g., for systemic administration) with an amount of 28 mg of ASO per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated for delivery (e.g., for systemic administration) with an amount of 12 mg of ASO per dose. In some such embodiments, the volume of administration is 5 mL.

[0302] In some such embodiments, an ASO (e.g., an SMN2 ASO), alone or together with a recombinant SMN1 gene and / or a small molecule such as risdiplam or branaplam that increases SMN function, is formulated for delivery (e.g., for systemic administration) in the range of 0.1 mg / kg to 200 mg / kg (ASO / patient body weight). In some embodiments, the dosage is from 0.1 mg / kg to 100 mg / kg. In some embodiments, the dosage is from 0.5 mg / kg to 100 mg / kg. In some embodiments, the dosage is from 1 mg / kg to 100 mg / kg. In some embodiments, the dosage is from 1 mg / kg to 50 mg / kg. In some embodiments, the dosage is from 1 mg / kg to 25 mg / kg. In some embodiments, the dosage is from 0.1 mg / kg to 25 mg / kg. In some embodiments, the dosage is from 0.1 mg / kg to 10 mg / kg. In some embodiments, the dosage is from 1 mg / kg to 10 mg / kg. In some embodiments, the dosage is from 1 mg / kg to 5 mg / kg.

[0303] In some embodiments, administration of the subject by the ASO is divided into an induction phase and a maintenance phase. In some such embodiments, the dosage administered during the induction phase is more than the dosage administered during the maintenance phase. In some embodiments, the dosage administered during the induction phase is less than the dosage administered during the maintenance phase. In some embodiments, the induction phase is achieved by a bolus injection and the maintenance phase is achieved by a continuous infusion. In some embodiments, the combination formulation is used during the induction phase.

[0304] In some embodiments, the pharmaceutical composition is administered as a bolus injection. In some such embodiments, the dose of the bolus injection contains a total of 5 mg to 60 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose. In some such embodiments, the dose of the bolus injection contains a total of 5 mg to 20 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose. In some such embodiments, the dose of the bolus injection contains a total of 12 mg to 50 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose. In some such embodiments, the dose of the bolus injection contains a total of 12 mg to 48 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose. In some such embodiments, the dose of the bolus injection contains a total of 12 mg to 36 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose. In some such embodiments, the dose of the bolus injection contains a total of 28 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose. In some such embodiments, the dose of the bolus injection contains a total of 12 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose. In some such embodiments, the volume of administration is 5 mL.

[0305] In some embodiments, the pharmaceutical composition is administered as a bolus injection. In some such embodiments, the dose of the bolus injection is 0.01 to 25 milligrams of antisense compound per kilogram of the subject's body weight. In some such embodiments, the dose of the bolus injection is 0.01 to 10 milligrams of antisense compound per kilogram of the subject's body weight. In some embodiments, the dose is 0.05 to 5 milligrams of antisense compound per kilogram of the subject's body weight. In some embodiments, the dose is 0.1 to 2 milligrams of antisense compound per kilogram of the subject's body weight. In some embodiments, the dose is 0.5 to 1 milligram of antisense compound per kilogram of the subject's body weight.

[0306] In some embodiments, such dosages are administered twice monthly. In some embodiments, such dosages are administered once a month. In some embodiments, such dosages are administered every two months. In some embodiments, such dosages are administered every six months. In some embodiments, such dosages are administered by bolus injection into the CSF. In some embodiments, such dosages are administered by intrathecal bolus injection. In some embodiments, such dosages are administered by bolus systemic injection (e.g., subcutaneous, intramuscular, or intravenous injection). In some embodiments, the subject receives a bolus injection into the CSF and a bolus systemic injection. In such embodiments, the dosages of the CSF bolus and the systemic bolus may be the same as or different from each other. In some embodiments, the CSF and systemic dosages are administered at different frequencies. In some embodiments, the present invention provides a dosing regimen comprising at least one bolus intrathecal injection and at least one bolus subcutaneous injection.

[0307] In some embodiments, the pharmaceutical composition is administered by continuous infusion (e.g., where the dose can be administered over a period of time, such as a 24-hour period). Such continuous infusion can be achieved by an infusion pump that delivers the pharmaceutical composition to the CSF. In some embodiments, such an infusion pump delivers the pharmaceutical composition to the IT or ICV. In some such embodiments, the dose administered is 5 mg to 60 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the dose administered is 5 mg to 20 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the dose administered is 12 mg to 50 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the dose administered is 12 mg to 48 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the dose administered is 12 mg to 36 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the dose administered is 28 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the dose administered is 12 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the volume administered is 5 mL.

[0308] In some embodiments, the dosage administered is from 0.05 to 25 milligrams of the antisense compound per kilogram of the subject's body weight per day. In some embodiments, the dosage administered is from 0.1 to 10 milligrams of the antisense compound per kilogram of the subject's body weight per day. In some embodiments, the dosage administered is from 0.5 to 10 milligrams of the antisense compound per kilogram of the subject's body weight per day. In some embodiments, the dosage administered is from 0.5 to 5 milligrams of the antisense compound per kilogram of the subject's body weight per day. In some embodiments, the dosage administered is from 1 to 5 milligrams of the antisense compound per kilogram of the subject's body weight per day.

[0309] In some embodiments, the present invention provides an administration regimen comprising injection into the CNS and at least one bolus systemic injection. In some embodiments, the present invention provides an administration regimen comprising injection into the CNS and at least one bolus subcutaneous injection. In some embodiments, the dosage is adjusted to achieve or maintain a concentration of from 0.1 to 100 micrograms of the antisense compound per gram of CNS tissue, regardless of whether it is a bolus or an injection. In some embodiments, the dosage is adjusted to achieve or maintain a concentration of from 1 to 10 micrograms of the antisense compound per gram of CNS tissue, regardless of whether it is a bolus or an injection. In some embodiments, the dosage is adjusted to achieve or maintain a concentration of from 0.1 to 1 microgram of the antisense compound per gram of CNS tissue, regardless of whether it is a bolus or an injection.

[0310] Accordingly, in some aspects, the present invention provides a pharmaceutical composition comprising one or more therapeutic molecules, such as one or more recombinant nucleic acids (e.g., packaged in a viral vector, e.g., within rAAV) and / or antisense compounds. In some aspects, such a pharmaceutical composition comprises a sterile aqueous saline solution and one or more therapeutic molecules. In some aspects, such a pharmaceutical composition consists of a sterile aqueous saline solution and one or more therapeutic molecules. In some aspects, the therapeutic molecule can be mixed with pharmaceutically acceptable active and / or inactive substances for the preparation of the pharmaceutical composition or formulation. The compositions and methods for formulating the pharmaceutical composition depend on many criteria including, but not limited to, the route of administration, the extent of the disease or the dose administered. In some aspects, the therapeutic molecule can be utilized in a pharmaceutical composition by combining such a therapeutic molecule with a suitable pharmaceutically acceptable diluent or carrier. In some aspects, a pharmaceutically acceptable diluent includes phosphate buffered saline (PBS). PBS is a suitable diluent for use in compositions delivered parenterally. Accordingly, in some aspects, used in the methods described herein is a pharmaceutical composition comprising one or more therapeutic molecules and a pharmaceutically acceptable diluent. In some aspects, the pharmaceutically acceptable diluent is PBS. The pharmaceutical composition comprising one or more therapeutic molecules described in the present application encompasses any pharmaceutically acceptable salt, ester, or salt of such an ester. In some aspects, a pharmaceutical composition comprising an ASO includes one or more oligonucleotides capable of (directly or indirectly) providing its biologically active metabolite or residue upon administration to an animal, including a human. Accordingly, in some aspects, provided are pharmaceutically acceptable salts of ASO, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0311] In some embodiments, the prodrug may include the incorporation of additional nucleosides at one or both ends of the oligomeric compound that are cleaved by endogenous nucleases in the body to form the active antisense oligomeric compound. Lipid-based vectors have been used in nucleic acid therapy in a variety of ways. For example, in one approach, the nucleic acid is introduced into preformed liposomes or lipoplexes composed of a mixture of cationic and neutral lipids. In another approach, DNA complexes with mono- or poly-cationic lipids are formed in the absence of neutral lipids. Some preparations are described in Akinc et al., Nature Biotechnology 26, 561-569 (1 May 2008), which is incorporated herein by reference in its entirety.

[0312] Kit In some embodiments, a kit is provided that includes a small molecule, a recombinant SMN1 gene (e.g., in rAAV), and / or an SMN2 ASO, e.g., in a pharmaceutical composition, for increasing SMN function. In some embodiments, such a kit further includes additional therapeutic agents such as one or more immunosuppressive agents. In some embodiments, such a kit further includes a means of delivery, e.g., a syringe or infusion pump.

[0313] The following examples are illustrative only and are not intended to limit the invention.

Examples

[0314] (Example 1) rAAV vector containing the hSMN1 gene A recombinant neurotropic AAV virus carrying codon-optimized human SMN1 cDNA was constructed.

[0315] (Example 2) ASO that increases full-length SMN2 mRNA (e.g., by promoting exon 7 inclusion in hSMN2 mRNA) An antisense oligonucleotide (ASO) that increases full-length SMN2 mRNA (e.g., promotes exon 7 inclusion in SMN2 mRNA) was prepared (Figure 3).

[0316] (Example 3) Administration and biodistribution of an rAAV vector containing the hSMN1 gene and an ASO that increases full-length SMN2 mRNA (e.g., promotes exon 7 inclusion in SMN2 mRNA) The rAAV of E...

Claims

A pharmaceutical composition for use in treating spinal muscular atrophy (SMA) in a subject, comprising: (i) risdiplam or branaplam; and (ii) nusinersen, A pharmaceutical composition comprising.

2. Further, (iii) an rAAV comprising a recombinant nucleic acid encoding the survival motor neuron 1 (SMN1) protein, The pharmaceutical composition according to claim 1, comprising.

3. The pharmaceutical composition according to claim 1 or 2, wherein the subject has a deletion or mutation in each survival motor neuron 1 (SMN1) allele.

4. The pharmaceutical composition according to claim 3, wherein the subject is homozygous for the mutation in the SMN1 gene.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject has one or more symptoms of SMA.

6. The pharmaceutical composition according to claim 5, wherein the symptoms include atrophy of limb muscles, difficulty walking or inability to walk, or difficulty breathing.

7. The pharmaceutical composition according to any one of claims 2 to 6, wherein the rAAV further comprises an AAV9 capsid protein.

8. The pharmaceutical composition according to any one of claims 2 to 7, wherein risdiplam or branaplam, the rAAV, and nusinersen are administered simultaneously.

9. The pharmaceutical composition according to any one of claims 2 to 7, wherein risdiplam or branaplam, the rAAV, and nusinersen are administered together.

10. The pharmaceutical composition according to any one of claims 2 to 7, wherein risdiplam or branaplam, the rAAV, and nusinersen are administered sequentially.

11. The pharmaceutical composition according to any one of claims 1 to 7 and 10, wherein risdiplam or branaplam is administered to the subject before nusinersen is administered to the subject.

12. The pharmaceutical composition according to any one of claims 2 to 7, 10 and 11, wherein risdiplam or branaplam is administered to the subject before the rAAV is administered to the subject.

13. The pharmaceutical composition according to any one of claims 2 to 7 and 10 to 12, wherein risdiplam or branaplam and the rAAV are administered to the subject before nusinersen is administered to the subject. The pharmaceutical composition according to any one of claims 2 to 7 and 10 to 13, characterized in that risdiplam or branaplam is administered to the subject before nusinersen and the rAAV are administered to the subject.

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