Combination therapy for spinal muscular atrophy

Abstract

To provide a combination therapy for spinal muscular atrophy.SOLUTION: Aspects of the application relate to compositions and methods for treating spinal muscular atrophy in a subject. In particular, this application provides therapeutic combinations of a small molecule that promotes SMN function and / or a recombinant nucleic acid that encodes the survival of motor neuron 1 (SMN1) protein (e.g., in a viral vector), and / or an antisense oligonucleotide (ASO) that increases full-length survival of motor neuron 2 (SMN2) mRNA (e.g., that is targeted to a nucleic acid molecule encoding the survival of motor neuron 2 (SMN2) and promotes the inclusion of exon 7 in SMN2 mRNA).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 887,579, filed August 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 technology]

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

[0004] The SMN gene product is intracellular, and its deficiency results in selective toxicity to lower motor neurons, leading to progressive neuronal loss and muscle weakness. Disease severity is modified by the copy number of a homologous gene (SMN2) with a splice-site mutation resulting in the production of only small amounts of full-length SMN transcripts. Patients with one or two 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 three copies of SMN2 generally exhibit a milder form of the disease, typically manifesting after six months of age. While many never achieve walking, patients rarely progress to respiratory failure and often survive into adulthood. Patients with four copies of SMN2 may not develop the disease until adulthood, with gradual onset of muscle weakness. Summary of the Invention [Problem to be solved by the invention]

[0005] Although several treatments for SMA have been developed, there remains a need for treatments that increase intracellular SMN activity in motor neurons involved in spinal muscular atrophy for patients with different levels of disease severity. [Means for solving the problem]

[0006] overview In some aspects, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject with 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.

[0007] In some aspects, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject with 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 with 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.

[0009] In some embodiments, the present application relates to combination therapies for spinal muscular atrophy (SMA) that include administering (e.g., simultaneously or sequentially) to a subject with SMA 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. In some embodiments, the small molecule that increases SMN function is a small molecule that increases full-length SMN2 mRNA in the subject. In some embodiments, the recombinant nucleic acid encoding SMN1 is provided in a viral vector, e.g., a recombinant adeno-associated virus (rAAV). In some embodiments, 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 embodiments, the present application relates to a combination therapy for spinal muscular atrophy (SMA) comprising administering (e.g., simultaneously or sequentially) to a subject with SMA a small molecule that increases SMN function and an oligomeric compound that modulates exon skipping (e.g., promotes exon 7 inclusion) in a recombinant nucleic acid encoding survival motor neuron 1 (SMN1) and / or a nucleic acid encoding survival motor neuron 2 (SMN2) mRNA. In some embodiments, the small molecule that increases SMN function is a small molecule that increases full-length SMN2 mRNA in the subject. In some embodiments, the recombinant nucleic acid encoding SMN1 is provided in a viral vector, e.g., a recombinant adeno-associated virus (rAAV). In some embodiments, the oligomeric compound that induces exon skipping in a nucleic acid encoding SMN2 is an antisense oligonucleotide (ASO) that modulates exon skipping (e.g., promotes exon 7 inclusion) in SMN2 pre-mRNA.

[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 branapram.

[0012] In some embodiments, the small molecule that increases SMN function (e.g., risdiplam or branapram) and the recombinant nucleic acid (e.g., in a viral vector such as rAAV) and / or SMN2 ASO (e.g., nusinersen) are provided as separate compositions but are administered to a subject together (e.g., simultaneously or contemporaneously, e.g., simultaneously). In some embodiments, the small molecule that increases SMN function (e.g., risdiplam or branapram) and the recombinant nucleic acid (e.g., in a viral vector such as rAAV) and / or SMN2 ASO (e.g., nusinersen) are provided as separate compositions and administered to the subject sequentially during separate visits (e.g., at different times, e.g., on different days) over the course of treatment (e.g., during a 1-week, 2-4-week, 1-month, 1-12-month, 1-year, 2-5-year, or longer treatment regimen). In some embodiments, the small molecule that increases SMN function (e.g., risdiplam or branapram) is administered before and / or after the recombinant nucleic acid (e.g., rAAV) and / or SMN2 ASO (e.g., nusinersen). In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branapram) and a recombinant nucleic acid (e.g., in a viral vector such as rAAV) and / or an SMN2 ASO (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 comprising either a small molecule that increases SMN function (e.g., risdiplam or branapram), a recombinant nucleic acid (e.g., in a viral vector such as rAAV), or an ASO, where the compositions are administered at different frequencies (e.g., together or sequentially).

[0013] In some embodiments, two or more different SMN function-increasing small molecules (e.g., risdiplam or branapram) are 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 with SMA (e.g., a human subject) comprises administering to the subject a small molecule that increases SMN function (e.g., risdiplam or branapram) and a recombinant nucleic acid encoding SMN1 (also referred to as a recombinant SMN1 gene) (e.g., in an 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 the subject an effective amount of a small molecule that increases SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV) and / or an SMN2 ASO (e.g., nusinersen).

[0015] In some embodiments, a subject with SMA has one or more symptoms of SMA (e.g., limb muscle atrophy, difficulty or inability to walk, difficulty breathing, or other symptoms of SMA). In some embodiments, a subject with SMA has two mutant 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 a group of children and adults. In some embodiments, the subject is over 18 years old or equal to 18 years old (e.g., 18 years old or older). In some embodiments, the subject is younger than 18 years old, younger than 10 years old, or younger than 6 years old. 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 a 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 flanking AAV inverted terminal repeats (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, AAVrhlO, or 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 survival motor neuron 2 (SMN2) pre-mRNA. In some embodiments, the SMN2 ASO promotes the inclusion of exon 7 in 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 (e.g., the SMN2 gene or SMN2 pre-mRNA) encoding the SMN2 protein. In some embodiments, the SMN2 ASO comprises a sequence complementary to intron 6 of a nucleic acid molecule (e.g., the SMN2 gene or SMN2 pre-mRNA) encoding the SMN2 protein. In some embodiments, the SMN2 ASO comprises a sequence complementary to intron 7 of a nucleic acid molecule (e.g., the SMN2 gene or SMN2 pre-mRNA) encoding the SMN2 protein. 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 nucleobase or backbone modifications.

[0019] In some embodiments, a recombinant SMN1 gene (e.g., in a viral vector) is administered (e.g., once or multiple times) to a subject who has previously been treated with a small molecule that increases SMN function and / or an SMN2 ASO (e.g., nusinersen) therapy. In some embodiments, a recombinant SMN1 gene (e.g., in a viral vector such as rAAV) is administered (e.g., once or multiple times) to a subject who is currently being treated with a small molecule that increases SMN function (e.g., risdiplam or branapram) and / or an SMN2 ASO (e.g., nusinersen) therapy. In some embodiments, a subject is initiated on a therapy comprising the concurrent or sequential administration of a small molecule that increases SMN function (e.g., risdiplam or branapram) 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, small molecules that increase SMN function (e.g., risdiplam or branapram) and a) rAAV containing a recombinant SMN1 gene (also referred to as SMN1 rAAV) and / or b) SMN2 ASO (e.g., nusinersen) are administered simultaneously. In some embodiments, small molecules that increase SMN function (e.g., risdiplam or branapram) and SMN1 rAAV and / or SMN2 ASO are administered together. In some embodiments, small molecules that increase SMN function (e.g., risdiplam or branapram) and SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) are administered separately in different compositions. In some embodiments, small molecules that increase SMN function (e.g., risdiplam or branapram) and SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) are administered sequentially. In some embodiments, the small molecule that increases SMN function (e.g., risdiplam or branapram) and the SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) are administered at different frequencies. In some embodiments, the small molecule that increases SMN function (e.g., risdiplam or branapram) is administered 1-6 times per year or more frequently (e.g., weekly or 2-4 times per month). In some embodiments, the SMN1 rAAV is administered once. In some embodiments, the SMN2 ASO is administered 1-6 times per year. In some embodiments, two or more subsequent administrations of the small molecule that increases SMN function (e.g., risdiplam or branapram) alone and / or with the SMN2 ASO (e.g., nusinersen) are administered after the initial administration of the SMN1 rAAV and SMN2 ASO (e.g., nusinersen). In some embodiments, the subject receives one or more booster doses of SMN1 rAAV. In some embodiments, the first and second doses of SMN1 rAAV are provided to the subject more than six months apart, or more than one year apart. In some embodiments, the first and second SMN1 rAAV compositions comprise the same rAAV capsid protein. In some embodiments, the first and second SMN1 rAAV compositions comprise different rAAV capsid proteins.

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

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

[0023] In some embodiments, the small molecule is administered via a suitable route (e.g., orally), 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 branapram), an SMN1 rAAV, and / or an SMN2 ASO (e.g., nusinersen) is administered to the intrathecal space of a subject. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branapram), an SMN1 rAAV, and / or an SMN2 ASO (e.g., nusinersen) is administered to the intracisternal space of a subject. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and / or an SMN2 ASO (e.g., nusinersen) is administered to the intracisternal space of a subject. The initial and / or subsequent doses of the ASO (e.g., nusinersen) are administered intravenously or intramuscularly.

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

[0025] In some embodiments, SMN protein expression in a subject with SMA is increased by administering to the subject (e.g., concomitantly or sequentially) an effective amount of a small molecule that increases SMN function (e.g., risdiplam or branapram) and an SMN1 rAAV and / or an SMN2 ASO (e.g., nusinersen). In some embodiments, the subject has previously been treated with a small molecule that increases SMN function (e.g., risdiplam or branapram). In some embodiments, the subject has previously been administered an SMN1 rAAV. In some embodiments, the subject has previously been treated with an SMN2 ASO (e.g., nusinersen). In some embodiments, SMN protein expression in a subject previously treated with an SMN1 rAAV is increased by administering to the subject an effective amount of a small molecule that increases SMN function (e.g., risdiplam or branapram) 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 an effective amount of a small molecule that increases SMN function (e.g., risdiplam or branapram) and / or an SMN1 rAAV. In some embodiments, SMN protein expression in a subject previously treated with a small molecule that increases SMN function (e.g., risdiplam or branapram) is increased by administering to the subject an effective amount of an 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 branapram). In some embodiments, the composition comprises a recombinant SMN1 gene (e.g., in an 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 branapram) is administered to a subject via any known route suitable for administering small molecule drugs (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 branapram), an SMN1 rAAV, and / or an SMN2 ASO (e.g., nusinersen) (e.g., as two or three separate compositions) are administered to a subject (e.g., a human subject) via an intrathecal route. In some embodiments, one or more of a small molecule that increases SMN function (e.g., risdiplam or branapram), an SMN1 rAAV, and / or an SMN2 ASO (e.g., as two or three separate compositions) are administered to the spinal canal, subarachnoid space, ventricles, or lumbar CSF by suboccipital puncture, or by other suitable route (e.g., via injection, infusion, using a pump and catheter, or via other suitable technique).In some embodiments, one or more of a small molecule that increases SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and / or an SMN2 ASO (e.g., as two or three separate compositions) are administered to a subject (e.g., a human subject) intracranially, intracerebroventricularly, intracerebrally, intraparenchymally, intravenously, or via other suitable routes. In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branapram) is administered to a subject via oral administration, while an SMN1 rAAV and / or an SMN2 ASO (e.g., nusinersen) (e.g., as two or three separate compositions) are administered to a subject (e.g., a human subject) via injection (e.g., intravenously, intrathecally, intramuscularly, intracranially, intracerebroventricularly, intracerebrally, or intraparenchymally). In some embodiments, a small molecule that increases SMN function (e.g., risdiplam or branapram) is administered to a subject via oral administration, while the SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) (e.g., as two or three separate compositions) are administered to the spinal canal, subarachnoid space, ventricles, or lumbar CSF via suboccipital puncture, or by other suitable route (e.g., via injection, infusion, using a pump and catheter, or via other suitable technique). Whether administered together or sequentially, each of the small molecule that increases SMN function (e.g., risdiplam or branapram), the SMN1 rAAV, and the 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 branapram), the SMN1 rAAV, and the 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, small molecules that increase SMN function (e.g., risdiplam or branapram), SMN1 rAAV and / or SMN2 ASO (e.g., nusinersen) are used in the manufacture of medicaments (e.g., as two or three separate medicaments) for treating diseases or conditions associated with survival motor neuron protein (SMN), such as spinal muscular atrophy (SMA).

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

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

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

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

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

[0033] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present application, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein. [Brief explanation of the drawings]

[0034] [Figure 1]Figure 1 illustrates increased levels of SMN activity in a greater number of motor neurons in subjects receiving combined treatment with a recombinant nucleic acid encoding SMN1 and an antisense oligonucleotide (e.g., nusinersen) that increases full-length SMN2 mRNA (e.g., promotes exon 7 inclusion in SMN2 mRNA).

[0035] [Figure 2] FIG. 2 is a schematic representation of a non-limiting example of a nucleic acid encoding SMN1.

[0036] [Figure 3-1] FIG. 3 illustrates the chemical structure of nusinersen, a non-limiting example of an antisense oligonucleotide that increases full-length SMN2 mRNA (e.g., promotes exon 7 inclusion in SMN2 mRNA). [Figure 3-2] FIG. 3 illustrates the chemical structure of nusinersen, a non-limiting example of an antisense oligonucleotide that increases full-length SMN2 mRNA (e.g., promotes exon 7 inclusion in SMN2 mRNA).

[0037] [Figure 4A-4B] Figures 4A-4B show the distribution of rAAV after different modes of administration in non-human primates. Figure 4A shows the distribution of rAAV in the cervical, thoracic, and lumbar spinal cord after lumbar puncture (LP) or intracisternal puncture (ICM) injection of rAAV encoding SMN1. Figure 4B shows the distribution of rAAV in the cervical, thoracic, and lumbar spinal cord after lumbar puncture (LP), intracisternal puncture (ICM), or intravenous injection of rAAV encoding SMN1.

[0038] [Figures 5A-5E]Figures 5A-5E illustrate the physical and biological compatibility of recombinant nucleic acids encoding SMN1 and antisense oligonucleotides that increase full-length SMN2 mRNA (e.g., promote exon 7 inclusion in SMN2 mRNA). Figure 5A shows SEC-HPLC analysis of rAAV encoding SMN1. Figure 5B shows SEC-HPLC analysis of an ASO that increases full-length SMN2. Figure 5C shows SEC-HPLC analysis of rAAV encoding SMN1 and an ASO that increases full-length SMN2. Figure 5D provides data on the infectivity of SMN1 rAAV in cells in vitro, either delivered with the SMN1 rAAV vector alone or delivered with the SMN1 rAAV vector and the SMN2 ASO. The results show that the infectivity of SMN1 rAAV is not significantly affected by the presence of the SMN2 ASO in the co-formulation. Figure 5E shows intracellular SMN protein expression levels and GEM formation in cells after treatment with SMN1 rAAV, SMN2 ASO, or both.

[0039] [Figures 6A-6B] Figures 6A-6B show that administration of either the SMN1 gene (e.g., in an rAAV vector) or the SMN2 ASO (e.g., nusinersen, e.g., in a single dose) partially rescues motor function by postnatal day (PND) 8** and fully rescues motor function by PND 16. They also show that body weight lags behind WT controls. Figure 6A is a series of graphs showing the righting reflex (RR) of four separate groups 8 and 16 days after ASO (nusinersen). Figure 6B is a series of graphs showing the body weight of four separate groups 8 and 16 days after ASO (nusinersen). The partial rescue of RR (PND 7-16) and body weight provides an opportunity for the additional benefit of combination therapy in this preclinical model.

[0040] [Figures 7A-7B]Figures 7A-7C show the results of the first study using weight and RR as primary endpoints for treatment with SMN1 gene therapy (in an rAAV vector) and SMN2 ASO (nusinersen). Figure 7A is a graph showing weight change over time (in days). Figure 7B is a graph showing RR change over time (in days). Figure 7C is a chart outlining the conditions for the three study groups. [Figure 7C] Figures 7A-7C show the results of the first study using weight and RR as primary endpoints for treatment with SMN1 gene therapy (in an rAAV vector) and SMN2 ASO (nusinersen). Figure 7A is a graph showing weight change over time (in days). Figure 7B is a graph showing RR change over time (in days). Figure 7C is a chart outlining the conditions for the three study groups.

[0041] [Figure 8A] Figures 8A-8C show the results of a second study using weight and RR as primary endpoints for treatment with SMN1 gene therapy (in an rAAV vector) and SMN2 ASO (nusinersen). Figure 8A is a chart outlining the conditions for the three study groups. Figure 8B is a graph showing weight change over time (in days). Figure 8C is a graph showing RR change over time (in days). [Figure 8B-8C] Figures 8A-8C show the results of a second study using weight and RR as primary endpoints for treatment with SMN1 gene therapy (in an rAAV vector) and SMN2 ASO (nusinersen). Figure 8A is a chart outlining the conditions for the three study groups. Figure 8B is a graph showing weight change over time (in days). Figure 8C is a graph showing RR change over time (in days).

[0042] [Figure 9A-9B]Figures 9A-9B show a comparison of the % change in body weight from PND7 to PND13. Figure 9A shows the % change in body weight at a dose of 1x10 GC / ASO (nusinersen): 1µg for gene therapy (rAAV). Figure 9B shows the % change in body weight at a dose of 3x10 GC / ASO (nusinersen): 3µg for gene therapy (rAAV).

[0043] [Figures 10A-10B] Figures 10A-10B show a comparison of the % change in RR from PND7 to PND13. Figure 10A shows the % change in RR at a dose of 1x10 GC / ASO (nusinersen): 1µg for gene therapy (rAAV). Figure 10B shows the % change in RR at a dose of 3x10 GC / ASO (nusinersen): 3µg for gene therapy (rAAV).

[0044] [Figure 11] Figure 11 illustrates a model showing complementarity in neuronal and non-neuronal cells using combination therapy to treat SMA. For example, therapy 1 can be an ASO (e.g., an SMN2 ASO), a small molecule that increases SMN function, or a combination therapy of an ASO and a small molecule that increases SMN function (e.g., administered together or sequentially). Therapy 2 can be an SMN1 gene therapy, a small molecule that increases SMN function, or a combination therapy of SMN1 gene therapy and a small molecule that increases SMN function (e.g., administered together or sequentially). For example, in some embodiments, therapy 1 is an ASO (e.g., an SMN2 ASO), and therapy 2 is a small molecule that increases SMN function. Therapies 1 and 2 can be any other therapy or combination therapy, including therapies not used in therapy 1 or therapy 2. DETAILED DESCRIPTION OF THE INVENTION

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

[0046] In some aspects, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject with 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 some aspects, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject with 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 some aspects, the present application relates to a method of treating spinal muscular atrophy (SMA) in a subject with 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 spinal muscular atrophy (SMA) in a subject, for example, a human subject with SMA, using combination therapy.

[0050] In some embodiments, the combination therapy involves administering to a subject with SMA a small molecule that increases SMN function in the subject (e.g., risdiplam or branapram) and (e.g., concurrently or sequentially) a) a recombinant nucleic acid that expresses the SMN1 gene (e.g., in a viral vector such as an rAAV that encodes SMN1) and / or b) an antisense oligonucleotide (ASO) that increases full-length SMN2 mRNA (e.g., an ASO that promotes the inclusion of exon 7 in SMN2 mRNA, such as nusinersen). "Combination therapy," "combined treatment," "combined therapy," or "combined 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., a recombinant SMN1 gene, an SMN2 ASO, a 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 branapram) and a recombinant nucleic acid expressing SMN1 (e.g., in an 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 with elevated intracellular survival motor neuron (SMN) protein levels, compared to treatment with either the recombinant nucleic acid, the SMN2 ASO (e.g., nusinersen), or the small molecule that increases SMN function (e.g., risdiplam or branapram) alone.

[0052] Small molecules that can increase SMN function (e.g., risdiplam or branapram) and / or recombinant nucleic acids that express SMN1 (e.g., in rAAV) and / or ASOs that increase full-length SMN2 mRNA (e.g., SMN2, such as nusinersen) Methods and compositions for administration of an ASO that promotes the inclusion of exon 7 in mRNA may also be useful for providing therapeutically effective levels of SMN protein in subjects with SMA and for treating subjects with different levels of disease severity.

[0053] Spinal muscular atrophy, or proximal spinal muscular atrophy (SMA), is an inherited neurodegenerative disorder characterized by the loss of spinal motor neurons. SMA is an early-onset, autosomal recessive disorder and is currently the leading cause of death among young children. The severity of SMA varies among patients, and therefore, it is classified into different types depending on the age of onset and motor developmental milestones. The designation SMA0 has been proposed to reflect prenatal onset and severe joint contractures, facial diplegia, and respiratory failure. Three postnatal forms of SMA have been designated. Type 1 SMA (also known as Werdnig-Hoffmann disease) is the most severe form, with onset at birth or within the first six months of life, and typically leads to death within two years. Children with type 1 SMA are unable to sit or walk and have severe respiratory impairment. Type 2 SMA is an intermediate form with onset within the first two years. Children with type 2 SMA can sit but are unable to stand or walk. Type III (also known as Kugelberg-Welander disease) begins after 18 months to 2 years of age (Lefebvre et al., Hum. Mol. Genet., 1998, 7, 1531-1536) and usually progresses chronically. Children with Type III SMA are able to stand and walk independently, at least during infancy. The adult form (Type IV) is the mildest form of SMA, with onset after age 30, and only a few cases have been reported. Types III and IV SMA are also known as late-onset SMA.

[0054] The molecular basis of SMA results from the loss of both copies of the survival motor neuron gene 1 (SMN1), also known as the SMN telomeric protein, which is part of a multiprotein complex thought to be involved in snRNP biogenesis and recycling. A nearly identical gene, SMN2, also known as the SMN centromeric gene, resides in a duplicated region on chromosome 5q13 and modulates disease severity. Normal SMN1 gene expression alone results in the expression of the survival motor neuron (SMN) protein. While SMN1 and SMN2 potentially encode the same protein, 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 This results in approximately 10-20% SMN protein and 80-90% unstable / non-functional SMN delta7 protein. 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 homozygous loss of both functional copies of the SMN1 gene. However, the SMN2 gene encodes the same protein as SMN1 and therefore 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 incomplete inclusion of exon 7 in the SMN2 transcript. Therefore, the predominant form of SMN2 lacks exon 7 and is unstable and inactive. The full-size protein produced by the SMN2 gene is identical to that produced by a similar gene called SMN1. However, only 10–15 percent of all functional SMN protein is produced by the SMN2 gene (the remainder is produced by the SMN1 gene). Typically, people have two copies of the SMN1 gene and one to two copies of the SMN2 gene in each cell. However, the number of SMN2 gene copies varies, with some people having up to eight copies. The more copies of the SMN2 gene a person has, the more SMN protein they produce. Extra copies of the SMN2 gene can modify the severity of SMA. Because all individuals with spinal muscular atrophy have mutations in both copies of the SMN1 gene, which results in little or no SMN protein being produced from SMN1, the SMN2 gene can help replace some of the missing 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 occur later in life. Affected individuals with one or two functional copies of the SMN2 gene generally have severe muscle weakness that begins at birth or in early childhood. 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 treatments described herein can be administered to different subjects with different numbers of SMN2 genes.

[0056] In some embodiments, intracellular SMN protein levels can be increased by contacting motor neurons with a small molecule capable of increasing SMN function (e.g., risdiplam or branapram) and 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 containing exon 7, thereby resulting in increased expression of full-length SMN protein from cellular SMN2 transcripts. In some embodiments, the combination therapy includes administering a small molecule capable of increasing 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, for example, by promoting the inclusion of exon 7 in 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 can 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, a small molecule that increases SMN function and a recombinant SMN1 gene are administered together.In some embodiments, a small molecule that increases SMN function and an SMN2 ASO are administered together.In some embodiments, a small molecule that increases SMN function, a recombinant SMN1 gene, and an SMN2 ASO are administered together.In some embodiments, a small molecule that increases SMN function and a recombinant SMN1 gene are administered sequentially.In some embodiments, a small molecule that increases SMN function and an SMN2 ASO are administered sequentially.In some embodiments, a small molecule that increases SMN function, a recombinant SMN1 gene, and an SMN2 ASO are administered sequentially.

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

[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 a recombinant SMN1 gene. In some embodiments, the pharmaceutical composition comprising 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) to treat SMA is by injection (e.g., via intravenous injection, direct injection into the CNS, or any other suitable route).

[0059] In some embodiments, the small molecule that increases SMN function (e.g., risdiplam or branapram) 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 liquids). The administration of the small molecule that increases SMN function can be via any known route suitable for administering small molecule drugs (e.g., oral administration). In some embodiments, the small molecule that increases SMN function is given to a subject by oral administration.

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

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

[0062] The pharmaceutical compositions used in the present methods may include pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover).

[0063] Pharmaceutical compositions to be used for in vivo administration can be sterile, which can be accomplished by any means known in the art, including, but not limited to, filtration through sterile filtration membranes.

[0064] The pharmaceutical compositions described herein may be in any suitable unit dosage form known in the art, such as, 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., concurrently 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., concurrently 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., concurrently 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 concurrently as defined herein. In some embodiments, the first, second, and third compositions are administered concurrently as defined herein. In some embodiments, the first and second compositions are administered sequentially to a subject as defined herein. In some embodiments, the first, second, and third compositions are administered sequentially to a subject as defined herein.

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

[0067] Sequential administration, as used herein, refers to the administration of two or more of the therapies described herein for treating SMA (e.g., recombinant SMN1 gene, SMN2 ASO, or small molecules that increase SMN function) under different administration schedules. For example, the treatments may be administered on different days, weeks, months, or years during different clinic visits. The therapies 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 involves the administration of each of the recombinant SMN1 gene, SMN2 ASO, and / or small molecules that increase SMN function described herein at different frequencies or administration schedules.

[0068] Thus, in some embodiments, the first and second compositions described herein are administered separately to a subject at different times (e.g., at different times of the 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 separately to a subject at different times (e.g., at different times of the 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 an rAAV) is administered less frequently than a composition comprising a small molecule that increases SMN function (e.g., risdiplam or branapram). In some embodiments, a composition comprising a recombinant SMN1 gene (e.g., in an 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 branapram).

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

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

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

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

[0074] In some embodiments, small molecule drugs that increase SMN function can modulate splicing, stabilize, and / or increase the transcription or translation of an SMN gene (e.g., SMN1 or SMN2). In some embodiments, small molecule drugs that increase SMN function can improve the activity (e.g., potency and / or efficacy) 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 splice modulator. In some embodiments, the splice modulator is an SMN2 splice modulator. In some embodiments, the splice modulator is a 7-disubstituted phenyltetracycline. Non-limiting examples of 7-substituted phenyltetracycline SMN2 splice modulators are described in International Publication No. 2013 / 181391, the contents of which are incorporated herein by reference. In some embodiments, the splice modulator is a substituted isoindolinone. Non-limiting examples of substituted isoindolinone SMN2 splice modulators are described in U.S. Patent Application Publication No. 2009 / 0031435, the contents of which are incorporated herein by reference. In some embodiments, the splice modulator is a substituted carbazole derivative. Non-limiting examples of substituted carbazole derivatives that act as SMN2 splice modulators are described in International Publication No. 2005 / 023255, the contents of which are incorporated herein by reference. In some embodiments, the SMN2 splice modulator is a substituted 1,4-diazepane. Non-limiting examples of substituted 1,4-diazepanes that act as SMN2 splice modulators are described in International Publication No. 2019 / 028440, the contents of which are incorporated herein by reference. In some embodiments, the SMN2 splice modulator is a substituted pyridazine. Non-limiting examples of substituted pyridazines that act as SMN2 splice modulators are described in WO 2015 / 017589, WO 2014 / 028459, U.S. Pat. No. 10,195,196, U.S. Pat. No. 9,545,404, U.S. Pat. No. 8,729,263, and WO 2015 / 173181, the contents of each of which are incorporated herein by reference.

[0076] In some embodiments, the substituted pyridazine is a compound of formula (I'): [ka] or a pharmaceutically acceptable salt thereof [In the formula, A is C1-C4 alkyl (wherein two C1-C4 alkyl groups, combined with the atoms to which they are attached, can form a 5- or 6-membered ring and is substituted with 0 or 1 substituent selected from oxo, oxime, and hydroxy), haloC1-C4 alkyl, dihaloC1-C4 alkyl, trihaloC1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkoxy-, C3-C7 cycloalkyl, haloC1-C4 alkoxy, dihaloC1-C4 alkoxy, trihaloC ... HaloC1-C4 alkoxy, hydroxy, cyano, halogen, amino, mono- and di-C1-C4 alkylamino, heteroaryl, C1-C4 alkyl substituted with hydroxy, C1-C4 alkoxy substituted with aryl, amino, -C(O)NH, C1-C4 alkyl, -heteroaryl, -NHC(O)-, C1-C4 alkyl-, heteroaryl, C1-C4 alkyl-C(O)NH-, heteroaryl, C1-C4 alkylNHC(O)-heteroaryl, 3- to 7-membered cyclohexyl 2-hydroxy-phenyl substituted by 0, 1, 2 or 3 substituents independently selected from arylalkyl, 5-7 membered cycloalkenyl, or a 5, 6 or 9 membered heterocycle containing 1 or 2 heteroatoms independently selected from S, O and N, where heteroaryl has 5, 6 or 9 ring atoms, 1, 2 or 3 ring heteroatoms selected from N, O and S, and is substituted by oxo, hydroxy, nitro, halogen, C1-C4 alkyl, C1-C4 alkenyl, or substituted with 0, 1, or 2 substituents independently selected from C1-C4 alkoxy, C3-C7 cycloalkyl, C1-C4 alkyl-OH, trihaloC1-C4 alkyl, mono- and di-C1-C4 alkylamino, -C(O)NH2, -NH2, -NO2, hydroxyC1-C4 alkylamino, hydroxyC1-C4 alkyl, 4- to 7-membered heterocyclylC1-C4 alkyl, aminoC1-C4 alkyl, and mono- and di-C1-C4 alkylaminoC1-C4 alkyl; or A is 2-naphthyl optionally substituted at the 3-position with hydroxy and further substituted with 0, 1 or 2 substituents selected from hydroxy, cyano, halogen, C1-C4 alkyl, C2-C4 alkenyl, C1-C5 alkoxy, wherein said alkoxy is unsubstituted or substituted with hydroxy, C1-C4 alkoxy, amino, N(H)C(O)C1-C4 alkyl, N(H)C(O)2C1-C4 alkyl, alkylene 4-7 membered heterocycle, 4-7 membered heterocycle, and mono- and di-C1-C4 alkylamino; or A is a 6-membered heteroaryl having 1 to 3 ring nitrogen atoms, said 6-membered heteroaryl being substituted by phenyl or heteroaryl having 5 or 6 ring atoms, 1 or 2 ring heteroatoms independently selected from N, O and S, and substituted with 0, 1 or 2 substituents independently selected from C1-C4 alkyl, mono- and di-C1-C4 alkylamino, hydroxyC1-C4 alkylamino, hydroxyC1-C4 alkyl, aminoC1-C4 alkyl and mono- and di-C1-C4 alkylaminoC1-C4 alkyl; or A is a bicyclic heteroaryl having 9-10 ring atoms and 1, 2 or 3 ring heteroatoms independently selected from N, O or S, wherein said bicyclic heteroaryl is substituted with 0, 1 or 2 substituents independently selected from cyano, halogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy and C1-C4 alkoxy substituted with hydroxy, C1-C4 alkoxy, amino and mono- and di-C1-C4 alkylamino; or 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, said tricyclic heteroaryl being substituted with 0, 1, or 2 substituents independently selected from cyano, halogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy, C1-C4 alkoxy substituted with hydroxy, C1-C4 alkoxy, amino, mono- and di-C1-C4 alkylamino, and heteroaryl, wherein said heteroaryl has 5, 6, or 9 ring atoms and 1, 2, or 3 ring heteroatoms independently selected from N, O, and S. and having 1, 2, or 3 ring heteroatoms selected from the group consisting of oxo, hydroxy, nitro, halogen, C1-C4 alkyl, C1-C4 alkenyl, C1-C4 alkoxy, C3-C7 cycloalkyl, C1-C4 alkyl-OH, trihaloC1-C4 alkyl, mono- and di-C1-C4 alkylamino, -C(O)NH2, -NH2, -NO2, hydroxyC1-C4 alkylamino, hydroxyC1-C4 alkyl, 4- to 7-membered heterocyclylC1-C4 alkyl, aminoC1-C4 alkyl, and mono- and di-C1-C4 alkylaminoC1-C4 alkyl; B is a group of the formula: [ka] [In the formula, m, n, and p are independently selected from 0 or 1; R, R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, C1-C4 alkyl, wherein said alkyl is optionally substituted with hydroxy, amino, or mono- and di-C1-C4 alkylamino; R5 and R6 are independently selected from hydrogen and fluorine; or R and R3 combine to form a fused 5- or 6-membered heterocyclic ring having 0 or 1 additional ring heteroatoms selected from N, O, or S; R1 and R3 combine to form a C1-C3 alkylene group; R1 and R5 combine to form a C1-C3 alkylene group; R3 and R4, combined with the carbon atom to which they are attached, form a spirocyclic C3-C6 cycloalkyl; X is CR A R B , O, NR7 or a bond; R7 is hydrogen or C1-C4 alkyl; R A and R B are independently selected from hydrogen and C1-C4 alkyl, or R A and R B combine to form a divalent C2-C5 alkylene group; Z is CR8 or N; when Z is N, X is a bond; R8 is hydrogen or combines with R6 to form a double bond. or B is a group of the formula: [ka] [In the formula, p and q are independently selected from the group consisting of 0, 1 and 2; R9 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 10 and R 14 are independently selected from hydrogen, amino, mono- and di-C1-C4 alkylamino, and C1-C4 alkyl, wherein said alkyl is optionally substituted with hydroxy, amino, or mono- and di-C1-C4 alkylamino; R 11 is hydrogen, C1-C4 alkyl, amino or mono- and di-C1-C4 alkylamino; R 12 is hydrogen or C1-C4 alkyl; or R9 and R 10are combined to form a saturated azacycle having 4 to 7 ring atoms, said saturated azacycle being optionally substituted with 1 to 3 C1 to C4 alkyl groups; or R 11 and R 12 are combined to form a saturated azacycle having 4 to 7 ring atoms, said saturated azacycle being optionally substituted with 1 to 3 C1-C4 alkyl groups. and; C is H or absent, as valence permits.

[0077] In some embodiments, the substituted pyridazine is a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof [In the formula, A is C1-C4 alkyl (wherein two C1-C4 alkyl groups, combined with the atoms to which they are attached, can form a 5- or 6-membered ring and is substituted with 0 or 1 substituent selected from oxo, oxime, and hydroxy), haloC1-C4 alkyl, dihaloC1-C4 alkyl, trihaloC1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkoxy-, C3-C7 cycloalkyl, haloC1-C4 alkoxy, dihaloC1-C4 alkoxy, trihaloC ... HaloC1-C4 alkoxy, hydroxy, cyano, halogen, amino, mono- and di-C1-C4 alkylamino, heteroaryl, C1-C4 alkyl substituted with hydroxy, C1-C4 alkoxy substituted with aryl, amino, -C(O)NH, C1-C4 alkyl, -heteroaryl, -NHC(O)-, C1-C4 alkyl-, heteroaryl, C1-C4 alkyl-C(O)NH-, heteroaryl, C1-C4 alkylNHC(O)-heteroaryl, 3- to 7-membered cyclohexyl 2-hydroxy-phenyl substituted by 0, 1, 2 or 3 substituents independently selected from arylalkyl, 5-7 membered cycloalkenyl, or a 5, 6 or 9 membered heterocycle containing 1 or 2 heteroatoms independently selected from S, O and N, where heteroaryl has 5, 6 or 9 ring atoms, 1, 2 or 3 ring heteroatoms selected from N, O and S, and is substituted by oxo, hydroxy, nitro, halogen, C1-C4 alkyl, C1-C4 alkenyl, or substituted with 0, 1, or 2 substituents independently selected from C1-C4 alkoxy, C3-C7 cycloalkyl, C1-C4 alkyl-OH, trihaloC1-C4 alkyl, mono- and di-C1-C4 alkylamino, -C(O)NH2, -NH2, -NO2, hydroxyC1-C4 alkylamino, hydroxyC1-C4 alkyl, 4- to 7-membered heterocyclylC1-C4 alkyl, aminoC1-C4 alkyl, and mono- and di-C1-C4 alkylaminoC1-C4 alkyl; or A is 2-naphthyl optionally substituted at the 3-position with hydroxy and further substituted with 0, 1 or 2 substituents selected from hydroxy, cyano, halogen, C1-C4 alkyl, C2-C4 alkenyl, C1-C5 alkoxy, wherein said alkoxy is unsubstituted or substituted with hydroxy, C1-C4 alkoxy, amino, N(H)C(O)C1-C4 alkyl, N(H)C(O)2C1-C4 alkyl, alkylene 4-7 membered heterocycle, 4-7 membered heterocycle, and mono- and di-C1-C4 alkylamino; or A is a 6-membered heteroaryl having 1 to 3 ring nitrogen atoms, said 6-membered heteroaryl being substituted by phenyl or heteroaryl having 5 or 6 ring atoms, 1 or 2 ring heteroatoms independently selected from N, O and S, and substituted with 0, 1 or 2 substituents independently selected from C1-C4 alkyl, mono- and di-C1-C4 alkylamino, hydroxyC1-C4 alkylamino, hydroxyC1-C4 alkyl, aminoC1-C4 alkyl and mono- and di-C1-C4 alkylaminoC1-C4 alkyl; or A is a bicyclic heteroaryl having 9-10 ring atoms and 1, 2 or 3 ring heteroatoms independently selected from N, O or S, wherein said bicyclic heteroaryl is substituted with 0, 1 or 2 substituents independently selected from cyano, halogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy and C1-C4 alkoxy substituted with hydroxy, C1-C4 alkoxy, amino and mono- and di-C1-C4 alkylamino; or 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, wherein said tricyclic heteroaryl is substituted with 0, 1, or 2 substituents independently selected from cyano, halogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy, C1-C4 alkoxy substituted with hydroxy, C1-C4 alkoxy, amino, mono- and di-C1-C4 alkylamino, and 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 substituted with 0, 1, or 2 substituents independently selected from oxo, hydroxy, nitro, halogen, C1-C4 alkyl, C1-C4 alkenyl, C1-C4 alkoxy, C3-C7 cycloalkyl, C1-C4 alkyl-OH, trihaloC1-C4 alkyl, mono- and di-C1-C4 alkylamino (mono- and di-Ci-C4 alkylamino), -C(O)NH2, -NH2, -NO2, hydroxyC1-C4 alkylamino, hydroxyC1-C4 alkyl, 4- to 7-membered heterocycleC1-C4 alkyl, aminoC1-C4 alkyl, and mono- and di-C1-C4 alkylaminoC1-C4 alkyl; B is a group of the formula: [ka] [In the formula, m, n, and p are independently selected from 0 or 1; R, R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, C1-C4 alkyl, wherein said alkyl is optionally substituted with hydroxy, amino, or mono- and di-C1-C4 alkylamino; R5 and R6 are independently selected from hydrogen and fluorine; or R and R3 combine to form a fused 5- or 6-membered heterocyclic ring having 0 or 1 additional ring heteroatoms selected from N, O, or S; R1 and R3 combine to form a C1-C3 alkylene group; R1 and R5 combine to form a C1-C3 alkylene group; R3 and R4, combined with the carbon atom to which they are attached, form a spirocyclic C3-C6 cycloalkyl; X is CR A R B , O, NR7 or a bond; R7 is hydrogen or C1-C4 alkyl; R A and R B are independently selected from hydrogen and C1-C4 alkyl, or R A and R B combine to form a divalent C2-C5 alkylene group; Z is CR8 or N; when Z is N, X is a bond; R8 is hydrogen or combines with R6 to form a double bond. or B is a group of the formula: [ka] [In the formula, p and q are independently selected from the group consisting of 0, 1 and 2; R9 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 10 and R 14 are independently selected from hydrogen, amino, mono- and di-C1-C4 alkylamino, and C1-C4 alkyl, wherein said alkyl is optionally substituted with hydroxy, amino, or mono- and di-C1-C4 alkylamino; R 11 is hydrogen, C1-C4 alkyl, amino or mono- and di-C1-C4 alkylamino; R 12 is hydrogen or C1-C4 alkyl; or R9 and R 10are combined to form a saturated azacycle having 4 to 7 ring atoms, said saturated azacycle being optionally substituted with 1 to 3 C1 to C4 alkyl groups; or R 11 and R 12 are combined to form a saturated azacycle having 4 to 7 ring atoms, said saturated azacycle being optionally substituted with 1 to 3 C1-C4 alkyl groups. is.

[0078] In some embodiments, A is selected from the group consisting of C1-C4 alkyl (wherein two C1-C4 alkyl groups, combined with the atoms to which they are attached, can form a 5-6 membered ring and is substituted with 0 or 1 substituent selected from oxo, oxime, and hydroxy), haloC1-C4 alkyl, dihaloC1-C4 alkyl, trihaloC1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkoxy-, C3-C7 cycloalkyl, haloC1-C4 alkoxy, dihaloC1-C4 alkyl. Coxy, trihaloC1-C4 alkoxy, hydroxy, cyano, halogen, amino, mono- and di-C1-C4 alkylamino, heteroaryl, C1-C4 alkyl substituted with hydroxy, C1-C4 alkoxy substituted with aryl, amino, -C(O)NH, C1-C4 alkyl, -heteroaryl, -NHC(O)-, C1-C4 alkyl-, heteroaryl, C1-C4 alkyl-C(O)NH-, heteroaryl, C1-C4 alkylNHC(O)-heteroaryl, 3 and 2-hydroxy-phenyl substituted with 0, 1, 2, or 3 substituents independently selected from 1 to 7-membered cycloalkyl, 5 to 7-membered cycloalkenyl, or 5, 6, or 9-membered heterocycle containing 1 or 2 heteroatoms independently selected from S, O, and N, where heteroaryl has 5, 6, or 9 ring atoms, 1, 2, or 3 ring heteroatoms selected from N, O, and S, and is substituted with oxo, hydroxy, nitro, halogen, C1-C4 alkyl, C1-C4 alkene. In some embodiments, A is substituted with 0, 1, or 2 substituents independently selected from nyl, C1-C4 alkoxy, C3-C7 cycloalkyl, C1-C4 alkyl-OH, trihaloC1-C4 alkyl, mono- and di-C1-C4 alkylamino, -C(O)NH2, -NH2, -NO2, hydroxyC1-C4 alkylamino, hydroxyC1-C4 alkyl, 4- to 7-membered heterocyclylC1-C4 alkyl, aminoC1-C4 alkyl, and mono- and di-C1-C4 alkylaminoC1-C4 alkyl. [ka] [In the formula, R 16is a 5-membered heteroaryl having one ring nitrogen atom and 0 or 1 additional ring heteroatoms selected from N, O, or S, where the heteroaryl is optionally substituted with C1-C4 alkyl. In some embodiments, A is a group of the formula: [ka] In some embodiments, A is of the formula: [ka] [In the formula, R 16 is a 5-membered heteroaryl having one ring nitrogen atom and 0 or 1 additional ring heteroatoms selected from N, O, or S, where the heteroaryl is optionally substituted with C1-C4 alkyl. In some embodiments, A is a group of the formula: [ka] is.

[0079] In some embodiments, A is 2-naphthyl optionally substituted at the 3-position with hydroxy and further substituted with 0, 1, or 2 substituents selected from hydroxy, cyano, halogen, C1-C4 alkyl, C2-C4 alkenyl, and C1-C5 alkoxy, wherein said alkoxy is unsubstituted or substituted with hydroxy, C1-C4 alkoxy, amino, N(H)C(O)C1-C4 alkyl, N(H)C(O)2C1-C4 alkyl, alkylene 4-7 membered heterocycle, 4-7 membered heterocycle, and mono- and di-C1-C4 alkylamino.

[0080] In some embodiments, A is a 6-membered heteroaryl having 1 to 3 ring nitrogen atoms, said 6-membered heteroaryl being substituted by phenyl or heteroaryl having 5 or 6 ring atoms, 1 or 2 ring heteroatoms independently selected from N, O, and S, and substituted with 0, 1, or 2 substituents independently selected from C1-C4 alkyl, mono- and di-C1-C4 alkylamino, hydroxyC1-C4 alkylamino, hydroxyC1-C4 alkyl, aminoC1-C4 alkyl, and mono- and di-C1-C4 alkylaminoC1-C4 alkyl.

[0081] In some embodiments, A is a bicyclic heteroaryl having 9-10 ring atoms and 1, 2, or 3 ring heteroatoms independently selected from N, O, or S, wherein said bicyclic heteroaryl is substituted with 0, 1, or 2 substituents independently selected from cyano, halogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy, and C1-C4 alkoxy substituted with hydroxy, C1-C4 alkoxy, amino, and mono- and di-C1-C4 alkylamino.

[0082] In some embodiments, 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, wherein said tricyclic heteroaryl is substituted with 0, 1, or 2 substituents independently selected from cyano, halogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy, C1-C4 alkoxy substituted with hydroxy, C1-C4 alkoxy, amino, mono- and di-C1-C4 alkylamino, and heteroaryl, wherein said heteroaryl has 5, 6, or 9 ring atoms, N, O, and S. and is substituted with 0, 1, or 2 substituents independently selected from oxo, hydroxy, nitro, halogen, C1-C4 alkyl, C1-C4 alkenyl, C1-C4 alkoxy, C3-C7 cycloalkyl, C1-C4 alkyl-OH, trihaloC1-C4 alkyl, mono- and di-C1-C4 alkylamino, -C(O)NH2, -NH2, -NO2, hydroxyC1-C4 alkylamino, hydroxyC1-C4 alkyl, 4- to 7-membered heterocyclylC1-C4 alkyl, aminoC1-C4 alkyl, and mono- and di-C1-C4 alkylaminoC1-C4 alkyl.

[0083] In some embodiments, B is a group of the formula: [ka] [In the formula, m, n, and p are independently selected from 0 or 1; R, R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, C1-C4 alkyl, wherein said alkyl is optionally substituted with hydroxy, amino, or mono- and di-C1-C4 alkylamino; R5 and R6 are independently selected from hydrogen and fluorine; or R and R3 combine to form a fused 5- or 6-membered heterocyclic ring having 0 or 1 additional ring heteroatoms selected from N, O, or S; R1 and R3 combine to form a C1-C3 alkylene group; R1 and R5 combine to form a C1-C3 alkylene group; R3 and R4, combined with the carbon atom to which they are attached, form a spirocyclic C3-C6 cycloalkyl; X is CR A R B , O, NR7 or a bond; R7 is hydrogen or C1-C4 alkyl; R A and R B are independently selected from hydrogen and C1-C4 alkyl, or R A and R B combine to form a divalent C2-C5 alkylene group; Z is CR8 or N; when Z is N, X is a bond; R8 is hydrogen or combines with R6 to form a double bond. is.

[0084] In some embodiments, B is a group of the formula: [ka] [In the formula, p and q are independently selected from the group consisting of 0, 1 and 2; R9 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 10 and R 14 are independently selected from hydrogen, amino, mono- and di-C1-C4 alkylamino, and C1-C4 alkyl, wherein said alkyl is optionally substituted with hydroxy, amino, or mono- and di-C1-C4 alkylamino; R 11 is hydrogen, C1-C4 alkyl, amino or mono- and di-C1-C4 alkylamino; R 12 is hydrogen or C1-C4 alkyl; or R9 and R10 are combined to form a saturated azacycle having 4 to 7 ring atoms, said saturated azacycle being optionally substituted with 1 to 3 C1 to C4 alkyl groups; or R 11 and R 12 are combined to form a saturated azacycle having 4 to 7 ring atoms, said saturated azacycle being optionally substituted with 1 to 3 C1-C4 alkyl groups. is.

[0085] In some embodiments, B is [ka] In some embodiments, B is [ka] In some embodiments, B is [ka] In some embodiments, B is [ka] In some embodiments, B is [ka] [In the formula, R 17 is H or unsubstituted methyl. In some embodiments, B is [ka] [In the formula, R 17 is H or unsubstituted methyl. In some embodiments, B is [ka] [In the formula, R 17 is H or unsubstituted methyl. In some embodiments, B is [ka] In some embodiments, B is [ka] In some embodiments, B is [ka] is.

[0086] In some embodiments, the substituted pyridazine of formula (I') has formula (II'): [ka] or a pharmaceutically acceptable salt thereof [In the formula, R 16 is a 5-membered heteroaryl having one ring nitrogen atom and 0 or 1 additional ring heteroatoms selected from N, O, or S, wherein the heteroaryl is optionally substituted with C1-C4 alkyl.

[0087] In some embodiments, the substituted pyridazine of formula (I) is of formula (II): [ka] or a pharmaceutically acceptable salt thereof [In the formula, R 16 is a 5-membered heteroaryl having one ring nitrogen atom and zero or one additional ring heteroatom selected from N, O, or S, wherein said heteroaryl is optionally substituted with C1-C4 alkyl. In some embodiments, R 16 is thiophene, furan, pyrrole, dihydropyrrole, imidazole, pyrazole, pyrazine, isothiazole, isoxazole, triazole, tetrazole, oxazole, isoxazole, thiazole, isothiazole. 16 is pyrazole. In some embodiments, R 16 teeth, [ka] is.

[0088] In some embodiments, the substituted pyridazine of formula (I) has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0089] In some embodiments, the substituted pyridazine of formula (I) has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0090] In some embodiments, the substituted pyridazine is a compound of formula (III): [ka] or a pharmaceutically acceptable salt thereof [In the formula, R 1 is hydrogen or C 1~7 is alkyl; R 2 is hydrogen, cyano, C 1~7 Alkyl, C 1~7 Haloalkyl or C 3~8 is cycloalkyl; R 3 is hydrogen, C 1~7 Alkyl or C 3~8 is cycloalkyl; A is N-heterocycloalkyl or NR 12 R 13 wherein N-heterocycloalkyl contains 1 or 2 nitrogen ring atoms and optionally R 14 substituted with 1, 2, 3 or 4 substituents selected from: R 12 is heterocycloalkyl containing one nitrogen ring atom, where the heterocycloalkyl optionally contains R 14 substituted with 1, 2, 3 or 4 substituents selected from: R 13 is hydrogen, C 1~7 Alkyl or C 3~8 is cycloalkyl; R 14 is hydrogen, C 1~7 Alkyl, Amino, Amino-C 1~7 Alkyl, C 3~8 cycloalkyl and heterocycloalkyl, or two R 14 Let's go together 1~7 Forming alkylene; provided that when A is an N-heterocycloalkyl containing only one nitrogen ring atom, at least one R 14 The substituents are amino or amino-C 1~7 alkyl] is.

[0091] In some embodiments, the compound of formula (III) has the formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, R 1 is hydrogen or C 1~7 is alkyl; R 2 is hydrogen, cyano, C 1~7 Alkyl, C 1~7 Haloalkyl or C 3~8 is cycloalkyl; R 3 is hydrogen, C 1~7 Alkyl or C 3~8 is cycloalkyl; A is an N-heterocycloalkyl containing 1 or 2 nitrogen ring atoms, where the N-heterocycloalkyl optionally contains R 14 substituted with 1, 2, 3 or 4 substituents selected from: R 14 is hydrogen, C 1~7 Alkyl, Amino, Amino-C 1~7 Alkyl, C 3~8cycloalkyl and heterocycloalkyl, or two R 14 Let's go together 1~7 Forming alkylene; provided that when A is an N-heterocycloalkyl containing only one nitrogen ring atom, at least one R 14 The substituents are amino or amino-C 1~7 alkyl] is.

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

[0093] In some embodiments, R 2 is hydrogen or C 1~7 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 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 13 wherein N-heterocycloalkyl contains 1 or 2 nitrogen ring atoms and optionally R 14 substituted with 1, 2, 3 or 4 substituents selected from: R 12 is heterocycloalkyl containing one nitrogen ring atom, where the heterocycloalkyl optionally contains R 14 substituted with 1, 2, 3 or 4 substituents selected from: R 13 But hydrogen, C 1~7 Alkyl or C 3~8 is cycloalkyl; R 14 But hydrogen, C 1~7 Alkyl, Amino, Amino-C 1~7 Alkyl, C 3~8 cycloalkyl and heterocycloalkyl, or two R 14 Let's go together 1~7 Forming alkylene; provided that when A is an N-heterocycloalkyl containing only one nitrogen ring atom, at least one R 14 The substituents are amino or amino-C 1~7 It is alkyl.

[0096] In some embodiments, R 12 If necessary, R 14 and piperidinyl substituted with 1, 2, 3 or 4 substituents selected from:

[0097] In some embodiments, A has the formula: [ka] [In the formula, X is N or CH; R 4 is hydrogen, C 1~7 Alkyl or -(CH2) m -NR 9 R 10 and; R 5 is hydrogen or C 1~7 is alkyl; R 6 is hydrogen or C 1~7 is alkyl; R 7 is hydrogen or C 1~7 is alkyl; R 8 is hydrogen or C 1~7 is alkyl; R 9 and R10 is hydrogen, C 1~7 Alkyl and C 3~8 independently selected from cycloalkyl; R 13 is hydrogen, C 1~7 Alkyl or C 3~8 is cycloalkyl; n is 0, 1 or 2; m is 0, 1, 2 or 3; Alternatively, R 4 and R 5 Let's go together 1~7 Forming alkylene; Alternatively, R 4 and R 7 Let's go together 1~7 Forming alkylene; Alternatively, R 5 and R 6 Let's go together 2~7 Forming alkylene; Alternatively, R 5 and R 7 Let's go together 1~7 Forming alkylene; Alternatively, R 5 and R 9 Let's go together 1~7 Forming alkylene; Alternatively, R 7 and R 8 Let's go together 2~7 Forming alkylene; Alternatively, R 7 and R 9 Let's go together 1~7 Forming alkylene; Alternatively, R 9 and R 10 Let's go together 2~7 Forming alkylene; However, when X is CH, R 4 is -(CH2) m -NR 9 R 10 and; where X is N and R 4 But -(CH2) m -NR 9 R10 , m is 2 or 3] It is of the type.

[0098] In some embodiments, A has the formula: [ka] [In the formula, X is N or CH; R 4 is hydrogen, C 1~7 Alkyl or -(CH2) m -NR 9 R 10 and; R 5 is hydrogen or C 1~7 is alkyl; R 6 is hydrogen or C 1~7 is alkyl; R 7 is hydrogen or C 1~7 is alkyl; R 8 is hydrogen or C 1~7 is alkyl; R 9 and R 10 is hydrogen, C 1~7 Alkyl and C 3~8 independently selected from cycloalkyl; n is 0, 1 or 2; m is 0, 1, 2 or 3; Alternatively, R 4 and R 5 Let's go together 1~7 Forming alkylene; Alternatively, R 4 and R 7 Let's go together 1~7 Forming alkylene; Alternatively, R 5 and R 6 Let's go together 2~7 Forming alkylene; Alternatively, R 5 and R 7 Let's go together 1~7 Forming alkylene; Alternatively, R 5 and R 9 Let's go together 1~7 Forming alkylene; Alternatively, R 7 and R 8 Let's go together 2~7 Forming alkylene; Alternatively, R 7 and R 9 Let's go together 1~7 Forming alkylene; Alternatively, R 9 and R 10 Let's go together 2~7 Forming alkylene; However, when X is CH, R 4 is -(CH2) m -NR 9 R 10 and; where X is N and R 4 But -(CH2) m -NR 9 R 10 , m is 2 or 3] It is of the type.

[0099] In some embodiments, X is N.

[0100] In some embodiments, n is 1.

[0101] In some embodiments, R 6 is hydrogen, methyl or -(CH2) 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 [ka] In some embodiments, A is [ka] In some embodiments, A is [ka] In some embodiments, A is [ka] In some embodiments, A is [ka] In some embodiments, A is [ka] In some embodiments, A is [ka] In some embodiments, A is [ka] In some embodiments, A is [ka] is.

[0106] In some embodiments, A is [ka] is.

[0107] In some embodiments, the substituted pyridazine of formula (III) has the formula: [ka] or a pharmaceutically acceptable salt thereof.

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

[0109] In some embodiments, the small molecule drug that increases SMN function modulates the activity of mRNA decapping enzymes. In some embodiments, the small molecule drug inhibits the activity of mRNA decapping enzymes. In some embodiments, the small molecule drug is a DcpS inhibitor. In some embodiments, the DcpS inhibitor is a C5-substituted 2,4-diaminoquinazoline (2,4-DAQ). In some embodiments, the 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 SMN function is an HDAC inhibitor. In some embodiments, the HDAC inhibitor is a cinnamic acid compound and its derivatives. Non-limiting examples of cinnamic acid compounds that act as HDAC inhibitors are described in U.S. 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 indan derivative. Non-limiting examples of hydroxamic acid indan derivatives that act as HDAC inhibitors are described in WO 2017 / 218,950, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is a 3-spiro-7-hydroxamic acid tetralin. Non-limiting examples of 3-spiro-7-hydroxamic acid tetralins that act as HDAC inhibitors are described in WO 2016 / 168660, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is a 3-alkylbicyclic [4,5,0] hydroxamic acid. Non-limiting examples of 3-alkylbicyclic [4,5,0] hydroxamic acids that act as HDAC inhibitors are described in International Publication No. WO 2016 / 126722, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is a fused pyrimidine hydroxamate derivative. Non-limiting examples of fused pyrimidine hydroxamate derivatives that act as HDAC inhibitors are described in U.S. 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 tetrahydroindoles and tetrahydroindazoles that act as HDAC inhibitors are described in International Publication No. WO 2009114470. A2, the contents of which are incorporated herein by reference. In some embodiments, the HDAC inhibitor is a benzimidazole. Non-limiting examples of benzimidazoles that act as HDAC inhibitors are described in WO 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 contents of which are 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 WO 2010 / 028193, the contents of which are 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 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 International Publication No. 2006 / 101456 and U.S. Patent Application Publication No. 2010 / 0261710, the contents of each 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 contents of each 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 WO 2006 / 101454, the contents of which are 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 contents of which are 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 contents of which are 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 contents of which are 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 contents of which are incorporated herein by reference.

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

[0113] Other non-limiting examples of small molecule drugs that are HDAC inhibitors are described in the following, the contents of each of which are incorporated herein by reference: WO 2018 / 165520, U.S. Patent Application Publication No. 2017 / 0050984, U.S. Patent Application Publication No. 2007 / 0219244, U.S. Patent Application Publication No. 2017 / 0305900, U.S. Patent Application Publication No. 2017 / 0224684 A1, U.S. Patent Application Publication No. 2008 / 0312175, WO 2018 / 129533, WO 2018 / 119362, WO 2018 / 017858, WO 2018 / 009531, WO 2017 / 004522, U.S. Patent Application Publication No. 2018 / 0057 456, International Publication No. 2016 / 020369, International Publication No. 2014 / 143666, Japanese Patent No. 6336562, U.S. Patent Application Publication No. 2011 / 0300134, U.S. Patent Application Publication No. 2011 / 0218221, U.S. Patent No. 8,008,344, European Patent Application Publication No. 2045247, Japanese Patent Application Publication No. 2009507829 No. 102271668, U.S. Patent No. 9,855,267, U.S. Patent Application Publication No. 2018 / 0362472, U.S. Patent Application Publication No. 2017 / 0349573, Japanese Patent No. 5838157, International Publication No. 2019 / 007836, Taiwanese Patent Application Publication No. 200911230, Australian Patent Application No. 2007 / 21678.

[0114] Exemplary HDAC inhibitors also 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 artificially created (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, heterocyclic rings, and the like). 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 the 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 are also possible (e.g., at least about 200 g / mol and at most about 500 g / mol). In certain embodiments, the small molecule is a therapeutically active 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 (CFR)). The small molecule may also be complexed 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, and more preferably humans. In certain embodiments, the small molecule is a drug. Preferably, but not necessarily, the drug is one that has already been deemed safe and effective for use in humans or animals by an appropriate governmental or regulatory agency. For example, drugs approved for human use are listed by the FDA under 21 CFR §§ 330.5, 331-361, and 440-460, which are incorporated herein by reference, and drugs for veterinary use are listed by the FDA under 21 CFR §§ 500-589, which are incorporated herein by reference. All listed drugs are considered acceptable for use according to the present invention.

[0116] Definitions of certain functional groups and chemical terms are described in more detail below. Chemical elements are defined in Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. The functional groups are generally defined as described therein, and are identified according to the Periodic Table of the Elements, inside cover. In addition, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in detail 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 rdEdition, Cambridge University Press, Cambridge, 1987.

[0117] The compounds described herein may contain one or more asymmetric centers and therefore may exist in various stereoisomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be isolated from 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 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, EL See Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., University 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 a form that involves the formal migration of at least one hydrogen atom and at least one change in valence (e.g., the transition from a single bond to a double bond). Tautomerization refers to two or more interconvertible compounds resulting from a double bond, a triple bond and a 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 tautomeric pair) may be catalyzed by acids or bases. The compounds described herein may include one or more tautomeric forms and thus may exist as tautomers.

[0119] Exemplary tautomerizations include keto-enol, amide-imide, lactam-lactim, enamine-imine, and enamine-(different enamine) tautomerizations. For example, keto-enol tautomerizations can include: [ka]

[0120] In the formula, the bond [ka] is a single bond, and the dashed line [ka] is a single bond or is absent, and [ka] is a single or double bond.

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

[0122] When a range of values ​​("range") is listed, it is intended to encompass each value and subrange within the range. A range includes the two endpoints of the range unless otherwise stated. For example, "C1~6 "Alkyl" refers to C1, C2, C3, C4, C5, C6, 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 is intended to be included.

[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 the radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms ("C 1~20 In some embodiments, the alkyl group has 1 to 12 carbon atoms ("C 1~12 In some embodiments, the alkyl group has 1 to 10 carbon atoms ("C 1~10 In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C 1~9 In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C 1~8 In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C 1~7 In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C 1~6 In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C 1~5 In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C 1~4 In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C 1~3In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C 1~2 In some embodiments, the alkyl group has 1 carbon atom ("C 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 (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tert-amyl), and hexyl (C6) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), n-dodecyl (C9), and hexyl (C10). 12 ) and the like. Unless otherwise specified, each example of an alkyl group is independently unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents (e.g., halogen, such as F). In certain embodiments, an alkyl group is an unsubstituted C 1~12 Alkyl (unsubstituted C 1~6 Alkyl, for example, -CH3(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 a substituted C 1~12 Alkyl (substituted C 1~6 alkyl, 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, such as 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, such as fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkyl moiety has 1 to 12 carbon atoms ("C 1~12 In some embodiments, the haloalkyl moiety has 1 to 10 carbon atoms ("C 1~10 In some embodiments, the haloalkyl moiety has 1 to 9 carbon atoms ("C 1~9 In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms ("C 1~8 In some embodiments, the haloalkyl moiety has 1 to 7 carbon atoms (“C 1~7 In some embodiments, the haloalkyl moiety has 1 to 6 carbon atoms ("C 1~6 In some embodiments, the haloalkyl moiety has 1 to 5 carbon atoms ("C 1~5 In some embodiments, the haloalkyl moiety has 1 to 4 carbon atoms (“C 1~4 In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms (“C 1~3 In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C 1~2 In some embodiments, all of the hydrogen atoms of a haloalkyl are independently replaced with fluoro to provide a "perfluoroalkyl" group. In some embodiments, all of the hydrogen atoms of a haloalkyl are independently replaced with chloro to provide a "perchloroalkyl" group. Examples of haloalkyl groups include -CHF, -CHF, -CF, -CHCF, -CFCF, -CFCF, -CFCFCF, -CCl, -CFCl, -CFCl, and the like.

[0126] The term "heteroalkyl" refers to an alkyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within the parent chain (e.g., inserted between adjacent carbon atoms thereof) and / or disposed at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having 1 to 12 carbon atoms and one or more heteroatoms within the parent chain ("heteroC 1~12 In some embodiments, heteroalkyl groups are saturated groups having 1 to 11 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~11 In some embodiments, heteroalkyl groups are saturated groups having 1 to 10 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~10 In some embodiments, heteroalkyl groups are saturated groups having 1 to 9 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~9 In some embodiments, heteroalkyl groups are saturated groups having 1 to 8 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~8 In some embodiments, heteroalkyl groups are saturated groups having 1 to 7 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~7 In some embodiments, heteroalkyl groups are saturated groups having 1 to 6 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1~6 In some embodiments, heteroalkyl groups are saturated groups having 1 to 5 carbon atoms and 1 or 2 heteroatoms in the parent chain ("heteroC 1~5 In some embodiments, heteroalkyl groups are saturated groups having 1 to 4 carbon atoms and 1 or 2 heteroatoms in the parent chain ("heteroC 1~4 In some embodiments, heteroalkyl groups are saturated groups having 1 to 3 carbon atoms and 1 heteroatom in the parent chain ("heteroC 1~3In some embodiments, heteroalkyl groups are saturated groups having 1 to 2 carbon atoms and 1 heteroatom in the parent chain ("heteroC 1~2 In some embodiments, heteroalkyl groups are saturated groups having 1 carbon atom and 1 heteroatom ("heteroC1 alkyl"). In some embodiments, heteroalkyl groups are saturated groups having 2 to 6 carbon atoms and 1 or 2 heteroatoms in the parent chain ("heteroC 2~6 Unless otherwise specified, each example of a heteroalkyl group is independently unsubstituted (an "unsubstituted heteroalkyl") or substituted with one or more substituents (a "substituted heteroalkyl"). In certain embodiments, a heteroalkyl group is an unsubstituted heteroC 1~12 In certain embodiments, the heteroalkyl group is a substituted heteroC 1~12 It is alkyl.

[0127] The term "alkenyl" refers to the 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, an alkenyl group has 1 to 12 carbon atoms ("C 1~12 In some embodiments, an alkenyl group has 1 to 11 carbon atoms ("C 1~11 In some embodiments, an alkenyl group has 1 to 10 carbon atoms ("C 1~10 In some embodiments, an alkenyl group has 1 to 9 carbon atoms ("C 1~9 In some embodiments, an alkenyl group has 1 to 8 carbon atoms ("C 1~8 In some embodiments, an alkenyl group has 1 to 7 carbon atoms ("C 1~7 In some embodiments, an alkenyl group has 1 to 6 carbon atoms ("C 1~6 In some embodiments, an alkenyl group has 1 to 5 carbon atoms ("C 1~5In some embodiments, an alkenyl group has 1 to 4 carbon atoms ("C 1~4 In some embodiments, an alkenyl group has 1 to 3 carbon atoms ("C 1~3 In some embodiments, an alkenyl group has 1 to 2 carbon atoms ("C 1~2 In some embodiments, the alkenyl group has one carbon atom ("C1 alkenyl"). The 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 alkenyl groups include methylidenyl (C1), ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. 1~6 Examples of alkenyl groups include the above-mentioned C 2~4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents ("substituted alkenyl"). In certain embodiments, an alkenyl group is an unsubstituted C 1~12 In certain embodiments, the alkenyl group is a substituted C 1~12 Alkenyl groups have a C=C double bond with no defined stereochemistry (e.g., -CH=CHCH3 or [ka] ) can be in the (E)- or (Z)-configuration.

[0128] The term "heteroalkenyl" refers to an alkenyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within the parent chain (e.g., inserted between adjacent carbon atoms thereof) and / or disposed at one or more terminal positions of the parent chain. In certain embodiments, a 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 ("heteroalkenyl"). 1~12 In certain embodiments, a heteroalkenyl group refers to a group having 1 to 11 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 1~11 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 in the parent chain ("heteroC 1~10 In some embodiments, heteroalkenyl groups have 1 to 9 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 1~9 In some embodiments, heteroalkenyl groups have 1 to 8 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 1~8 In some embodiments, heteroalkenyl groups have 1 to 7 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 1~7 In some embodiments, heteroalkenyl groups have 1 to 6 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 1~6 In some embodiments, heteroalkenyl groups have 1 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 1~5 In some embodiments, heteroalkenyl groups have 1 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 1~4In some embodiments, heteroalkenyl groups have 1 to 3 carbon atoms, at least one double bond, and one heteroatom in the parent chain ("heteroC 1~3 In some embodiments, heteroalkenyl groups have 1 to 2 carbon atoms, at least one double bond, and one heteroatom in the parent chain ("heteroC 1~2 In some embodiments, heteroalkenyl groups have 1 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 1~6 Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an "unsubstituted heteroalkenyl") or substituted (a "substituted heteroalkenyl") with one or more substituents. In certain embodiments, a heteroalkenyl group is an unsubstituted heteroC 1~20 In certain embodiments, the heteroalkenyl group is a substituted heteroC 1~20 It is alkenyl.

[0129] The term "alkynyl" refers to the radical of a straight or branched chain hydrocarbon group having from 1 to 10 carbon atoms ("C 1~10 In some embodiments, an alkynyl group has 1 to 9 carbon atoms ("C 1~9 In some embodiments, an alkynyl group has 1 to 8 carbon atoms ("C 1~8 In some embodiments, an alkynyl group has 1 to 7 carbon atoms ("C 1~7 In some embodiments, an alkynyl group has 1 to 6 carbon atoms ("C 1~6 In some embodiments, an alkynyl group has 1 to 5 carbon atoms ("C 1~5 In some embodiments, an alkynyl group has 1 to 4 carbon atoms ("C 1~4 In some embodiments, an alkynyl group has 1 to 3 carbon atoms ("C 1~3 In some embodiments, an alkynyl group has 1 to 2 carbon atoms ("C 1~2In some embodiments, the alkynyl group has one carbon atom ("C1 alkynyl"). The one or more carbon-carbon triple bonds can be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of C1-4 alkynyl groups include, but are not limited to, methylidinyl (C1), ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. C 1~6 Examples of alkynyl groups include the aforementioned C 2~4 Alkynyl groups include pentynyl (C), hexynyl (C), and the like. Additional examples of alkynyl include heptynyl (C), octynyl (C), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents (a "substituted alkynyl").

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

[0131] The term "carbocyclyl" or "carbocyclic" refers to a ring system having 3 to 10 ring carbon atoms ("C 3~10 "Carbocyclyl" refers to the radical of a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms ("C 3~10 In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms ("C 3~8 In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms ("C 3~7 In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C3~6 In some embodiments, the carbocyclyl group has 4 to 6 ring carbon atoms ("C 4~6 In some embodiments, the carbocyclyl group has 5 to 6 ring carbon atoms ("C 5~6 In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms ("C 5~10 carbocyclyl). Exemplary C 3~6 Carbocyclyl groups include cyclopropyl (C), cyclopropenyl (C), cyclobutyl (C), cyclobutenyl (C), cyclopentyl (C), cyclopentenyl (C), cyclohexyl (C), cyclohexenyl (C), cyclohexadienyl (C), and the like. 3~8 As the carbocyclyl group, the aforementioned C 3~6 Included are carbocyclyl groups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C 3~10 As the carbocyclyl group, the aforementioned C 3~8 Carbocyclyl groups, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ) and other examples. 3~8 As the carbocyclyl group, the aforementioned C 3~10As the foregoing examples illustrate, in certain embodiments, carbocyclyl groups are either monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing fused, bridged, or spiro ring systems, such as bicyclic systems ("bicyclic carbocyclyl") or tricyclic systems ("tricyclic carbocyclyl")) and can be saturated or contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems in which a carbocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, and the point of attachment is on the carbocyclyl ring; in such cases, the number of carbons still refers to the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an "unsubstituted carbocyclyl") or substituted with one or more substituents (a "substituted carbocyclyl"). In certain embodiments, a carbocyclyl group is an unsubstituted C 3~10 In certain embodiments, the carbocyclyl group is a substituted C 3~10 In some embodiments, the cycloalkyl group has 3 to 10 ring carbon atoms ("C 3~10 In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C 3~8 In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ("C 3~6 In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms ("C 4~6 In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms ("C 5~6 In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms ("C 5~10 Cycloalkyl). C 5~6 Examples of cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). 3~6 Examples of cycloalkyl groups include the aforementioned C 5~6Cycloalkyl groups include cyclopropyl (C3) and cyclobutyl (C4). 3~8 Examples of cycloalkyl groups include the aforementioned C 3~6 Cycloalkyl groups include cycloalkyl groups, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each example of a carbocyclyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, a cycloalkyl group is an unsubstituted C 3~14 In certain embodiments, the cycloalkyl group is a substituted C 3~14 In certain embodiments, a carbocyclyl contains, where valences allow, 0, 1, or 2 C=C double bonds in the carbocyclic ring system.

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

[0133] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur ("5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur ("5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur ("5- to 6-membered heterocyclyl"). In some embodiments, a 5- to 6-membered heterocyclyl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, a 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 one 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 include azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-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-nanaphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, Examples include 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, and 1,2,3,4-tetrahydro-1,6-naphthyridinyl.

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

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

[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 the cyclic arrangement) having ring carbon atoms and 1 to 4 heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 14-membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon atom or a nitrogen atom, valence permitting. Heteroaryl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more carbocyclyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring; in such cases, the number of ring members still refers to the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which the heteroaryl ring defined above is fused with one or more aryl groups, and the point of attachment is on either the aryl ring or the heteroaryl ring; in such cases, the number of ring members refers to the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. In polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, for example, on a ring containing a heteroatom (e.g., 2-indolyl) or on a ring without a heteroatom (e.g., 5-indolyl). In certain embodiments, the 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, the heteroaryl is a substituted or unsubstituted 9- or 10-membered bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur.

[0138] In some embodiments, heteroaryl groups are 5- to 10-membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 10-membered heteroaryl"). In some embodiments, heteroaryl groups are 5- to 8-membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 8-membered heteroaryl"). In some embodiments, heteroaryl groups are 5- to 6-membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 6-membered heteroaryl"). In some embodiments, 5- to 6-membered heteroaryls have 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, 5- to 6-membered heteroaryls have 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has one ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("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, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, 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, where the point of attachment is on the alkyl portion.

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

[0142] The terms "unsaturated" or "partially unsaturated" refer to a moiety that contains at least one double or triple bond.

[0143] The terms "saturated" or "fully saturated" refer to a moiety that contains no double or triple bonds, for example, a moiety that contains only single bonds.

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

[0145] Groups are optionally substituted unless expressly stated otherwise. The term "optionally substituted" refers to being substituted or unsubstituted. In certain embodiments, alkyl groups, alkenyl groups, alkynyl groups, heteroalkyl groups, heteroalkenyl groups, heteroalkynyl groups, carbocyclyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups are optionally substituted. "Optionally substituted" refers to a group that can be substituted or unsubstituted (e.g., a "substituted" or "unsubstituted" alkyl group, a "substituted" or "unsubstituted" alkenyl group, a "substituted" or "unsubstituted" alkynyl group, a "substituted" or "unsubstituted" heteroalkyl group, a "substituted" or "unsubstituted" heteroalkenyl group, a "substituted" or "unsubstituted" heteroalkynyl group, a "substituted" or "unsubstituted" carbocyclyl group, a "substituted" or "unsubstituted" heterocyclyl group, a "substituted" or "unsubstituted" aryl group, or a "substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted" means that at least one hydrogen atom present in a group is replaced with an acceptable substituent, e.g., a substituent that, upon substitution, results in a stable compound, e.g., a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group; when more than one position in any given structure is substituted, the substituents are either the same or different at each position. The term "substituted" is intended to include substitution with all acceptable substituents of organic compounds, including any of the substituents described herein that result in the formation of a stable compound. The present invention contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms, such as nitrogen, may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result 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 carbon atom substituents include halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -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 , -CO2H, -CHO, -C(OR cc )2, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R 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 SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R 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 アルキル、C 1~20 パーハロアルキル、C 1~20 アルケニル、C 1~20Alkynyl, Hetero C 1~20 Alkyl, Hetero C 1~20 Alkenyl, Hetero C 1~20 Alkynyl, C 3~10 Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 and aryl and 5- to 14-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd substituted with a group; - is the counterion; Alternatively, two geminal hydrogens on a carbon atom can be bonded to the groups =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb or =NOR cc Replaced by; Each R aa Examples of, independently, 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 Carbocyclyl, 3-14 membered heterocyclyl, C 6~14 aryl and 5- to 14-membered heteroaryl, or two R aa The groups, when joined, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd substituted with a group; Each Rbb Examples of - are, independently, hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR 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 Carbocyclyl, 3-14 membered heterocyclyl, C 6~14 aryl and 5- to 14-membered heteroaryl, or two R bb The groups, when joined, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd substituted with a group; Each R cc Examples of are independently hydrogen, C 1~20 Alkyl, C 1~20 Perhaloalkyl, C 1~20 Alkenyl, C 1~20 Alkynyl, Hetero C1~20 Alkyl, Hetero C 1~20 Alkenyl, Hetero C 1~20 Alkynyl, C 3~10 Carbocyclyl, 3-14 membered heterocyclyl, C 6~14 aryl and 5- to 14-membered heteroaryl, or two R cc The groups, when joined, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd substituted with a group; Each R dd Examples of are, independently, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -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 , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R 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(Rff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R 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, Hetero C 1~10 Alkyl, Hetero C 1~10 Alkenyl, Hetero C 1~10 Alkynyl, C 3~10 Carbocyclyl, 3- to 10-membered heterocyclyl, C 6~10 aryl, and 5- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg substituted with a group or two geminal R dd The substituents may be linked to form =O or =S; - is the counterion; Each R ee Examples of, independently, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 1~10 Alkenyl, C 1~10 Alkynyl, Hetero C1~10 Alkyl, Hetero C 1~10 Alkenyl, Hetero C 1~10 Alkynyl, C 3~10 Carbocyclyl, C 6~10 aryl, 3- to 10-membered heterocyclyl, and 3- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg substituted with a group; Each R ff Examples of are, independently, 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 Carbocyclyl, 3- to 10-membered heterocyclyl, C 6~10 aryl and 5- to 10-membered heteroaryl, or two R ff The groups, when joined, form a 3- to 10-membered heterocyclyl ring or a 5- to 10-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R gg substituted with a group; Each R gg Examples of are, independently, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1~6 Alkyl, -ON(C 1~6 alkyl)2, -N(C 1~6 alkyl)2, -N(C 1~6 Alkyl)3 + X - , -NH(C 1~6 alkyl)2 + X - , -NH2(C 1~6 alkyl)+ X - , -NH3 + X - , -N(OC 1~6 Alkyl)(C 1~6 alkyl), -N(OH)(C 1~6 alkyl), -NH(OH), -SH, -SC 1~6 Alkyl, -SS(C 1~6 alkyl), -C(=O)(C 1~6 alkyl), -CO2H, -CO2(C 1~6 alkyl), -OC(=O)(C 1~6 alkyl), -OCO2(C 1~6 alkyl), -C(=O)NH2, -C(=O)N(C 1~6 alkyl)2, -OC(=O)NH(C 1~6 alkyl), -NHC(=O)(C 1~6 alkyl), -N(C 1~6 alkyl)C(=O)(C 1~6 alkyl), -NHCO2(C 1~6 alkyl), -NHC(=O)N(C 1~6 alkyl)2, -NHC(=O)NH(C 1~6 alkyl), -NHC(=O)NH2, -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)NH2, -OC(=NH)N(C 1~6 alkyl)2, -OC(NH)NH(C 1~6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1~6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1~6 alkyl), -SO2N(C 1~6 alkyl)2, -SO2NH(C 1~6 alkyl), -SO2NH2, -SO2C 1~6 Alkyl, -SO2OC 1~6 Alkyl, -OSO2C 1~6 Alkyl, -SOC 1~6Alkyl, -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)NH2, -C(=O)S(C 1~6 alkyl), -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 Carbocyclyl, C 6~10 aryl, 3- to 10-membered heterocyclyl, or 5- to 10-membered heteroaryl; or two geminal R gg The substituents can be linked to form =O or =S; and Each X - is the counter ion.

[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, -NO2, -C(=O)R aa , -CO2R aa , -C(=O)N(R bb )2, -OC(=O)R aa , -OCO2R 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 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, -NO2, -C(=O)R aa , -CO2R aa , -C(=O)N(R bb )2, -OC(=O)R aa , -OCO2R 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, where R aa is hydrogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 alkyl, an oxygen protecting group when attached to an oxygen atom (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl), or a sulfur protecting group when attached to a sulfur atom (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridinesulfenyl, 2-pyridinesulfenyl, or triphenylmethyl); each R bb are independently hydrogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 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 , -SRaa , -N(R bb )2, -CN, -SCN, or -NO2. 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 or -NO2, wherein R aa is hydrogen, substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 alkyl, an oxygen protecting group when attached to an oxygen atom (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl), or a sulfur protecting group when attached to a sulfur atom (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridinesulfenyl, 2-pyridinesulfenyl, or triphenylmethyl); each R is independently hydrogen, a substituted (e.g., substituted with one or more halogens) or unsubstituted C 1~10 It is an alkyl or nitrogen protecting group (eg, Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts).

[0148] In certain embodiments, the molecular weight of the carbon atom substituent 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 carbon atom substituent is composed of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, nitrogen, and / or silicon atoms. In certain embodiments, the carbon atom substituent is composed of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, and / or nitrogen atoms. In certain embodiments, the carbon atom substituent is composed of carbon, hydrogen, fluorine, chlorine, bromine, and / or iodine atoms. In certain embodiments, the carbon atom substituent is composed of carbon, hydrogen, fluorine, chlorine, bromine, and / or iodine 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 an -OH group. The term "substituted hydroxyl" or "substituted hydroxyl" in turn refers to a hydroxyl group in which the oxygen atom directly attached to the parent molecule has been replaced with a group other than hydrogen, such as -OR aa , -ON(R bb )2, -OC(=O)SR aa , -OC(=O)R aa , -OCO2R 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 , -OSO2R 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, wherein X - , R aa , R bb and R cc is as defined herein.

[0151] The term "thiol" or "thio" refers to an -SH group. The term "substituted thiol" or "substituted thio" in turn refers to a thiol group in which the sulfur atom directly attached to the parent molecule has been replaced with a group other than hydrogen, such as -SRaa , -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 wherein R aa and R cc is as defined herein.

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

[0153] The term "monosubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced with one hydrogen and one non-hydrogen group, and includes -NH(R bb ), -NHC(=O)R aa , -NHCO2R aa , -NHC(=O)N(R bb )2, -NHC(=NR bb )N(R bb )2, -NHSO2R aa , -NHP(=O)(OR cc )2 and -NHP(=O)(N(R bb )2)2)2, wherein R aa , R bb and R cc is as defined herein, and —NH(R bb ) group R bb is not hydrogen.

[0154] The term "disubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced with two groups other than hydrogen, such as -N(R bb )2, -NR bb C(=O)R aa , -NRbb CO2R aa , -NR bb C(=O)N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -NR bb SO2R aa , -NR bb P(=O)(OR cc )2 and -NR bb P(=O)(N(R bb )2)2)2, wherein R aa , R bb and R cc is as defined herein, with the proviso that the nitrogen atom directly attached to the parent molecule is not replaced with a hydrogen.

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

[0156] The term "sulfonyl" refers to -SO2N(R bb )2, -SO2R aa and -SO2OR aa wherein R aa and R bb is as defined herein.

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

[0158] The term "acyl" refers to a group having the general formula -C(=O)R X1 , -C(=O)OR X1 , -C(=O)-OC(=O)R X1 , -C(=O)SRX1 , -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 , and -C(=NR X1 )N(R X1 )2, wherein R X1 is 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 or two R are substituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphatic amino, mono- or di-heteroaliphatic amino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or diarylamino, or mono- or di-heteroarylamino; or two R are substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphatic amino, mono- or di-heteroaliphatic amino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or diarylamino, or mono- or di-heteroarylamino; X1The groups taken together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-COH), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. 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, azido, nitro, hydroxyl, thiol, halo, aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, etc., each of which may or may not be further substituted).

[0159] The term "carbonyl" refers to the carbon directly bonded to the parent molecule. 2 Groups that are hybridized and substituted with oxygen, nitrogen or sulfur atoms, such as ketones (-C(=O)R aa ), carboxylic acid (-CO2H), aldehyde (-CHO), ester (-CO2R aa , -C(=O)SR aa , -C(=S)SR aa ), amide (-C(=O)N(R bb )2, -C(=O)NR bb SO2R aa , -C(=S)N(R bb )2) and imine (-C(=NR bb )R aa , -C(=NR bb ) OR aa ), -C(=NR bb )N(R bb ) 2) wherein R aa and Rbb is as defined herein.

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

[0161] The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of this invention and that are typically not biologically or otherwise undesirable. In many cases, the compounds of this invention are capable of forming acid and / or base salts by virtue of 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, such as acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, glucose, etc. The salts may be octadecanoate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate and trifluoroacetate.

[0163] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0164] Organic acids from which salts can 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, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, etc. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0165] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I-XII of the periodic table. In certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper, with particularly preferred salts including ammonium, potassium, sodium, calcium, and magnesium salts.

[0166] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0167] The pharmaceutically acceptable salts of the present disclosure can be synthesized from the basic or acidic moiety of the parent compound by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of an appropriate base (e.g., hydroxide, carbonate, bicarbonate, etc. of Na, Ca, Mg, or K), 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 or an organic solvent, or a mixture of the two. Generally, the use of non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable, if available. Further lists 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] Recombinant nucleic acid encoding SMN1 In some embodiments, a combination therapy for treating SMA includes administration (e.g., concurrently or sequentially) of a recombinant nucleic acid encoding SMN1 (e.g., administered in a viral vector such as rAAV) in addition to other therapies described herein (e.g., an SMN2 ASO or a small molecule that increases SMN function). In some embodiments, the recombinant nucleic acid encoding SMN1 (also referred to herein as a recombinant SMN1 gene) comprises the SMN1 gene operably linked to a promoter (e.g., to 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., in 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 an AAV capsid particle.

[0169] In some embodiments, recombinant SMN1 gene is administered to a subject by a viral vector. In some embodiments, recombinant SMN1 gene is administered in a recombinant AAV genome that comprises adjacent AAV inverted terminal repeats (ITRs). Thus, in some embodiments, recombinant viral particles (e.g., rAAV particles) that comprise a gene encoding SMN1 are administered to a subject together with SMN2 ASO.

[0170] Figure 2 provides a non-limiting example of a recombinant viral genome comprising an SMN1 gene operably linked to a promoter. Figure 2 illustrates the SMN1 gene flanked by AAV ITRs. The SMN1 gene comprises a human SMN1 codon-optimized SMN1 open reading frame and is operably linked to the CB7 promoter (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 in 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 aspects of the recombinant SMN1 gene are described in more detail in the following paragraphs.

[0173] SMN1 coding sequence: In some embodiments, a coding sequence (e.g., an SMN1 cDNA sequence) encoding a wild-type human SMN protein 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 Nos. NM_001297715.1; NM_000344.3; NM_022874.2., DQ894095, NM_000344, NM_022874, and BC062723. A non-limiting example of an amino acid sequence for wild-type human SMN protein is provided in UniProtKB / Swiss-Prot:Q16637.1. For other publications that describe SMN1 coding sequences, see, for example, WO2010129021A1 and WO2009151546A2, the entire contents of which are incorporated herein by reference.

[0174] In some embodiments, a coding sequence is provided that encodes a functional SMN protein, hi some embodiments, the amino acid sequence of the functional SMN1 is that of the human SMN1 protein or a sequence that shares 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 the residues in two sequences that are the same when aligned for correspondence. The length of sequence identity comparison can be over the entire length of the genome, preferably the entire length of the gene coding sequence, or a fragment of at least about 500-5000 nucleotides. However, identity over smaller fragments, e.g., 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 desired.

[0177] An "aligned" sequence or "alignment" refers to multiple nucleic acid sequences or protein (amino acid) sequences, often containing corrections for base or amino acid deletions or additions, compared to a reference sequence.

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

[0179] Several 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 can be used. Many algorithms known in the art 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 alignment and percent sequence identity of the regions of optimal overlap between the query sequence and the search sequence. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ provided in GCG version 6.1, incorporated herein by reference, with its default parameters (word size 6 and NOP AM coefficients for the scoring matrix).

[0180] In some embodiments, the modified hSMN1 coding sequence is a codon-optimized sequence that is optimized for expression in a target species.As used herein, a "subject" is a mammal, such as a human, a mouse, a rat, a guinea pig, a dog, a cat, a horse, a cow, a pig, or a non-human primate such as a monkey, a chimpanzee, a baboon, or a 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] Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be carried out using online available methods (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, the entire contents of which are incorporated herein by reference. 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] Many options are available for making the actual codon changes or synthesizing the codon-optimized coding region designed as described herein. Such modifications or synthesis can be performed using standard and routine molecular biological procedures well known to those skilled in the art. In one approach, a series of complementary oligonucleotide pairs spanning the length of the desired sequence, each 80-90 nucleotides in length, are synthesized by standard methods. These oligonucleotide pairs are synthesized so that, upon annealing, they form 80-90 base pair double-stranded fragments containing cohesive ends; for example, each oligonucleotide in the pair is synthesized so that it extends 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region complementary to the other oligonucleotide in the pair. The single-stranded end of each pair of oligonucleotides is designed to anneal to the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal, and then approximately 5-6 of these double-stranded fragments are allowed to anneal together via the cohesive single-stranded ends, which are then ligated together and inserted into a standard bacterial cloning vector, such as Invitrogen. The constructs are then cloned into a TOPO® vector available from Biosciences Biosciences Corporation, Carlsbad, Calif. The constructs are then sequenced by standard methods. Several of these constructs are prepared, consisting of 5-6 80-90 base pair fragments ligated together so that the entire desired sequence is represented in a series of plasmid constructs, i.e., approximately 500 base pair fragments. 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 may also be used (e.g., including commercially available gene synthesis services).

[0184] In some embodiments, the SMN1 cDNA sequence can be synthetically produced in vitro using techniques known in the art, such as PCR-based accurate synthesis of long DNA sequences, as described in Xiong et al., PCR-based accurate synthesis of long DNA sequences, Nature Protocols 1, 791-797 (2006). A method combining duplex asymmetric PCR and overlap extension PCR may be used. A method combining duplex asymmetric PCR and overlap extension PCR is described by Young and Dong, Two-step total gene synthesis method, Nucleic Acids Res. 2004; 32(7): e59. 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; see also Gene Seq. 2012 Apr;6(1):10-21 for the following patents for oligonucleotide synthesis and gene synthesis: U.S. Pat. No. 8,008,005 and U.S. Pat. No. 7,985,565. Each of these documents is incorporated herein by reference. Additionally, kits and protocols for generating DNA via PCR are commercially available. These include, but are not limited to, Taq polymerase; OneTaq® (New England Biolabs); Q5® High-Fidelity This includes the use of polymerases, including DNA Polymerase (New England Biolabs); and GoTaq® G2 Polymerase (Promega). DNA may also be produced from cells transfected with a plasmid containing the hSMN sequence described herein. Kits and protocols are known and commercially available, including, but not limited to, QIAGEN Plasmid Kit; Chargeswitch® Pro Filter Plasmid. Kits (Invitrogen); and GenElute™ Plasmid Kits (Sigma-Aldrich) are available. Other techniques useful herein include sequence-specific isothermal amplification methods that eliminate 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 can also be generated from RNA molecules by amplification through the use of reverse transcriptase (RT), an RNA-dependent DNA polymerase. RT polymerizes a strand of DNA complementary to the original RNA template, called cDNA. This cDNA can then be further amplified by PCR or isothermal methods as outlined above. Custom DNA can also be commercially generated from companies including, but not limited to, GenScript; GENEWIZ®; GeneArt® (Life Technologies); and Integrated DNA Technologies.

[0185] "Functional SMN1" refers to a gene encoding a native SMN protein, or a gene encoding another SMN protein that provides at least about 50%, at least about 75%, at least about 80%, at least about 90%, or approximately the same or more than 100% of the biological activity level of the native survival motor neuron protein, or a naturally occurring variant or polymorphism thereof that is not associated with disease. In addition, the SMN1 homolog SMN2 also encodes the SMN protein, but processes the functional protein less efficiently. Based on the copy number of SMN2, subjects lacking a functional hSMN1 gene demonstrate different degrees of SMA. Therefore, 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 functional SMN has a sequence that is about 95% or more identical to the native protein, or about 97% or more, or about 99% identical at the amino acid level. Such functional SMN proteins may also include naturally occurring polymorphisms. Identity can be determined by preparing an alignment of the sequences and using various algorithms and / or computer programs known in the art or commercially available (e.g., BLAST, ExPASy; ClustalO; FASTA; e.g., using the Needleman-Wunsch algorithm, Smith-Waterman algorithm).

[0187] Percent identity can be readily determined for amino acid sequences spanning the entire length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or peptide fragments 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 can be up to about 700 amino acids. Generally, when referring to "identity," "homology," or "similarity" between two different sequences, the "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., vector), such as naked DNA, phage, transposon, cosmid, episome, etc., that transfers the SMN1 sequence carried therein and is useful for generating viral vectors and / or for delivery to host cells. The selected vector can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods used to generate such constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY stomach.

[0189] In some embodiments, an expression cassette comprising an SMN1 (e.g., hSMN1) nucleic acid sequence(s) is provided. As used herein, "expression cassette" refers to a nucleic acid molecule comprising an SMN1 sequence operably linked to a promoter and optionally including other regulatory sequences. In some embodiments, the expression cassette is packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such expression cassettes for producing viral vectors contain 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 signal is the 5' inverted terminal repeat (ITR) and the 3' ITR. When packaged into an AAV capsid, the ITRs together with the expression cassette are referred to herein 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" particularly 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 in the ribosomes by which an mRNA chain controls the assembly of amino acid sequences to produce proteins or peptides.

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

[0193] In some embodiments, the expression cassette contains a promoter sequence as part of the expression control sequence, e.g., located 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, e.g., 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 promoters. See Hioki et al., Gene Therapy, June 2007, 14(11):872-82, incorporated herein by reference. Other neuron-specific promoters include those for 67 kDa glutamic acid decarboxylase (GAD67), homeobox Dlx5 / 6, glutamate receptor 1 (GluRl), preprotachykinin 1 (Tacl), neuron-specific enolase (NSE), and dopaminergic receptor 1 (Drdla). See, e.g., Delzor et al., Human Gene Therapy Methods. See August 2012, 23(4): 242-254. In another embodiment, the promoter is the GUSb promoter at http: / / www.jci.Org / articles / view / 41615#B30.

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

[0195] In addition to a promoter, the expression cassette and / or vector may contain one or more other appropriate transcription initiation, termination, and 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 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 CNS indications. In some embodiments, the expression cassette contains one or more expression enhancers. In some embodiments, the expression cassette contains two or more expression enhancers. These enhancers may be the same or different from each other. For example, the enhancer may include a CMV immediate-early enhancer. This enhancer may exist in two copies located adjacent to each other. Alternatively, the double copies of the enhancer may be separated by one or more sequences. In yet another embodiment, the expression cassette further contains an intron, such as a chicken beta-actin intron. Other suitable introns include those known in the art and described, for example, in WO 2011 / 126808. In some embodiments, an 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 to stabilize the mRNA. An example of such a sequence is a modified WPRE sequence, which may be engineered 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 both to expression control sequences that are adjacent to a gene of interest and to expression control sequences that act in trans or at a distance to regulate the gene of interest.

[0197] Recombinant viral vectors: In some embodiments, an adeno-associated virus vector is provided that includes an AAV capsid and at least one expression cassette. In some embodiments, the at least one expression cassette includes 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 gene can also include AAV ITR sequences. In some embodiments, the ITRs are derived from an AAV serotype different from the serotype of the capsid protein used to package the rAAV genome. In some embodiments, the ITR sequences are derived from AAV2 or its deleted versions (AITRs), which can be used for convenience and to accelerate regulatory approval. However, ITRs from other AAV sources may also be selected. When the ITRs are derived from AAV2 and the AAV capsid is derived from another AAV source, the resulting vector may be referred to as pseudotyped. Typically, the rAAV vector genome includes the AAV 5' ITR, the SMN1 coding sequence and any regulatory sequences, and the AAV 3' ITR. However, other arrangements of these elements may be suitable. A shortened version of the 5' ITR, called the AITR, has been described in which the D-sequence and 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 more than one serotype. In some embodiments, ITR sequences and plasmids containing 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™). 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 particle comprises 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 including the SMN1 gene and an inverted terminal repeat (ITR) sequence (e.g., a wild-type ITR sequence or an engineered ITR sequence) flanking the expression construct. In some embodiments, the nucleic acid is encapsidated by the viral capsid.

[0200] Thus, in some embodiments, the AAV particle comprises 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 at a ratio of approximately 1:1:10, respectively.

[0201] In some embodiments, recombinant adeno-associated viruses (rAAVs) are AAV DNase-resistant particles with an AAV protein capsid in which a nucleic acid sequence is packaged for delivery to target cells. In some embodiments, the AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. AAV capsids may be selected from those known to those skilled in the art, including their 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, AAVrhlO, AAV5, AAVhull, AAV8DJ, AAVhu32, AAVhu37, AAVpi2, AAVrh8, AAVhu48R3, AAVhu68 and variants thereof. WO2018160585A2, WO2018160582A1, Royo, et al, Brain Res, 2008 January, 1190: 15-22; Petrosyan et al, Gene Therapy, 2014 December, 21(12):991-1000; Holehonnur et al, BMC Neuroscience, 2014, 15:28; and Cearley et al, Mol Ther. 2008 October; each of which is incorporated herein by reference. 16(10): 1710-1718. Other AAV capsids useful herein include AAVrh39, AAVrh20, AAVrh25, AAV10, AAVbb1 and AAVbb2, and their variants. For example, other AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAVrh64R1, AAVrh64R2, AAVrh8, and any variant of known or mentioned AAV or AAV that has not yet been discovered, can be selected as the source for the capsid of AAV viral vector (DNase-resistant viral particle). For example, see US Patent Application Publication No. 2007-0036760A1; US ​​Patent Application Publication No. 2009-0197338A1; European Patent Application Publication No. 1310571. Also see International Publication No. 2003 / 042397 (AAV7 and other monkey AAVs), US Patent No. 7790449 and US Patent No. 7282199 (AAV8), International Publication No. 2005 / 033321 and US Patent No. 7,906,111 (AAV9), and International Publication No. 2006 / 110689, and International Publication No. 2003 / 042397 (rh10). Alternatively, recombinant AAV based on any of the listed AAVs can be used as the origin for AAV capsid.These documents also describe other AAVs that can be selected to produce AAV, and are incorporated herein by reference. In some embodiments, the AAV cap for use in viral vectors can be produced by mutagenesis (for example, by insertion, deletion, or substitution) of one of the above-mentioned AAV Caps or its encoding nucleic acid. In some embodiments, the AAV capsid is a chimera that comprises domains derived from two, three, four, or more of the above-mentioned 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 comprises two or more of the above-mentioned Caps.As used herein, with respect to AAV, the term variant refers to any AAV sequence derived from a known AAV sequence, including 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 more sequence identity across the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes variants that may contain up to about 10% variation with any of the described or known AAV capsid sequences. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with an AAV capsid 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 percent identity of an AAV capsid, 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 AAVs are provided. The abbreviation "sc" in this context refers to self-complementary. "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV assemble to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, DM McCarty et al., "Self-complementary AAVs: A Self-Complementary AAV" (ScAAV). "recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), See Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated by reference herein in its entirety.

[0203] The method for producing and isolating AAV virus vector suitable for delivery to subject is known in the art.For example, refer to US Patent Application Publication No. 2007 / 0036760 (February 15, 2007), US Patent No. 7,790,449; US Patent No. 7,282,199; International Publication No. 2003 / 042397; International Publication No. 2005 / 033321, International Publication No. 2006 / 110689; and US Patent No. 7,588,772 B2.In one system, producer cell line is transiently transfected with the construct that codes for the transgene flanked by ITR and the construct(s) that codes for rep and cap.In the second system, the packaging cell line that stably supplies rep and cap is transiently transfected with the construct that codes for the transgene flanked by ITR. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, requiring the separation of rAAV from contaminating viruses. Systems have also been developed that do not require helper virus infection for AAV recovery, 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 constructs encoding the necessary helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In yet another system, a transgene flanked by ITRs and the rep / cap genes are introduced into insect cells by infection with a baculovirus-based vector.For a review of these production systems, see generally, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entirety: 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] If necessary, the SMN1 gene described herein can be used to produce a viral vector other than rAAV, which can also be used in combination therapy with SMN2 ASO.Such other viral vectors can include any virus suitable for gene therapy, including but not limited to adenovirus; herpesvirus; lentivirus; retrovirus etc.Preferably, when one of these other vectors is produced, it is produced as a replication-defective viral vector.

[0205] A "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 in 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 produce 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 "weak" and contain only the transgene of interest flanked by signals necessary for amplification and packaging of the artificial genome), although these genes can be supplied during production. Thus, they are considered safe for use in gene therapy, as replication and infection by progeny virions cannot occur unless the viral enzymes required for replication are present. Such replication-defective viruses can be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrating or non-integrating), or another suitable viral origin.

[0206] Also provided is a host cell that comprises at least one of the disclosed AAV particles, expression constructs or nucleic acid vectors.Such host cell includes mammalian host cell, for example, human host cell, and can be isolated in either cell culture or tissue culture.In the case of genetically modified animal model (for example, mouse), the host cell that is transformed can be contained in the body of non-human animal itself.

[0207] Oligomeric compounds that increase production of full-length SMN2 mRNA In some embodiments, a combination therapy for treating SMA includes administering (e.g., concomitantly or sequentially) an ASO complementary to the pre-mRNA encoding SMN2 (also referred to in the present application as an SMN2 ASO) in addition to other therapies described herein (e.g., a recombinant SMN1 gene and / or a small molecule that increases SMN function). In some embodiments, the ASO increases full-length SMN2 mRNA. In some embodiments, the ASO alters splicing of SMN2 pre-mRNA. In some embodiments, the ASO promotes exon 7 inclusion in SMN2 mRNA. Some sequences and regions useful for altering splicing of SMN2 can be found in PCT / US06 / 024469 (published as WO 2007 / 002390) and WO 2018014041 A2, which are incorporated by reference in their entireties for any purpose.

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

[0209] ASO that effectively modulates the expression of SMN2 mRNA to produce functional SMN protein is considered to be active ASO. The modulation of SMN2 expression can be measured in bodily fluids, which may or may not contain animal cells, tissues, or organs. Methods for obtaining samples for analysis, such as bodily fluids (e.g., sputum, serum, CSF), tissues (e.g., biopsies), or organs, and methods for preparing samples to enable analysis are well known to those skilled in the art. The effect of treatment can be evaluated by measuring biomarkers related to target gene expression in one or more biological fluids, tissues, or organs collected from animals that have been contacted with one or more compositions described in the present 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 a reference level, e.g., the level of full-length SMN2 mRNA in a control (e.g., a subject not administered an SMN2 ASO). An 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, an increase in full-length SMN2 mRNA can be determined by examining the external characteristics of a 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 level of full-length SMN2 mRNA in a sample obtained from a subject receiving SMN2 ASO treatment can be compared to the level of full-length SMN2 mRNA in a subject not treated with SMN2 ASO to determine the extent to which the SMN2 ASO has increased full-length SMN2 mRNA. The reference level of mRNA is obtained from the same subject before receiving the SMN2 ASO. In some embodiments, the reference level of full-length SMN2 mRNA is a range determined by a population of subjects who have not received the SMN2 ASO.

[0212] In some embodiments, the increased level of full-length SMN2 mRNA is, for example, greater 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 more, above the reference value.

[0213] In some embodiments, it can be determined whether an SMN2 ASO has resulted in an increase in full-length SMN2 mRNA by comparing the ratio of full-length SMN2 mRNA to a shorter SMN2 mRNA (e.g., SMN2 mRNA without exon 7) in a subject receiving SMN2 ASO administration with a reference ratio. In some embodiments, the reference ratio is the ratio of full-length SMN2 mRNA to a shorter SMN2 mRNA (e.g., SMN2 mRNA without exon 7) before administration of the SMN2 ASO. In some embodiments, the ratio of full-length SMN2 mRNA to short SMN2 mRNA (e.g., SMN2 mRNA without exon 7) in a subject receiving an SMN2 ASO is, for example, greater 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 more, 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 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 with SMA or at risk of having SMA before treatment. In some embodiments, production of exon 7-containing SMN protein is increased in subjects receiving SMN2 ASO administration, with an increase in exon 7-containing SMN protein levels of at least about, e.g., more 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 more, compared to the reference value. Also contemplated are methods of contacting a bodily fluid, organ, or tissue with an effective amount of one or more of the compositions described herein. The bodily fluid, organ, or tissue can be contacted with one or more of the compositions, resulting in modulation of SMN1 expression and SMN2 expression in cells of the bodily fluid, organ, or tissue. An effective amount of the composition is administered to a subject or contacted with cells of recombinant SMN1 and SMN2 genes. This can be determined by monitoring the effect of the ASO on the expression of functional SMN protein.

[0215] 1. Antisense oligonucleotides (ASOs) In some embodiments, ASOs comprising a sequence complementary to a nucleic acid encoding human SMN2 are provided for use in treating diseases or conditions associated with survival motor neuron protein (SMN), such as spinal muscular atrophy (SMA) (e.g., with a recombinant SMN1 gene and / or small molecules that increase SMN function). In some embodiments, ASOs comprising a sequence complementary to a nucleic acid encoding human SMN2 are provided for use in treating diseases or conditions associated with survival motor neuron protein (SMN) (e.g., with a recombinant SMN1 gene and / or small molecules that increase SMN function) by administering the ASO directly to the central nervous system (CNS) or CSF.

[0216] As used herein, the term "oligomeric compound" refers to a compound comprising an oligonucleotide. In some embodiments, an oligomeric compound consists of an oligonucleotide. As used herein, the term "oligonucleotide" refers to a compound comprising a phosphate linking group, a heterocyclic base moiety, and a sugar moiety. In some embodiments, an oligomeric compound further comprises one or more conjugate groups and / or terminal groups. In some embodiments, an 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, an ASO increases full-length SMN2 mRNA in a subject. In some embodiments, an ASO that increases full-length SMN2 mRNA is an antisense oligonucleotide complementary to a nucleic acid encoding SMN2. In some embodiments, an ASO increases full-length SMN2 mRNA by altering the splicing pattern of SMN2 pre-mRNA. In some embodiments, an ASO promotes exon skipping during splicing of SMN2 pre-mRNA. In some embodiments, an ASO promotes the inclusion of exon 7 in SMN2 mRNA. In some embodiments, an ASO is designed to target intron 6, intron 7, or the boundary between exon 7 and an adjacent intron of SMN2 pre-mRNA to promote the inclusion of exon 7 in SMN2 mRNA. In some embodiments, an ASO comprises a nucleobase sequence complementary to intron 6 of SMN2 pre-mRNA. In some embodiments, an 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 level of full-length SMN protein and / or full-length SMN2 mRNA. Non-limiting examples of sequences and regions useful for altering SMN2 splicing can be found in PCT / US06 / 024469, which is incorporated herein by reference in its entirety for any purpose. In some embodiments, the antisense oligonucleotide has a nucleobase sequence complementary to intron 7 of SMN2. Non-limiting examples of such nucleobase sequences are illustrated 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 (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 an equal length portion 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 boundary between exon 7 and the adjacent intron), 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 targeting intron 7 of the SMN2 pre-mRNA is nusinersen. An exemplary nucleotide sequence for nusinersen is 5'-UCACUUUCAUAAUGCUGG-3' (SEQ ID NO: 26). The active substance nusinersen (also referred to as ISIS 396443) 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 The SMN2 ASO is a uniformly modified 2'-O-(2-methoxyethyl) phosphorothioate antisense oligonucleotide consisting of 18 nucleotide residues with UGG-3' (SEQ ID NO: 25). 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-thiouridylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiocytidylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-P-thioadenylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiocytidylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-P-thioadenylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiocytidylyl, which corresponds to the molecular formula C234H323N61O128P17S17Na17. Dilyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiouridylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiouridylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiouridylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiocytidylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl) -P-thioadenylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiouridylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-P-thioadenylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-P-thioadenylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiouridylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)- P-thioguanylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiocytidylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-5-methyl-P-thiouridylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)-P-thioguanylyl-(3'-O→5'-O)-2'-O-(2-methoxyethyl)guanosine, which 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.Provided herein are antisense compounds useful for modulating gene expression through antisense action mechanisms, including target occupancy-based antisense mechanisms.In one aspect, provided herein are antisense compounds that modulate the splicing of target genes.Such modulation includes promoting or inhibiting exon inclusion.Further provided herein are antisense compounds that target cis splicing regulatory elements present in pre-mRNA molecules, including exon splicing enhancers, exon splicing silencers, intron splicing enhancers, and intron splicing silencers.Disruption of cis splicing regulatory elements is thought to change splice site selection, which can result in changes in the composition of splice products.

[0223] Eukaryotic pre-mRNA processing is a complex process requiring numerous signals and protein factors to achieve proper mRNA splicing. Exon definition by the spliceosome requires more than the canonical splicing signals that define intron-exon boundaries. One such additional signal is provided by cis-acting regulatory enhancer and silencer elements. Exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs), and intronic splicing silencers (ISSs) have been identified that either repress or enhance 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. USA). 101(44): 15700-15705). Proteins specific to these regulatory sequences (trans-factors) Binding of β-glucan directs the splicing process, promoting or inhibiting the use of specific splice sites, thus modulating the ratio of spliced ​​products (Scamborova et al. 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, antisense oligonucleotides comprise one or more modifications compared to naturally occurring oligomeric oligonucleotides such as DNA or RNA.Such modified antisense oligonucleotides may have one or more desirable properties.In some embodiments, the modifications change the antisense activity of antisense oligonucleotides, for example, by increasing the affinity of antisense oligonucleotides to their target nucleic acids, increasing their resistance to one or more nucleases, and / or changing the pharmacokinetics or tissue distribution of oligonucleotides.In some embodiments, modified antisense oligonucleotides comprise 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, antisense oligonucleotide comprises one or more modified nucleosides.Such modified nucleosides can comprise modified sugars and / or modified nucleobases.In some embodiments, the incorporation of such modified nucleosides into oligonucleotides leads to increased affinity and / or increased stability for target nucleic acid, including but not limited to, increased resistance to nuclease degradation, and / or improved toxicity and / or uptake properties of modified oligonucleotides.

[0226] i. Nucleobase The base moiety of naturally occurring nucleosides is a heterocyclic base, typically purine and pyrimidine.In addition to "unmodified" or "natural" nucleobases such as purine nucleobases adenine (A) and guanine (G) and pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), 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, modified nucleobases are nucleobases whose structure is similar to that of the parent nucleobase, such as 7-deazapurine, 5-methylcytosine or G-clamp.In some embodiments, nucleobase mimics include more complex structures, such as 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 optionally contain one or more nucleosides with a modified sugar moiety compared to the natural sugar. Oligonucleotides containing sugar-modified nucleosides can have enhanced nuclease stability, increased binding affinity, or some other advantageous biological properties. Such modifications include, but are not limited to, 2'-F-5'-methyl substituted nucleosides (see PCT International Application Publication No. 2008 / 101157, published August 21, 2008, for other disclosed 5',2'-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atom with S and an additional substitution at the 2' position (see U.S. Patent Application Publication No. 200501309, published June 16, 2005). 23) or 5'-substitution of alternative BNAs (LNAs are substituted, for example, with a 5'-methyl or 5'-vinyl group; see PCT International Application Publication No. 2007 / 134181, published November 22, 2007); bridging non-geminal ring atoms to form bicyclic nucleic acids (BNAs); substitution of S, N(R), or C(R)(R) (R = H, C-C) of ribosyl ring oxygen atoms; 12 alkyl or protecting groups), and combinations thereof.

[0228] Examples of nucleosides having modified sugar moieties include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, and 2'-O(CH)OCH substituents. The 2'-position substituent can also be allyl, amino, azido, thio, O-allyl, O-Ci ... 10 Alkyl, OCF3, O(CH2)SCH3, O(CH2)2-ON(R m )(R n ) and O-CH2-C(=O)-N(R m )(R n ) (wherein each R m and R n are independently H or substituted or unsubstituted C1 to C 10 It can also be selected from the group consisting of alkyl.

[0229] Examples of bicyclic nucleic acids (BNAs) include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the antisense compounds provided herein comprise one or more BNA nucleosides in which the bridge comprises one of the following formulas: 4'-beta-D-(CH2)-O-2' (beta-D-LNA); 4'-(CH2)-S-2: 4'-alpha-L-(CH2)-O-2' (alpha-L-LNA); 4'-(CH2)2-O-2' (ENA); 4'-C(CH3)2-O-2' (see PCT / US2008 / 068922); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2' (U.S. Patent No. 7,897,497, issued July 15, 2008). 399,845); 4'-CH2-N(OCH3)-2' (see PCT / US2008 / 064591); 4'-CH2-ON(CH3)-2' (see U.S. Patent Application Publication No. 2004-0171570, published September 2, 2004); 4'-CH2-N(R)-O-2' (see U.S. Patent No. 7,427,672, issued September 23, 2008); 4'-CH2-C(CH3)-2' and 4'-CH2-C(=CH2)-2' (see PCT / US2008 / 066154); where R is independently H, C1-C 12 alkyl, or a protecting group.

[0230] In some embodiments, modified nucleosides containing modified sugar moieties are not bicyclic sugar moieties. In some embodiments, the sugar ring of a 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 C1-C 10 Alkyl or C2-C 10 Alkenyl and C2-C 10 It can be alkynyl. In some embodiments, such a substituent is at the 2' position of the sugar.

[0231] In some embodiments, modified nucleosides include a substituent at the 2' position of the sugar. In some embodiments, such substituents include halide (including but not limited to F), allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ) or O-CH2-C(=O)-N(R m )(R n ) (wherein each R m and R n are independently H or substituted or unsubstituted C1 to C 10 alkyl).

[0232] In some embodiments, modified nucleosides suitable for use in the present invention are 2-methoxyethoxy, 2'-O methyl (2'-O CH3), 2'-fluoro (2'-F).

[0233] In some embodiments, O[(CH) n O] m CH3, O(CH2) n NH2, O(CH2)2CH3, O(CH2) n ONH2, OCH2C(=O)N(H)CH3 and O(CH2) n ON[(CH2) n

[0023] CH3]2, where n and m are from 1 to about 10. Other 2'-sugar substituents include C1 to C 10Included are alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligomeric compounds or groups for improving the pharmacodynamic properties of oligomeric compounds, and other substituents with similar properties.

[0234] In some embodiments, the modified nucleoside contains 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 and other modified nucleosides, such as 2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides with 2'-MOE substituents have also been shown to be antisense inhibitors of gene expression with promising characteristics 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 (up) or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F arabino (FANA). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar of the 3'-terminal nucleoside or 2'-5'-linked oligonucleotide, and the 5' position of the 5'-terminal nucleotide.

[0236] In some embodiments, suitable nucleosides have sugar substitutes, such as cyclobutyl, in place 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; 5,5 Nos. 76,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is incorporated herein by reference in its entirety.

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

[0238] B internucleoside linkage 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), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Oligonucleotides with non-phosphorus linking groups are referred to as oligonucleosides. Modified linkages compared to natural phosphodiester linkages can be used to change, typically increase, the nuclease resistance of oligonucleotides.In some embodiments, linkages with chiral atoms can be prepared as racemic mixtures, as separate enantiomers.Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates.Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

[0239] The antisense oligonucleotides described herein may contain one or more asymmetric centers and thus may give rise to enantiomers, diastereomers, and other stereoisomeric configurations which may be defined in terms of absolute stereochemistry as (R) or (S) for sugar anomers, etc., or (D) or (L) for amino acids, etc. The antisense compounds provided herein may 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 10 modified internucleoside linkages.In some embodiments, each internucleoside linkage of the antisense oligonucleotide is a modified internucleoside linkage.In some embodiments, such modified internucleoside linkage is a phosphorothioate linkage.

[0241] c. Length In some embodiments, the present invention provides the antisense oligonucleotide of any of various ranges of length.In some embodiments, antisense compound or antisense oligonucleotide comprises or consists of the linked nucleoside of XY, wherein 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, and is XY. For example, in some embodiments, the antisense compound or antisense oligonucleotide may be selected from the group consisting of 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, 1 0-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17,14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16 ~23, 16~24, 16~25, 16~26, 16~27, 16~28, 16~29, 16~30, 17~18, 17~19, 17~20, 17~21, 17~22, 17~23, 17~24, 17~25, 17~26, 17~27, 17~28, 17~29, 17~30, 18~19, 18~20, 18~21, 18~22, 18~23, 18~24, 18~25, 18~26, 18~27, 18~28, 18~29, 18~30, 19~20, 19~21 1, 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 linked nucleosides.

[0242] In some embodiments, antisense compounds or antisense oligonucleotides are 15 nucleosides in length. In some embodiments, antisense compounds or antisense oligonucleotides are 16 nucleosides in length. In some embodiments, antisense compounds or antisense oligonucleotides are 17 nucleosides in length. In some embodiments, antisense compounds or antisense oligonucleotides are 18 nucleosides in length. In some embodiments, antisense compounds or antisense oligonucleotides are 19 nucleosides in length. In some embodiments, antisense compounds or antisense oligonucleotides are 20 nucleosides in length.

[0243] d. oligonucleotide motif In some embodiments, antisense oligonucleotides have chemically modified subunits arranged in a specific direction along their length.In some embodiments, antisense oligonucleotides are fully modified.In some embodiments, antisense oligonucleotides are uniformly modified.In some embodiments, antisense oligonucleotides are uniformly modified, and each nucleoside comprises a 2-MOE sugar moiety.In some embodiments, antisense oligonucleotides are uniformly modified, and each nucleoside comprises a 2'-OMe sugar moiety.In some embodiments, antisense oligonucleotides are uniformly modified, and each nucleoside comprises a morpholino sugar moiety.

[0244] In some embodiments, the oligonucleotide comprises 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 comprises a single nucleoside.

[0245] In some embodiments, the oligonucleotide comprises 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 an alternating motif comprises more than a single nucleoside of a certain type. For example, an oligomeric compound may comprise the following nucleoside motif: [Table 2] 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 compounds In some embodiments, oligomeric compounds are composed solely of oligonucleotides.In some embodiments, oligomeric compounds comprise oligonucleotides and one or more conjugate groups and / or terminal groups.Such conjugate groups and / or terminal groups can be added to oligonucleotides having any of the chemical motifs described in this application.Thus, for example, oligomeric compounds comprising oligonucleotides having one or more regions of alternating nucleosides can comprise terminal groups.

[0248] a. conjugate group In some embodiments, oligonucleotides are modified by the attachment of one or more conjugate groups.Generally, conjugate groups modify one or more properties of the attached oligomeric compound, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance.Conjugate groups are routinely used in the chemical arts and are directly linked to parent compounds, such as oligomeric compounds, such as oligonucleotides, or optionally linked via conjugate linking moieties or conjugate linking groups.Conjugate groups may include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin and dyes. Certain conjugate groups have been previously described, for example: cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-27 ... cholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecane-diol 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), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264. 229-237), or octadecylamine moiety or hexylamino-carboxamide moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264. 229-237), The hydroxyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0249] In some embodiments, the conjugate group is a benzodiazepine, benzothiadiazine, benzothiazide ... Oligonucleotide-drug conjugates and their preparation are described in U.S. Patent Application Serial No. 09 / 334,130.

[0250] Representative U.S. patents that teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 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;5,416,203,5,451,463,5,510,47 5; 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. Conjugate groups can be attached to either or both ends of the oligonucleotide (terminal conjugate groups) and / or at any internal position.

[0251] b.Terminal group In some embodiments, the oligomeric compound comprises a terminal group at one or both ends. In some embodiments, the terminal group may comprise any of the conjugate groups described herein. In some embodiments, the terminal group may comprise an additional nucleoside and / or an inverted abasic nucleoside. In some embodiments, the terminal group is a stabilizing group.

[0252] In some embodiments, oligomeric compounds include one or more terminal stabilizing groups that enhance properties such as, for example, nuclease stability. Stabilizing groups include cap structures. The terms "cap structure" or "terminal cap moiety," as used herein, refer to a chemical modification that can be attached to one or both termini of an oligomeric compound. Certain terminal modifications can protect oligomeric compounds having terminal nucleic acid moieties from exonuclease degradation and aid in intracellular delivery and / or localization. The cap can be present at the 5'-end (5'-cap) or 3'-end (3'-cap), or can be present at both ends (for more non-limiting details, see Wincott et al., International PCT Publication No. 97 / 26270; Beaucage and Tyer, 1993, Tetrahedron 49, 1925; U.S. Patent Application Publication No. 2005 / 0020525; and WO 03 / 00444). (See issue 602).

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

[0254] C. oligomeric compound motif In some embodiments, the oligomeric compound comprises the motif: T-(Nu1) n1 ,-(Nu2) n2 -(Nu1) n3 -(Nu2) n4 -(Nu1) n5 -T2 [In the formula, Nu1 is the first type of nucleoside, Nu2 is a second type of nucleoside, Each of n1 and n5 independently represents 0 to 3; The sum of n2 and n4 is between 10 and 25, n3 is 0 to 5, each T1 and T2 is independently H, a hydroxyl protecting group, an optionally linked conjugate group, or a capping group. Includes:

[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, antisense compounds are synthesized under conditions where specific binding is desired (e.g., in An antisense compound specifically hybridizes to a target nucleic acid if there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences (under physiological conditions in the case of an in vivo assay or therapeutic treatment, and under conditions under which the assay is performed in the case of an in vitro assay).

[0259] Therefore, "stringent hybridization conditions" or "stringent conditions" refer to conditions under which an antisense compound hybridizes to a target sequence while minimizing the number of other sequences. Stringent conditions are sequence-dependent and vary under different circumstances, and the "stringent conditions" under which an antisense oligonucleotide hybridizes to a target sequence are determined by the nature and composition of the antisense oligonucleotide and the assay in which it is tested.

[0260] It is understood in the art that incorporating affinity modification of nucleotide can allow more mismatch number compared to unmodified compound.Similarly, certain nucleobase sequences may be more tolerant to mismatch than other nucleobase sequences.Those skilled in the art can determine the appropriate number of mismatch between oligonucleotides or between antisense oligonucleotide and target nucleic acid, for example, by determining melting temperature (Tm).Tm or ATM can be calculated by techniques known to those skilled in the art.For example, the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allows those skilled in the art to evaluate nucleotide modification for its ability to increase the melting temperature of RNA:DNA duplex.

[0261] B pre-mRNA processing In some embodiments, the antisense compounds provided herein are complementary to pre-mRNA.In some embodiments, such antisense compounds change the splicing of pre-mRNA.In some embodiments, the ratio of one variant of the mature mRNA corresponding to target pre-mRNA to another variant of this mature mRNA changes.In some embodiments, the ratio of one variant of the protein expressed from target pre-mRNA to another variant of the protein changes. Certain oligomeric compounds and nucleobase sequences that can be used to alter pre-mRNA splicing are described, for example, in U.S. Pat. No. 6,210,892; U.S. Pat. No. 5,627,274; U.S. Pat. No. 5,665,593; U.S. Pat. No. 5,916,808; U.S. Pat. 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; WO 2007 / 090073; WO 2007 / 047913; Hua et al., PLoS Biol 5(4):e73; Vickers et al., J. Immunol. 2006 Mar. 15; 176(6):3652-61; and Hua et al., American J. of Human Genetics (April 2008) 82, 1-15. and the like, each of which is incorporated herein by reference in its entirety for any purpose. In some aspects, the splicing-altering antisense sequences 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. WO 2018 / 014043 (PCT / US2017 / 042465), International Publication No. WO 2018 / 014042 (PCT / US2017 / 042464), or International Publication No. WO 2018 / 014041 (PCT / US2017 / 042463), the contents of which are incorporated herein in their entireties.

[0263] Administration and Treatment In some embodiments, a "therapeutically effective" amount of a small molecule capable of increasing SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in a viral vector, e.g., in an rAAV), and / or an SMN2 ASO (e.g., nusinersen) is delivered to a subject as described herein (e.g., via parallel or sequential administration) to achieve a desired result, e.g., treatment of SMA or one or more symptoms thereof. In some embodiments, SMA is assessed by clinical symptoms such as weight loss, decreased muscle strength, decreased muscle tone, the presence of scoliosis, tremors or spasms, and / or decreased respiratory health. In some embodiments, SMA is assessed by age- and ability-appropriate motor function scales, as well as electrophysiological measurements of motor unit health. In some embodiments, a subject's motor neuron function is assessed using the Children's Hospital of Philadelphia Infant Neuromuscular Disease Examination (CHOP INTEND) (e.g., Glanzman AM, et al. The ... 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 contents of which regarding CHOP INTEND are incorporated herein by reference. In some embodiments, motor neuron function in subjects with late-onset SMA can be tested using the Expanded Hammersmith Functional Motor Scale (HFMSE) (see, e.g., Glanzman AM et al; the Pediatric Neuromuscular Clinical Research Network for Spinal Muscular Atrophy (PNCR), and the Muscle The Pediatric Neuromuscular Clinical Research Network for SMA. Expanded Hammersmith Functional Motor Scale for SMA (HFMSE). March 7, 2009. The contents of the HFMSE are incorporated herein by reference. In some embodiments, the electrophysiological function of motor neurons is assessed using compound muscle action potential (CMAP) and / or motor unit number estimation (MUNE). The CMAP response is a measure of the electrophysiological output from a specific muscle or muscle group following stimulation of the innervating nerve, as 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 contents of which are incorporated herein by reference. ... CMAP values ​​are decreased in subjects with SMA. In some embodiments, CMAP decreases before physical symptoms appear. Motor unit number estimation (MUNE) is an electrophysiology method for estimating the number of lower motor neurons innervating a group of muscles supplied by a nerve, and is well suited to assessing motor neuron loss in 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 contents of which are incorporated herein by reference. MUNE values ​​are calculated from the ratio of the maximum compound muscle action potential (CMAP) to the mean single motor unit potential (SMUP).

[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 spasms. Other desired endpoints may be determined by a physician.

[0265] In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV) are administered to a subject (e.g., concomitantly and sequentially) to increase body weight. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and an SMN2 gene (e.g., in an rAAV) are administered to a subject (e.g., concomitantly and sequentially) to increase body weight. An ASO (e.g., nusinersen) is administered to a subject (e.g., together and sequentially) to increase body weight. In some embodiments, a small molecule (e.g., risdiplam or branapram) for increasing SMN function, a recombinant SMN1 gene (e.g., in rAAV), and an SMN2 ASO are administered to a subject (e.g., together and sequentially) to increase body weight. In some embodiments, a small molecule (e.g., risdiplam or branapram) for increasing SMN function and a recombinant SMN1 gene (e.g., in rAAV) are administered to a subject (e.g., together and sequentially) to prevent or reduce muscle weakness. In some embodiments, a small molecule (e.g., risdiplam or branapram) for increasing SMN function and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to prevent or reduce muscle weakness. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to prevent or reduce muscle weakness. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV) are administered to a subject (e.g., together and sequentially) to increase muscle strength. In some embodiments, a small molecule for increasing SMN function and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to increase muscle strength. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to increase muscle strength. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene are administered to a subject (e.g., together and sequentially) to increase muscle tone.In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to increase muscle tone. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to increase muscle tone. In some embodiments, a small molecule for increasing SMN function and a recombinant SMN1 gene (e.g., in an rAAV) are administered to a subject (e.g., together and sequentially) to prevent or reduce scoliosis. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to prevent or reduce scoliosis. In some embodiments, a small molecule for increasing SMN function, a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to prevent or reduce scoliosis. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV) are administered to a subject (e.g., together and sequentially) to reduce tremors or spasms. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to reduce tremors or spasms. In some embodiments, a small molecule to increase SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., concomitantly and sequentially) to reduce tremors or spasms.In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV) are administered to a subject (e.g., together and sequentially) to maintain or increase respiratory health. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to maintain or increase respiratory health. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to maintain or increase respiratory health. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV) are administered to a subject (e.g., together and sequentially) to prevent or reduce neuronal loss. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to prevent or reduce neuronal loss. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV) and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to prevent or reduce neuronal loss. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV) are administered to a subject (e.g., together and sequentially) to prevent or reduce motor neuron loss. In some embodiments, a small molecule for increasing SMN function and an SMN2 ASO are administered to a subject (e.g., together and sequentially) to prevent or reduce motor neuron loss.In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to prevent or reduce motor neuron loss. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV) are administered to a subject (e.g., together and sequentially) to improve the scores of either motor neuron function tests and / or electrophysiological tests. In some embodiments, a small molecule for increasing SMN function and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., together and sequentially) to improve the scores of either motor neuron function tests and / or electrophysiological tests. In some embodiments, a small molecule to increase SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) are administered to a subject (e.g., concomitantly and sequentially) to improve scores on either motor neuron function tests and / or electrophysiological tests.

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

[0267] In some cases, SMA is detected in fetuses around 30-36 weeks of gestation. In this situation, it may be desirable to treat the newborn as soon as possible after delivery. It may also be desirable to treat the fetus in utero. Thus, provided are methods for rescuing and / or treating a newborn subject with SMA, comprising administering (e.g., simultaneously or sequentially) a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV), or a small molecule for increasing SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen), or a small molecule for increasing SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) to neuronal cells of a fetus and / or newborn subject (e.g., a human fetus and / or newborn). In some embodiments, methods are provided for rescuing and / or treating a fetus with SMA, comprising administering (e.g., simultaneously or sequentially) a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV), or a small molecule for increasing SMN function and an SMN2 ASO (e.g., nusinersen), or a small molecule for increasing SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene, and an SMN2 ASO (e.g., nusinersen) to neuronal cells of the fetus in utero. In some embodiments, the method comprises administering (e.g., simultaneously or sequentially) one or more compositions described herein via intrathecal injection.In some embodiments, in utero treatment is defined as administering (e.g., concurrently and sequentially) a small molecule to increase SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV) as described herein, or a small molecule to increase SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen), or a small molecule to increase SMN2 function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV) and an SMN2 ASO (e.g., nusinersen) following detection of SMA in the fetus. See, e.g., 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, neonatal treatment involves delivery of at least one dose of a small molecule to increase SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV), or a small molecule to increase SMN2 function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen), or a combination of a small molecule to increase SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an 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 other embodiments, 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 from about 24 hours to about 48 hours later.

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

[0270] In some embodiments, a small molecule to increase SMN2 function and a recombinant SMN1 gene (e.g., in an rAAV), or a small molecule to increase SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen), or a small molecule to increase SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV) and an SMN2 ASO (e.g., nusinersen) are re-administered at a later date.

[0271] In some embodiments, two or more readministrations are provided. Such readministration can include readministration of the recombinant SMN1 gene with the same type of viral vector, with a different viral vector (e.g., using an AAV capsid protein of a different serotype), 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 a recombinant SMN1 gene (e.g., in addition to receiving a small molecule (e.g., risdiplam or branapram) or a small molecule and an SMN2 ASO to increase SMN function), a second, different rAAV (e.g., rAAVhu68) encoding the recombinant SMN1 gene can be subsequently administered, or vice versa. Also, if the patient has neutralizing antibodies against the first rAAV serotype, a second, different rAAV serotype can then 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 to increase SMN function (e.g., risdiplam or branapram) and a recombinant SMN1 gene (e.g., in an rAAV), or a small molecule to increase SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen), or a small molecule to increase SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and an SMN2 ASO may require additional therapy, such as transient co-treatment with an immunosuppressant before, during, and / or after treatment with the compositions described herein. Such immunosuppressants for 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 temporary treatment may include a steroid (e.g., prednisone or prednisolone) administered once daily for seven days at a tapering dose starting at about 60 mg and tapering off by 10 mg / day (no dose on day 7). Other doses and immunosuppressants may also be selected.

[0273] In some embodiments, the subject has one or more indicators of SMA. In some embodiments, the subject has reduced electrical activity in 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 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 upright. 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 muscle electrophysiological measurements and / or muscle biopsy.

[0274] In some embodiments, the subject has SMA Type I. In some embodiments, the subject has SMA Type II. In some embodiments, the subject has SMA Type III. 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 over 15 years of age.

[0275] In some embodiments, the first dose of the pharmaceutical composition (e.g., a small molecule to increase SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), an SMN2 ASO (e.g., nusinersen), or both) is administered in utero. In some such embodiments, the first dose is administered before 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 formation of the blood-brain barrier. In some embodiments, the first dose is administered into the CSF.

[0276] In some embodiments, the first dose of the pharmaceutical composition (e.g., a small molecule to increase SMN function such as risdiplam or branapram, a recombinant SMN1 gene (e.g., in an 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 over 15 years old.

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

[0278] In some embodiments, administration of at least one pharmaceutical composition (e.g., of a small molecule to increase SMN function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and / or an SMN2 ASO (e.g., nusinersen)) results in a phenotypic change in the 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; improved muscle strength; improved electrical activity in at least one muscle; improved respiration; weight gain; and survival. In some embodiments, the at least one phenotypic change is detected in the subject's motor neurons. In some embodiments, administration of at least one pharmaceutical composition described herein enables the subject to sit up, stand, and / or walk. In some embodiments, administering at least one pharmaceutical composition allows the subject to eat, drink and / or breathe without assistance.In some embodiments, the effectiveness of treatment is evaluated by muscle electrophysiological assessment.In some embodiments, administering pharmaceutical composition improves at least one symptom of SMA, and has little or no inflammatory effect.In some embodiments, the absence of inflammatory effect is determined by the absence of significant increase in Aif1 level during treatment.

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

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

[0281] In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and a recombinant nucleic acid encoding SMN1 (e.g., in an 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 a clinic visit). In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram) and an SMN2 ASO (e.g., nusinersen) are administered together to a subject. In some embodiments, a small molecule for increasing SMN function (e.g., risdiplam or branapram), a recombinant nucleic acid encoding SMN1 (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) are administered together to a subject. Thus, in some embodiments, the combined administration of a small molecule for increasing SMN function (e.g., risdiplam or branapram), an SMN2 ASO (e.g., nusinersen), and a recombinant nucleic acid encoding SMN1 refers to administration during the same clinic visit (e.g., during the same clinic day). In some embodiments, the combined administration of a small molecule for increasing SMN function (e.g., risdiplam or branapram), an SMN2 ASO (e.g., nusinersen), and a recombinant nucleic acid encoding SMN1 refers to administration at different times during the same clinic visit (e.g., during the same clinic day). In some embodiments, the concurrent administration of a small molecule for increasing SMN function (e.g., risdiplam or branapram), an SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) represents the initiation of a new treatment. In other embodiments, the concurrent administration of a small molecule to increase SMN function (e.g., risdiplam or branapram), an SMN1 gene (e.g., in an rAAV), and an SMN2 ASO (e.g., nusinersen) is an add-on therapy to a subject currently being treated with a different composition.

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

[0283] In some embodiments, a small molecule to increase SMN2 function (e.g., risdiplam or branapram), an SMN2 ASO (e.g., nusinersen), and a recombinant SMN1 gene (e.g., in an rAAV) are administered at different frequencies. The ASO (e.g., nusinersen) or small molecule for increasing SMN2 function (e.g., risdiplam or branapram) is administered to the subject 1 to 6 times per year. In some embodiments, the recombinant SMN1 gene (e.g., in an rAAV) is administered once. In some embodiments, two or more subsequent administrations of a small molecule for increasing SMN2 function (e.g., risdiplam or branapram) and / or an SMN2 ASO (e.g., nusinersen) are administered after the initial administration of the SMN2 ASO (e.g., nusinersen) and recombinant SMN1 gene. In some embodiments, the SMN2 ASO (e.g., nusinersen) is administered to the subject prior to administration of a small molecule for increasing SMN2 function (e.g., risdiplam or branapram), an SMN2 ASO, and / or a recombinant SMN1 gene (e.g., in an rAAV). In some embodiments, the SMN2 ASO (e.g., nusinersen) is administered to the subject at a dose of 0.01 to 25 milligrams (e.g., 0.01 to 10 milligrams, 0.05 to 5 milligrams, 0.1 to 2 milligrams, or 0.5 to 1 milligram) per kilogram of the subject's body weight, and the recombinant SMN1 gene (e.g., in an 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 In some embodiments, the SMN2 ASO is administered to a subject at a dose of 0.001 to 25 milligrams (e.g., 0.001 to 10 milligrams, 0.005 to 5 milligrams, 0.01 to 2 milligrams, or 0.05 to 1 milligram) per kilogram of subject body weight, and the recombinant SMN1 gene (e.g., in the rAAV) is administered at a dose of 1 x 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), or, for example, for IV administration, about 3 x 1013 ~5×10 14 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) is administered to a subject per dose. In some embodiments, a total of 5 mg to 20 mg of SMN2 ASO (e.g., nusinersen) is administered to a subject per dose. In some embodiments, a total of 12 mg to 48 mg of SMN2 ASO (e.g., nusinersen) is administered to a subject per dose. In some embodiments, a total of 12 mg to 36 mg of SMN2 ASO (e.g., nusinersen) is administered to a subject per dose. In some embodiments, a total of 28 mg of SMN2 ASO (e.g., nusinersen) is administered to a subject per dose. In some embodiments, a total of 12 mg of SMN2 ASO (e.g., nusinersen) is administered to a subject per dose. In some embodiments, the SMN2 ASO (e.g., nusinersen) and / or recombinant SMN1 gene is administered to a subject intravenously or intramuscularly. In some embodiments, SMN2 ASO (e.g., nusinersen) and / or recombinant SMN1 gene are administered to the intrathecal space of a subject. In some embodiments, SMN2 ASO (e.g., nusinersen) and / or recombinant SMN1 gene are administered to the intracisternal space of a subject. In some embodiments, administration of SMN2 ASO (e.g., nusinersen) and recombinant nucleic acid increases intracellular SMN protein levels in a subject. In some embodiments, administration of SMN2 ASO (e.g., nusinersen) and recombinant nucleic acid increases intracellular SMN protein levels in the cervical, thoracic and lumbar spinal cord regions of motor neurons in a subject.

[0285] In some embodiments, the dose of a small molecule (e.g., risdiplam or branapram) for increasing SMN2 function, a recombinant SMN1 gene (e.g., in rAAV), and an SMN2 ASO (e.g., nusinersen) is administered by bolus injection into the CSF. In some embodiments, the dose is administered by LP and / or ICM bolus injection. In some embodiments, the dose is 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 CSF bolus and systemic bolus doses may be the same or different from each other. In some embodiments, the CSF and systemic doses are administered at different frequencies.

[0286] In some embodiments, pharmaceutical compositions are provided that include a small molecule for increasing SMN2 function (e.g., risdiplam or branapram), a recombinant SMN1 gene (e.g., in an rAAV), and / or an SMN2 ASO (e.g., nusinersen). The pharmaceutical compositions can be designed for delivery to a subject in need thereof by any appropriate route (e.g., by a different route 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 intracisternomagna (ICM) delivery).

[0287] In some embodiments, direct delivery to the CNS is desirable and may be achieved via intrathecal injection. The term "intrathecal administration" refers to delivery targeted to the cerebrospinal fluid (CSF). This may be achieved by direct injection into the ventricles or lumbar CSF, by suboccipital puncture, or by other suitable means. Meyer et al., Molecular Therapy (31 October 2014) states: demonstrated the effectiveness of direct CSF injection, which results in widespread transgene expression throughout the spinal cord of mice and non-human primates when using a 10-fold lower dose 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, incorporated herein by reference). See also Passini et al., Hum Gene Ther. 2014 Jul;25(7):619-30, incorporated herein by reference. In some embodiments, the composition is delivered via lumbar injection.

[0288] In some embodiments, the delivery vehicle and formulation are designed to avoid direct systemic delivery of a suspension containing the AAV composition(s) described herein. Advantageously, this can have the advantages of reduced systemic exposure compared to systemic administration, reduced toxicity, and / or reduced undesired immune responses to the AAV and / or transgene product.

[0289] Compositions comprising small molecules for increasing SMN2 function (e.g., risdiplam or branapram), recombinant SMN1 genes (e.g., in rAAV) and / or SMN2 ASOs (e.g., nusinersen) can be formulated for any suitable route of administration (e.g., oral, inhaled, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular and other parenteral routes).

[0290] In some embodiments, the recombinant SMN1 gene delivery constructs described herein 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-defective viruses (e.g., AAV, adenovirus, and / or lentivirus). Alternatively, delivery can be mediated by non-viral constructs, such as "naked DNA," "naked plasmid DNA," RNA, and mRNA, combined with various delivery compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, poly-glycan compositions and other polymer, lipid, and / or cholesterol-based nucleic acid conjugates, as well as other constructs, e.g., described herein or known in the art. See, for example, 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, both of which are incorporated herein by reference. Non-viral SMN1 delivery constructs may also be formulated for any suitable route of administration.

[0291] Viral vectors or non-viral DNA or RNA transfer moieties can be formulated with physiologically acceptable carriers for use in gene transfer and gene therapy applications.Many suitable purification methods can be selected.Examples of suitable purification methods for separating empty capsids from vector particles have been described, for example, the process described in International Patent Application No. PCT / US16 / 65976, entitled "Scalable Purification Method for AAV8," filed on December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,098, filed on April 13, 2016, and U.S. Patent Application No. 62 / 266,341, filed on December 11, 2015, which are incorporated herein by reference. International Patent Application No. PCT / US16 / 65974, filed December 9, 2016, and its priority documents, U.S. Patent Application Nos. 62 / 322,083, filed April 13, 2016, and 62 / 266,351, filed December 11, 2015 (AAV1), which are incorporated herein by reference; International Patent Application No. PCT / US16 / 66013, filed December 9, 2016, and its priority documents, See also the purification methods described in U.S. Provisional Application Nos. 62 / 322,055, filed April 13, 2016, and 62 / 266,347, filed December 11, 2015 (AAVrhlO); and International Patent Application No. PCT / US16 / 65970, filed December 9, 2016, and its priority applications, U.S. Provisional Application Nos. 62 / 266,357 and 62 / 266,357 (AAV9). Briefly, a two-step purification scheme is described that selectively captures and isolates genome-containing rAAV vector particles from clarified, concentrated supernatants of rAAV-producing cell cultures. The process utilizes an affinity capture method performed at high salt concentrations, 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 AAV viral vectors, the quantification of genome copies ("GC") can be used as a measure of the dose contained in the formulation. Any method known in the art can be used to determine the genome copy (GC) number of the replication-defective viral composition of the present invention. One method for titrating AAV GC number is as follows: A 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., polyA signal). Another suitable method for determining genome copies is quantitative PCR (qPCR), in particular 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-defective virus composition, in dosage units, is about 1.0 x 10, including all integer or fractional amounts within the range. 9 GC~approx. 1.0×10 15 GC range (e.g., to treat an average subject weighing 70 kg), preferably 1.0 x 10 for human patients. 12 GC~1.0×10 14 The composition may be formulated to contain a quantity of replication-defective virus of GC. The total dose administered to a subject may depend on the route of administration. In some embodiments, the composition may contain at least 1 x 10 per dose, including all integers or fractions within the range. 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 9In another embodiment, the composition is formulated to contain at least 1 x 10 GC 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 In another embodiment, the composition is formulated to contain at least 1 x 10 GC 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 In another embodiment, the composition is formulated to contain at least 1 x 10 GC 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 In another embodiment, the composition is formulated to contain at least 1 x 10 GC 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 In another embodiment, the composition is formulated to contain at least 1 x 10 GC 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 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integer or fractional amounts within the range. 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 In some embodiments, for human applications, the viral (e.g., rAAV) dose is 1 x 10 per dose, including all integer or fractional amounts within the range. 10 ~Approx. 1×10 12 It can be in the range of GC.

[0294] These above doses may be administered in various volumes of pharmaceutically acceptable carrier, excipient, or buffer formulations ranging from about 25 microliters to about 1,000 microliters, or to about 10 milliliters, or up to 20 milliliters, inclusive of all figures within the range, depending on the size of the area to be treated, the viral titer used, and the desired effect of the route and method of administration. 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 other embodiments, the volume is about 75 μl. In other embodiments, the volume is about 100 μl. In other embodiments, the volume is about 125 μl. In other embodiments, the volume is about 150 μl. In other embodiments, the volume is about 175 μl. In yet other embodiments, the volume is about 200 μl. In other embodiments, 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 about 1 μl to 150 mL may be selected, with larger volumes being selected for adults. Typically, for newborn infants, a suitable volume is about 0.5 mL to about 10 mL. For older infants, a volume of about 0.5 mL to about 15 mL may be selected. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, a volume of up to about 30 mL may be selected. For preadolescents and adolescents, a volume of up to about 50 mL may be selected. In still other embodiments, patients may receive intrathecal administration in a volume of about 5 mL to about 15 mL, or 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 may vary depending on the therapeutic application for which the recombinant vector is used.

[0296] A recombinant SMN1 gene, for example, 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 based on the route of administration. For example, one suitable carrier includes saline, which can be formulated with 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 other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the SMN1 rAAV, small molecules for increasing SMN function (e.g., risdiplam or branapram) and / or ASOs (e.g., nusinersen), and pharmaceutically acceptable carrier(s). 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 for increasing SMN function (e.g., risdiplam or branapram), an SMN1 rAAV, and / or an SMN2 ASO (e.g., nusinersen) may comprise a pharmaceutically acceptable carrier and / or may be mixed with a suitable excipient designed for delivery to a subject via injection, osmotic pump, intrathecal catheter, or 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 can be used in the preparation of medicaments (e.g., administered concomitantly or sequentially) to deliver SMN1 to a subject (e.g., a human patient) in need thereof to provide the subject with functional SMN, and / or to treat spinal muscular atrophy in combination therapy with one or more SMN2 ASOs.

[0300] In some embodiments, pharmaceutical compositions comprising rAAV and a pharmaceutically acceptable carrier (e.g., buffers, salts, and / or other components of a pharmaceutical formulation) are selected to include one or more components that prevent adhesion of the rAAV to the infusion tubing but do not interfere with the binding activity of the rAAV in vivo.

[0301] In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated for delivery (e.g., for systemic administration) in 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) in 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) in 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) in 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) in 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) in 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) in an amount of 12 mg of ASO per dose. In some such embodiments, the administration volume is 5 mL.

[0302] In some such embodiments, the ASO (e.g., an SMN2 ASO) (alone or with a recombinant SMN1 gene and / or a small molecule to increase SMN function, such as risdiplam or branapram) 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 dose is 0.1 mg / kg to 100 mg / kg. In some embodiments, the dose is 0.5 mg / kg to 100 mg / kg. In some embodiments, the dose is 1 mg / kg to 100 mg / kg. In some embodiments, the dose is 1 mg / kg to 50 mg / kg. In some embodiments, the dose is 1 mg / kg to 25 mg / kg. In some embodiments, the dose is 0.1 mg / kg to 25 mg / kg. In some embodiments, the dose is 0.1 mg / kg to 10 mg / kg. In some embodiments, the dose is 1 mg / kg to 10 mg / kg. In some embodiments, the dose is 1 mg / kg to 5 mg / kg.

[0303] In some embodiments, the administration of ASO to the subject is divided into induction stage and maintenance stage.In some such embodiments, the dose administered during induction stage is higher than the dose administered during maintenance stage.In some embodiments, the dose administered during induction stage is lower than the dose administered during maintenance stage.In some embodiments, induction stage is achieved by bolus injection, and maintenance stage is achieved by continuous infusion.In some embodiments, combination preparation is used during induction stage.

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

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

[0306] In some embodiments, such doses are administered twice monthly. In some embodiments, such doses are administered monthly. In some embodiments, such doses are administered every two months. In some embodiments, such doses are administered every six months. In some embodiments, such doses are administered by bolus injection into the CSF. In some embodiments, such doses are administered by intrathecal bolus injection. In some embodiments, such doses 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 CSF bolus and systemic bolus doses may be the same or different from each other. In some embodiments, the CSF and systemic doses 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 a dose can be administered over a period of time, e.g., 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 IT or ICV. In some such embodiments, the administered dose is 5 mg to 60 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the administered dose is 5 mg to 20 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the administered dose is 12 mg to 50 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the administered dose is 12 mg to 48 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the administered dose is 12 mg to 36 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the administered dose is 28 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the administered dose is 12 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose per day. In some such embodiments, the administered volume is 5 mL.

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

[0309] In some embodiments, the present invention provides a dosing regimen comprising CNS infusion and at least one bolus systemic injection. In some embodiments, the present invention provides a dosing regimen comprising CNS infusion and at least one bolus subcutaneous injection. In some embodiments, the dose is adjusted to achieve or maintain a concentration of 0.1 to 100 micrograms of antisense compound per gram of CNS tissue, whether by bolus or infusion. In some embodiments, the dose is adjusted to achieve or maintain a concentration of 1 to 10 micrograms of antisense compound per gram of CNS tissue, whether by bolus or infusion. In some embodiments, the dose is adjusted to achieve or maintain a concentration of 0.1 to 1 micrograms of antisense compound per gram of CNS tissue, whether by bolus or infusion.

[0310] Thus, in some embodiments, the present invention provides pharmaceutical compositions comprising one or more therapeutic molecules, e.g., one or more recombinant nucleic acids (e.g., packaged in a viral vector, e.g., rAAV) and / or antisense compounds. In some embodiments, such pharmaceutical compositions comprise a sterile saline solution and one or more therapeutic molecules. In some embodiments, such pharmaceutical compositions consist of a sterile saline solution and one or more therapeutic molecules. In some embodiments, the therapeutic molecules may be mixed with pharmaceutically acceptable active and / or inactive substances for the preparation of a pharmaceutical composition or formulation. The compositions and methods for formulating pharmaceutical compositions depend on many criteria, including, but not limited to, the route of administration, the extent of the disease, or the administered dose. In some embodiments, therapeutic molecules can be utilized in pharmaceutical compositions by combining such therapeutic molecules with a suitable pharmaceutically acceptable diluent or carrier. In some embodiments, pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in parenterally delivered compositions. Thus, in some embodiments, the methods described herein use pharmaceutical compositions comprising one or more therapeutic molecules and a pharmaceutically acceptable diluent. In some embodiments, the pharmaceutically acceptable diluent is PBS. Pharmaceutical compositions comprising one or more therapeutic molecules described herein include any pharmaceutically acceptable salts, esters, or salts of such esters. In some embodiments, pharmaceutical compositions comprising ASOs include one or more oligonucleotides that can provide (directly or indirectly) their biologically active metabolites or residues upon administration to animals, including humans. Thus, in some embodiments, pharmaceutically acceptable salts of ASOs, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents are provided. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0311] In some embodiments, prodrugs can include the incorporation of additional nucleosides at one or both ends of the oligomeric compound, which are cleaved by endogenous nucleases in the body to form active antisense oligomeric compounds. Lipid-based vectors have been used in nucleic acid therapy in various ways. For example, in one method, nucleic acids are introduced into preformed liposomes or lipoplexes composed of a mixture of cationic lipids and neutral lipids. In another method, DNA complexes with mono- or poly-cationic lipids are formed without the presence of neutral lipids. Some preparations are described in Akinc et al., Nature Biotechnology 26, 561-569 (1 May 2008), the entire contents of which are incorporated herein by reference.

[0312] kit In some embodiments, kits are provided that include small molecules for increasing SMN function, recombinant SMN1 genes (e.g., in rAAV), and / or SMN2 ASOs, e.g., in pharmaceutical compositions. In some embodiments, such kits further include additional therapeutic agents, such as one or more immunosuppressants. In some embodiments, such kits further include 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. [Example]

[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 ASOs that increase full-length SMN2 mRNA (e.g., by promoting exon 7 inclusion in hSMN2 mRNA) An ASO was prepared that increases full-length SMN2 mRNA (e.g., promotes exon 7 inclusion in SMN2 mRNA) (Figure 3).

[0316] Example 3 Administration and biodistribution of rAAV vectors containing the hSMN1 gene and ASOs that increase full-length SMN2 mRNA (e.g., promote exon 7 inclusion in SMN2 mRNA) The rAAV of Example 1 and the ASO of Example 2 are administered to animal SMA disease models and control animals, including mice, pigs, and non-human primates (e.g., macaques), in SMA disease models and control animal models.

[0317] rAAV and ASO are administered via different routes, including intrathecal and systemic routes (e.g., via lumbar puncture, intracisternal and intravenous delivery).

[0318] The distribution of rAAV and ASO is assessed in animal models, particularly within the spinal cord, to determine, for example, the relative amounts of rAAV and / or ASO in the cervical, thoracic, and lumbar regions of the spinal cord.

[0319] Figure 4. 3 x 10 M IgG4-dependent steroids administered via lumbar puncture or intracisternal delivery 13 Results using rAAV for GC and 2 x 10 i.v. 14 The results of using GC are shown in the figure.

[0320] Example 4 Co-formulation of an rAAV vector containing the hSMN1 gene with an ASO that increases full-length SMN2 mRNA (e.g., promotes exon 7 inclusion in SMN2 mRNA). Figure 5 shows an rAAV vector and hSMN1 gene. 1 illustrates non-limiting examples of the physical and biological characterization of a composition comprising both an ASO that increases full-length SMN2 mRNA (e.g., promotes exon 7 inclusion in SMN2 mRNA).

[0321] Figure 5A shows the SEC-HPLC profile of the rAAV vector alone. Figure 5B shows the SEC-HPLC profile of the ASO alone. Figure 5C shows the SEC-HPLC profiles of the rAAV vector and ASO when they are present in the same formulation. The HPLC profiles of the rAAV vector and ASO remain the same in Figure 5C, indicating that there is no significant incompatibility when rAAV and ASO are co-formulated.

[0322] Figure 5D provides data on the infectivity of rAAV in cells in vitro upon delivery of either the rAAV vector alone or the rAAV vector with an ASO. The results show that the infectivity of rAAV is not significantly affected by the presence of an ASO in the co-formulation.

[0323] Figure 5E shows intracellular SMN protein expression levels and GEM formation in cells after treatment with rAAV, ASO, or both.

[0324] Example 5 Intracerebroventricular (ICV) administration of nusinersen and AAV-SMN1 Nusinersen and AAV-SMN1 were delivered to the cerebrospinal fluid (CSF) through the right lateral ventricle in neonatal (P0-P1) SMA mice carrying the human SMN2 transgene using a micro-osmotic pump (ALZET Osmotic Pumps, Cupertino, Calif., USA). Low- or high-dose nusinersen (1 μg and 4 μg, respectively) was delivered with low- or high-dose AAV-SMN1 (1 × 10, respectively). 10 GC or 8 × 10 10 Mice are administered nusinersen (GC) along with AAV-SMN1 at birth (P0-P1). The body weight and righting reflex of the mice are measured and compared to the body weight and righting reflex of control mice of the same genotype receiving either nusinersen or AAV-SMN1 alone.

[0325] Mice administered both nusinersen and AAV-SMN1 have significantly higher body weights and faster righting reflexes compared to controls.

[0326] Studies show tha...

Claims

[Claim 1] The invention described in the specification.

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