Combination therapy for spinal muscular atrophy

Abstract

To provide a combination therapy for spinal muscular atrophy.SOLUTION: Aspects of the present application relate to compositions and methods for treating spinal muscular atrophy in a subject. In particular, this application provides therapeutically combinations of recombinant nucleic acids encoding Survival of Motor Neuron 1 (SMN1) proteins (e.g., in viral vectors) and antisense oligonucleotides (ASOs) that increase full-length Survival of Motor Neuron 2 (SMN2) mRNA (e.g., ASOs that target nucleic acids molecules encoding Survival of Motor Neuron 2 (SMN2) and promote inclusion of exon 7 in SMN2mRNA).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of the filing date of U.S. Provisional Application No. 62 / 764,893, entitled "Combination Therapy for Spinal Muscular Atrophy," filed August 15, 2018, and U.S. Provisional Application No. 62 / 783,189, entitled "Combination Therapy for Spinal Muscular Atrophy," filed December 20, 2018, the contents of each of which are incorporated herein by reference in their entirety.

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

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

[0004] The SMN gene product is intracellularly expressed, and its loss results in selective toxicity to lower motor neurons, leading to progressive neuronal loss and muscle weakness. A centromeric duplicated homolog (SMN2) exists, but SMN2 harbors a splice-site mutation that results in minimal production of full-length SMN transcripts. The severity of the disease varies depending on the copy number of SMN2. Patients with one or two copies of SMN2 present with 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 present with a milder form of the disease, typically manifesting after six months of age. While many never achieve walking, progression to respiratory failure is rare, and many patients survive into adulthood. Patients with four copies of SMN2 present with gradual muscle weakness but may not show symptoms until adulthood.

[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 in patients with different levels of disease severity. Summary of the Invention [Means for solving the problem]

[0006] In some embodiments, the present application relates to the treatment of spinal muscular atrophy (SMA), which involves co-administering to a subject with SMA a recombinant nucleic acid encoding survival motor neuron 1 (SMN1) and an oligomeric compound that increases full-length survival motor neuron 2 (SMN2) mRNA. 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 splicing of SMN2 pre-mRNA to increase inclusion of exon 7 in SMN2 mRNA).

[0007] In some embodiments, the present application relates to the treatment of spinal muscular atrophy (SMA), which involves co-administering to a subject with SMA a recombinant nucleic acid encoding survival motor neuron 1 (SMN1) and an oligomeric compound that induces exon skipping in a nucleic acid encoding survival motor neuron 2 (SMN2). 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 induces exon skipping in the SMN2 pre-mRNA.

[0008] In some embodiments, the recombinant nucleic acid (e.g., in a viral vector) and the ASO are combined and administered to the subject as a single composition. In some embodiments, the recombinant nucleic acid (e.g., in a viral vector) and the ASO are provided as separate compositions but are administered to the subject simultaneously (e.g., at the same time or for the same period of time (e.g., during the same medical visit, e.g., at the same time or on the same day)). In some embodiments, the recombinant nucleic acid (e.g., in a viral vector) and the ASO are provided as separate compositions and administered to the subject sequentially during separate medical visits (e.g., at different times, e.g., on different days) over the course of treatment (e.g., during a treatment regimen spanning 1 week, 2-4 weeks, 1 month, 1-12 months, 1 year, 2-5 years, or more). In some embodiments, the ASO is administered before and / or after administration of the recombinant nucleic acid. In some embodiments, the recombinant nucleic acid (e.g., in a viral vector) and / or the ASO are administered at different frequencies. In some embodiments, a subject is treated with a combination of a) a composition comprising both a recombinant nucleic acid (e.g., in a viral vector) and an ASO, and b) another composition comprising either a recombinant nucleic acid (e.g., in a viral vector) or an ASO. In some embodiments, two or more different recombinant SMN1 nucleic acids 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 and / or different SMN2 ASOs are administered to a subject during different medical visits.

[0009] Thus, in some embodiments, a method of treating SMA in a subject (e.g., a human subject) with SMA involves administering to the subject a recombinant nucleic acid encoding SMN1 (also referred to as a recombinant SMN1 gene) and an ASO that increases full-length SMN2 mRNA in the subject (also referred to as an SMN2 ASO). In some embodiments, a method of treating SMA in a subject includes administering to a subject with SMA effective amounts of the recombinant SMN1 gene and the SMN2 ASO.

[0010] In some embodiments, a subject with SMA has one or more SMA symptoms (e.g., limb muscle atrophy, difficulty or inability to walk, difficulty breathing, or other SMA symptoms). 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 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.

[0011] In some embodiments, the subject is a human subject. In some embodiments, the subject is selected from a pediatric population and an adult population. In some embodiments, the subject is 18 years of age or older (e.g., 18 years of age or older). In some embodiments, the subject is under 18 years of age, under 10 years of age, or under 6 years of age. In some embodiments, the subject is about 2 weeks old, about 1 month old, about 3 months old, about 6 months old, about 1 year old, about 2 years old, about 3 years old, about 4 years old, or about 5 years old.

[0012] 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 capsid protein, an AAVrhlO capsid protein, an 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. In some embodiments, the ASO alters the splicing pattern of survival motor neuron 2 (SMN2) pre-mRNA. In some embodiments, the ASO promotes 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 encoding the SMN2 protein. In some embodiments, the ASO comprises a sequence complementary to intron 6 of a nucleic acid molecule encoding the SMN2 protein. In some embodiments, the ASO comprises a sequence complementary to intron 7 of a nucleic acid molecule encoding the SMN2 protein. In some embodiments, the ASO comprises the sequence of SEQ ID NO: 1. In some embodiments, the ASO is nusinersen. In some embodiments, the ASO comprises one or more nucleobase modifications or backbone modifications.

[0013] In some embodiments, a recombinant SMN1 gene (e.g., in a viral vector) is administered (e.g., administered one or more times) to a subject who has previously been treated with SMN2 ASO therapy. In some embodiments, a recombinant SMN1 gene (e.g., in a viral vector) is administered (e.g., administered one or more times) to a subject who is currently undergoing treatment with SMN2 ASO therapy. In some embodiments, a subject is initiated on a treatment comprising simultaneous or sequential administration of a recombinant SMN1 gene (e.g., in a viral vector) and an SMN2 ASO.

[0014] In some embodiments, the rAAV containing the recombinant SMN1 gene (also referred to as SMN1 rAAV) and the SMN2 ASO are administered at the same time. In some embodiments, the SMN1 rAAV and SMN2 ASO are administered simultaneously. In some embodiments, the SMN1 rAAV and SMN2 ASO are administered together in a single composition. In some embodiments, the SMN1 rAAV and SMN2 ASO are administered separately. In some embodiments, the SMN1 rAAV and SMN2 ASO are administered sequentially. In some embodiments, the SMN1 rAAV and SMN2 ASO are administered at different frequencies. In some embodiments, the SMN1 rAAV is administered once. In some embodiments, the SMN2 ASO is administered 1 to 6 times per year. In some embodiments, an initial administration of the SMN1 rAAV and SMN2 ASO is followed by two or more subsequent doses of SMN2 ASO alone. In some embodiments, one or more booster doses of SMN1 rAAV are administered to the subject. In some embodiments, the SMN1 rAAV is administered to the subject more than six months or more than one year between the first and second administrations. In some embodiments, the first SMN1 rAAV composition and the second SMN1 rAAV composition comprise the same rAAV capsid protein. In some embodiments, the first SMN1 rAAV composition and the second SMN1 rAAV composition comprise different rAAV capsid proteins.

[0015] 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.

[0016] 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. In some embodiments, a total of 12 mg to 36 mg of SMN2 ASO is administered to a subject per dose. In some embodiments, a total of 28 mg of SMN2 ASO is administered to a subject per dose. In some embodiments, a total of 12 mg of SMN2 ASO is administered to a subject per dose. In some embodiments, the dose volume is 5 mL.

[0017] In some embodiments, the SMN1 rAAV and SMN2 ASO are administered intrathecally to the subject. In some embodiments, the SMN1 rAAV and SMN2 ASO are administered intracisternally to the subject. In some embodiments, the initial dose and / or subsequent doses of SMN2 ASO are administered intravenously or intramuscularly.

[0018] In some embodiments, administration of the SMN1 rAAV and SMN2 ASO increases intracellular SMN protein levels in the subject, ie, in the cervical, thoracic, and lumbar spinal cord of the subject (e.g., in motor neurons in the brain and / or spinal cord of the subject).

[0019] Thus, in some embodiments, administering an effective amount of a composition comprising an SMN1 rAAV and an SMN2 ASO to a subject with SMA increases SMN protein expression in the subject. 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. In some embodiments, administering an effective amount of an SMN2 ASO to a subject previously treated with an SMN1 rAAV increases SMN protein expression in the subject. In some embodiments, administering an effective amount of an SMN1 rAAV to a subject previously treated with an SMN2 ASO increases SMN protein expression in the subject. In some embodiments, the pharmaceutical composition is administered to the CNS or CSF of the subject. In some embodiments, the pharmaceutical composition is administered intravenously to the subject.

[0020] In some embodiments, the composition comprises both an SMN1 rAAV and an SMN2 ASO. In some embodiments, the pharmaceutical composition comprises both an SMN1 rAAV and an SMN2 ASO and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is administered to a subject in need thereof in a therapeutically effective amount.

[0021] In some embodiments, the SMN1 rAAV and SMN2 ASO (e.g., both together in a single composition or as two separate compositions) are co-administered one or more times to a subject (e.g., a human subject) via the intrathecal route. In some embodiments, the SMN1 rAAV and SMN2 ASO are co-administered one or more times to the spinal canal, subarachnoid space, ventricles, or lumbar CSF by suboccipital puncture or other appropriate route (e.g., via injection, via infusion, using a pump and catheter, or via other suitable techniques). In some embodiments, the SMN1 rAAV and SMN2 ASO (e.g., both together in a single composition or as two separate compositions) are co-administered one or more times to a subject (e.g., a human subject) via the intracranial route, intraventricular route, intracerebral route, intraparenchymal route, intravenous route, or other appropriate route. SMN1 rAAv and SMN2 rAAv, whether administered simultaneously or sequentially, can be administered by any suitable or appropriate means known in the art. Each of the ASOs can be administered (e.g., intrathecally, intravenously, etc.), and the SMN1 rAAV and SMN2 ASO can be administered by the same or different means (e.g., via the same or different routes of administration).

[0022] In some aspects, the SMN1 rAAV and / or SMN2 ASO are used in the manufacture of a medicament for treating a disease or condition associated with survival motor neuron protein (SMN), such as spinal muscular atrophy (SMA).

[0023] In some aspects, the disclosure relates to a method for treating spinal muscular atrophy (SMA) in a subject with SMA, the method comprising administering an effective amount of a composition comprising an rAAV encoding SMN1 to a subject who has previously been treated with an ASO that increases full-length SMN2 mRNA.

[0024] In some aspects, the disclosure relates to a method for treating spinal muscular atrophy (SMA) in a subject with SMA, the method comprising administering to a subject who has previously been administered an rAAV encoding SMN1 a composition comprising an ASO that increases full-length SMN2 mRNA in an effective amount.

[0025] Another aspect of the present disclosure relates to a composition comprising a rAAV encoding SMN1 and an ASO capable of increasing full-length SMN2 mRNA. In some embodiments, the rAAV comprises an AAV9 capsid protein. In some embodiments, the ASO is nusinersen. In some embodiments, the composition is a pharmaceutical composition and comprises a pharmaceutically acceptable carrier.

[0026] Other aspects and advantages of the present invention will become readily apparent from the detailed description of the invention that follows.

[0027] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present application, which can 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]

[0028] [Figure 1] Subjects receiving treatment with a recombinant nucleic acid encoding SMN1 in combination with an antisense oligonucleotide (e.g., nusinersen) that increases full-length SMN2 mRNA (e.g., promotes inclusion of exon 7 in SMN2 mRNA) show increased levels of SMN activity in a greater number of motor neurons. [Figure 2] 1 is a schematic representation of a non-limiting example of a nucleic acid encoding SMN1. [Figure 3-1] As a non-limiting example of an antisense oligonucleotide that increases full-length SMN2 mRNA (e.g., promotes inclusion of exon 7 in SMN2 mRNA), the chemical structure of nusinersen is shown. [Figure 3-2] Same as above. [Figure 4] 1 shows the distribution of rAAV after different modes of administration in non-human primates. [Figure 5] We demonstrate that recombinant nucleic acids encoding SMN1 and antisense oligonucleotides that increase full-length SMN2 mRNA (e.g., promote the inclusion of exon 7 in SMN2 mRNA) are physically and biologically compatible. [Figure 6] 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 at postnatal day (PND) 8** and fully rescues motor function at PND 16 after dosing. Delayed weight gain compared to WT controls is also shown. A is a series of graphs showing the righting reflex (RR) of four separate groups 8 and 16 days after administration of the ASO (nusinersen). B is a series of graphs showing the body weight of four separate groups 8 and 16 days after administration of the ASO (nusinersen). The partial rescue of RR (PND 7-16) and body weight provides a clue that combination therapy may offer additional benefit in this preclinical model. [Figure 7A]

[0033] Figure 1 shows the results of the first study using weight and RR as primary endpoints for combination treatment of SMN1 gene therapy (delivered via rAAV vector) and ASO (nusinersen). Graph showing weight change (in days) over time. [Figure 7B]

[0033] Figure 1 shows the results of the first study using weight and RR as primary endpoints for combination treatment of SMN1 gene therapy (delivered via rAAV vector) and ASO (nusinersen). Graph showing change in RR (in days) over time. [Figure 7C]

[0033] Figure 1 shows the results of the first study using weight and RR as primary endpoints for the combination of SMN1 gene therapy (delivered via rAAV vector) and ASO (nusinersen). Figure 2 shows a chart outlining the conditions for the three study arms. [Figure 8A]

[0033] Figure 1 shows the results of a second study using weight and RR as primary endpoints for the combination of SMN1 gene therapy (delivered via rAAV vector) and ASO (nusinersen). Figure 2 shows a chart outlining the conditions for the three study arms. [Figure 8B]

[0033] Figure 1 shows the results of a second study using weight and RR as primary endpoints for combination treatment of SMN1 gene therapy (delivered via rAAV vector) and ASO (nusinersen). Graph showing weight change (in days) over time. [Figure 8C]

[0033] Figure 1 shows the results of a second study using weight and RR as primary endpoints for combination treatment of SMN1 gene therapy (delivered via rAAV vector) and ASO (nusinersen). Graphs show change in RR (in days) over time. [Figure 9] Figure 1 shows a comparison of the % change in body weight from PND7 to PND13. (A) shows the % change in body weight at a fixed dose of gene therapy (rAAV): 1 x 10 GC / ASO (nusinersen): 1 μg. (B) shows the % change in body weight at a fixed dose of gene therapy (rAAV): 3 x 10 GC / ASO (nusinersen): 3 μg. [Figure 10] The figure shows a comparison of the % change in RR from PND7 to PND13. (A) shows the % change in RR at a fixed dose of gene therapy (rAAV): 1 x 10 GC / ASO (nusinersen): 1 μg. (B) shows the % change in RR at a fixed dose of gene therapy (rAAV): 3 x 10 GC / ASO (nusinersen): 3 μg. [Figure 11] Complementation in neuronal and non-neuronal cells to combination therapy is shown. DETAILED DESCRIPTION OF THE INVENTION

[0029] This application relates to compositions and methods for treating spinal muscular atrophy (SMA) in a subject (e.g., a human subject with SMA). In some aspects, the treatment involves administering to the subject with SMA both a recombinant nucleic acid (e.g., in a viral vector) that expresses the SMN1 gene and an antisense oligonucleotide (ASO) that increases full-length SMN2 mRNA in the subject (e.g., an ASO that promotes inclusion of exon 7 in SMN2 mRNA).

[0030] In some embodiments, the combination of a recombinant nucleic acid expressing SMN1 and an antisense oligonucleotide that increases full-length SMN2 mRNA (e.g., an ASO that promotes inclusion of exon 7 in SMN2 mRNA) can enhance intracellular SMN protein levels in some motor neurons and can also increase the number of motor neurons with elevated intracellular survival motor neuron (SMN) protein levels compared to treatment with either the recombinant nucleic acid or the ASO alone. This is shown in Figures 1 and 11. In addition to producing therapeutically effective levels of SMN protein in subjects with SMA, methods and compositions for administering a recombinant nucleic acid expressing SMN1 in combination with an ASO that increases full-length SMN2 mRNA in SMN2 (e.g., an ASO that promotes inclusion of exon 7 in SMN2 mRNA) can also be useful for treating subjects with different levels of disease severity.

[0031] Spinal muscular atrophy (SMA), also known as proximal spinal muscular atrophy (SMA), is a genetic 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 in infants. Because the severity of SMA varies among patients, it is classified into different types based on the age of onset and motor developmental milestones. The designation SMA0 has been proposed to reflect prenatal onset, which involves severe joint contractures, facial nerve paralysis, 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 occurring at birth or within the first six months of life and typically resulting in death within two years. Children with type 1 SMA are unable to sit or walk and have severe respiratory problems. Type 2 SMA is an intermediate form with onset occurring within the first two years of life. Children with type II SMA can sit but cannot 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 unaided, at least during infancy. The adult form (type IV) is the mildest form of SMA, with onset occurring after age 30, and few cases have been reported. Types III and IV SMA are also known as late-onset SMA.

[0032] The molecular basis of SMA is the loss of both copies of the survival motor neuron gene 1 (SMN1), which may also be known as telomeric SMN (a protein that is part of a multiprotein complex thought to be involved in the biogenesis and recycling of snRNPs). A nearly identical gene, SMN2 (also known as centromeric SMN), resides in a duplicated region on chromosome 5q13, and SMN2 modulates disease severity. Expression of the normal SMN1 gene alone results in expression of the survival motor neuron (SMN) protein. Although SMN1 and SMN2 potentially have the capacity to encode the same protein, SMN2 contains a translationally silent mutation at position +6 of exon 7, resulting in inefficient inclusion of exon 7 in SMN2 transcripts. Thus, the prevailing form of SMN2 is a truncated version lacking exon 7, which is unstable and inactive (Cartegni and Krainer, Nat. Genet., 2002, 30, 377-384). Expression of the SMN2 gene results in approximately 10-20% SMN protein and 80-90% unstable / nonfunctional SMN delta 7 protein. The SMN protein has a well-established role in spliceosome assembly and may also mediate mRNA transport in neuronal axons and nerve terminals.

[0033] Although SMA is caused by the homozygous loss of both functional copies of the SMN1 gene, the SMN2 gene encodes the same protein as SMN1 and therefore has the potential to overcome the genetic abnormality in SMA patients. SMN2 contains a translationally silent mutation (C→T) at position +6 of exon 7, resulting in inefficient inclusion of exon 7 in SMN2 transcripts. Therefore, certain forms of SMN2 lack exon 7 and are unstable and inactive.

[0034] In some embodiments, intracellular SMN protein levels may be increased by contacting motor neurons with both a recombinant nucleic acid encoding a recombinant SMN1 gene to promote intracellular expression of recombinant SMN protein and an ASO that modulates intracellular SMN2 splicing such that the percentage of cellular SMN2 transcripts containing exon 7 is increased, thereby increasing expression of full-length SMN protein from cellular SMN2 transcripts. In some embodiments, treatment is performed using a combination of both a recombinant nucleic acid encoding the SMN1 gene (also referred to herein as a recombinant SMN1 gene) and an ASO that increases full-length SMN2 mRNA (e.g., an ASO that increases intracellular levels of full-length SMN2 mRNA, e.g., by promoting the inclusion of exon 7 in SMN2 mRNA). In some embodiments, increasing intracellular levels of full-length SMN2 mRNA is useful in targeting multiple aspects of SMA and may be useful in treating a wide range of subjects with different disease severity, including patients with different types of SMA (including patients with different genomic copy numbers of the SMN2 gene).

[0035] In some embodiments, the recombinant SMN1 gene (e.g., an rAAV encoding SMN1) and the SMN2 ASO are administered simultaneously. In some embodiments, the recombinant SMN1 gene (e.g., an rAAV encoding SMN1) and the SMN2 ASO are administered sequentially.

[0036] In some embodiments, the combination treatment involves administering a composition that includes both a recombinant SMN1 gene and an SMN2 ASO formulated together. In some embodiments, the combination treatment involves administering a first composition comprising a recombinant SMN1 gene and a second separate composition comprising an SMN2 ASO. In some embodiments, the first composition and the second composition are administered simultaneously (at the same time or at different times during the same medical visit (e.g., the same visit to a hospital, clinic, or other medical facility where the subject is receiving treatment)). In some embodiments, the first composition and the second composition are administered sequentially to the subject, e.g., during consecutive medical visits in which either the first composition or the second composition is administered to the subject.

[0037] Thus, in some embodiments, the first composition and the second composition are administered separately to a subject at different times (e.g., different times of day, different days of the same week, or different weeks). In some embodiments, the first composition and the second composition are administered at different frequencies. In some embodiments, the composition comprising the recombinant SMN1 gene is administered less frequently than the composition comprising the SMN2 ASO.

[0038] Thus, in some embodiments, a recombinant SMN1 gene is administered to a subject before the subject is treated with an SMN2 ASO, while in other embodiments, the subject is treated with an SMN2 ASO before administration of the recombinant SMN1 gene.

[0039] In some embodiments, a subject may be treated with both i) a pharmaceutical composition containing a recombinant SMN1 gene and SMN2 ASO formulated together, and ii) a separate pharmaceutical composition containing either a recombinant SMN1 gene or SMN2 ASO. For example, in some embodiments, a subject may be first treated with a composition containing both a recombinant SMN1 gene and SMN2 ASO, followed by treatment with a composition containing either a recombinant SMN1 gene or SMN2 ASO. In some embodiments, a subject may be administered two or more doses of a composition containing both a recombinant SMN1 gene and SMN2 ASO, and two or more separate doses of a composition containing either a recombinant SMN1 gene or SMN2 ASO.

[0040] In some embodiments, an initial administration of the combination of the recombinant SMN1 gene and SMN2 ASO is followed by one, two, or more subsequent doses of the SMN2 ASO alone. In some embodiments, an initial administration of the combination of the recombinant SMN1 gene and SMN2 ASO is followed by one, two, or more subsequent doses of the recombinant SMN1 gene. In some embodiments, an initial administration of either the recombinant SMN1 gene or SMN2 ASO alone is followed by the administration of the combination of the recombinant SMN1 gene and SMN2 ASO.

[0041] The order and frequency of administration of compositions containing both recombinant nucleic acids and ASOs, and compositions containing either recombinant nucleic acids or ASOs, can be adjusted to suit individual treatments.

[0042] In some embodiments, pharmaceutical compositions comprising both a recombinant SMN1 gene (e.g., in a viral vector) and an SMN2 ASO, or pharmaceutical compositions comprising different doses of the recombinant SMN1 gene or SMN2 ASO, are provided.

[0043] A variety of assays exist for measuring SMN expression and activity levels in vitro. See, e.g., Tanguy et al., 2015, cited above. The methods described herein can also be used in conjunction with any other treatment for treating SMA or its symptoms. Wang et al., Consensus Statement for Standard of Care in Spinal Muscular See also Atropy (which discusses the current standard of care for SMA) and http: / / www.ncbi.nim.nih.gv / 3 / 4oc^s / IB 1352 / . For example, if nutrition 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 continuous positive airway pressure at night. Scoliosis surgery can be safely performed in individuals with SMA Type II and SMA Type III 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, which is incorporated herein by reference.

[0044] Recombinant nucleic acid encoding SMN1 In some embodiments, the combination therapy for treating SMA includes a recombinant nucleic acid encoding SMN1 (e.g., administered in a viral vector (such as rAAV)). In some embodiments, the recombinant nucleic acid encoding SMN1 (also referred to herein as a recombinant SMN1 gene) includes the SMN1 gene operably linked to a promoter (e.g., a promoter active in motor neurons). In some embodiments, the recombinant nucleic acid encoding SMN1 is provided in a non-viral vector (e.g., a non-viral plasmid). Meanwhile, in some embodiments, the recombinant nucleic acid encoding SMN1 is provided in a recombinant viral vector (e.g., 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.

[0045] In some embodiments, the recombinant SMN1 gene is administered to a subject in a viral vector. In some embodiments, the recombinant SMN1 gene is administered in a recombinant AAV genome that includes adjacent AAV inverted terminal repeats (ITRs). Thus, in some embodiments, recombinant viral particles (e.g., rAAV particles) that include a gene encoding SMN1 are administered to a subject together with SMN2 ASO.

[0046] Figure 2 provides a non-limiting example of a recombinant viral genome comprising an SMN1 gene operably linked to a promoter. Figure 2 shows the SMN1 gene flanked by AAV ITRs. The SMN1 gene contains a human SMN1 codon-optimized SMN1 open reading frame and is operably linked to the CB7 promoter (a chicken beta-actin promoter together 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 shown in Figure 2 is non-limiting; alternative SMN1 coding sequences, promoters, and other regulatory elements may be used.

[0047] In some embodiments, the rAAV genome is packaged within a viral capsid. In some embodiments, the capsid protein is a hu68 serotype capsid protein. However, other capsid proteins of other serotypes can also be used.

[0048] These and other aspects of the recombinant SMN1 gene are described in more detail in the following paragraphs.

[0049] SMN1 coding sequence: In some embodiments, a coding sequence encoding a wild-type human SMN protein (e.g., an SMN1 cDNA sequence) is provided. Nucleic acid sequences encoding human SMN1 are known in the art. For example, for non-limiting examples of nucleic acid sequences for 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 the amino acid sequence of a wild-type human SMN protein is provided in UniProtKB / Swiss-Prot:Q16637.1. For other publications describing SMN1 coding sequences, see, for example, WO2010129021A1 and WO2009151546A2 (the contents of which are incorporated herein by reference in their entireties).

[0050] In some embodiments, a coding sequence is provided that encodes a functional SMN protein, hi some embodiments, the functional SMN1 amino acid sequence is that of the human SMN1 protein or a sequence that shares 95% identity therewith.

[0051] In some embodiments, modified hSMN1 coding sequences are provided. In some embodiments, the modified hSMN1 coding sequence has less than about 80% identity to the full-length native hSMN1 coding sequence, preferably about 75% or less. 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., delivery using rAAV particles). In some embodiments, the modified hSMN1 coding sequence shares less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61%, or less.

[0052] The terms "percent identity (%)," "sequence identity," "percent sequence identity," or "percent identical," in the context of nucleic acid sequences, refer to the presence of matching residues in two sequences when aligned for correspondence. The length over which sequence identity is compared can span the entire genome, and preferably the entire length of a gene coding sequence or a fragment of at least about 500 to 5,000 nucleotides. However, identity between smaller fragments (e.g., fragments of at least about 9 nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 nucleotides, or more) may also be desired.

[0053] "Aligned" sequences or "alignment" refers to comparing multiple nucleic acid or protein (amino acid) sequences to a reference sequence, often including corrections for deletions or additions of bases or amino acids.

[0054] 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 the "Clustal X" program, the "MAP" program, the "PIMA" program, the "MSA" program, the "BLOCKMAKER" program, the "MEME" program, and the "Match-Box" program. Generally, any of these programs are used with default settings, although those skilled in the art can change these settings as needed. Alternatively, those skilled 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 these reference algorithms and programs. See, for example, J.D. Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

[0055] 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," and these programs are accessible via 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 that can be used to measure nucleotide sequence identity are also known in the art, including those included in the programs mentioned above. As another example, polynucleotide sequences can be compared using Fasta™ (a program included in GCG version 6.1). Fasta™ provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™, using the default Fasta™ parameters (string length 6 and NOP AM coefficient for scoring matrix) provided in GCG version 6.1 (incorporated herein by reference).

[0056] In some embodiments, the modified hSMN1 coding sequence is a codon-optimized sequence optimized for expression in the target species. As used herein, a "subject" is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate (such as a monkey, chimpanzee, baboon, or gorilla). In some embodiments, the subject is a human. Thus, in some embodiments, the SMN1 coding sequence is codon-optimized for expression in humans.

[0057] Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be performed using methods available online (e.g., GeneArt), published methods, or companies that provide codon optimization services (e.g., DNA2.0 (Menlo Park, CA)). One codon optimization method is described, for example, in U.S. International Patent Publication No. WO2015 / 012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. Patent Publication No. 2014 / 0032186 and U.S. Patent Publication No. 2006 / 0136184.

[0058] In some embodiments, the entire length of the open reading frame (ORF) is modified, while in some embodiments, only a fragment of the ORF is modified. By using one of these methods, a frequency can be applied to any given polypeptide sequence to obtain a nucleic acid fragment of the codon-optimized coding region that encodes the polypeptide. Thus, in some embodiments, a codon-optimized SMN1 coding sequence (e.g., codon-optimized hSMN1) is obtained. In some embodiments, the codons of one or more portions of the SMN1 coding sequence (e.g., up to the entire ORF) are optimized for expression in humans.

[0059] 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, each 80-90 nucleotides in length and spanning the length of the desired sequence, are synthesized by standard methods. These oligonucleotide pairs are synthesized so that, upon annealing, they form 80-90 base pair double-stranded fragments containing overhanging ends. For example, each oligonucleotide pair can be synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region complementary to the other oligonucleotide pair. The single-stranded end of each oligonucleotide pair is designed to anneal to the single-stranded end of another oligonucleotide pair. These oligonucleotide pairs are allowed to anneal, and then approximately five to six of these double-stranded fragments are allowed to anneal together via their overhanging single-stranded ends. The double-stranded fragments are then ligated together and cloned into a standard bacterial cloning vector (e.g., the TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif.). The constructs are then sequenced by standard methods. Several such constructs are prepared, each consisting of five to six 80-90 base pair fragments ligated together (i.e., approximately 500 base pair fragments), so that the entire desired sequence is present in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. This final construct is then cloned into a standard bacterial cloning vector and sequenced. Additional or alternative methods, including, for example, commercially available gene synthesis services, may also be used.

[0060] In some embodiments, the SMN1 cDNA sequence can be synthetically generated in vitro using techniques known in the art, such as PCR-based accurate synthesis (PAS) of long DNA sequences, as described in Xiong et al., PCR-based accurate synthesis (PAS). Accurate synthesis of long DNA sequences, Nature Protocols 1, 791-797 (2006). 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. See also Gordeeva et al, J Microbiol Methods. Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification. 2010 May; 81(2): 147-52. Epub 2010 Mar 10. See also the following patents relating to oligonucleotide synthesis and gene synthesis: Gene Seq. 2012 Apr; 6(1): 10-21, US8008005, and US7985565. Each of these documents is incorporated herein by reference. Additionally, kits and protocols for generating DNA via PCR are commercially available, including those that use polymerases including, but not limited to, Taq polymerase, OneTaq® (New England Biolabs), Q5® High-Fidelity DNA Polymerase (New England Biolabs), and GoTaq® G2 Polymerase (Promega). DNA can also be obtained from cells transfected with plasmids containing the hSMN sequences described herein.Kits and protocols are known and commercially available, including, but not limited to, QIAGEN Plasmid Kits, Chargeswitch® Pro Filter Plasmid Kits (Invitrogen), and GenElute™ Plasmid Kits (Sigma-Aldrich). Other techniques useful herein include sequence-specific isothermal amplification methods that eliminate the need for thermal cycling. These methods typically use a strand-displacing DNA polymerase, such as Bst DNA Polymerase, Large Fragment (New England Biolabs), instead of heat to separate double-stranded DNA. DNA can also be generated from RNA molecules by amplification using reverse transcriptase (RT), an RNA-dependent DNA polymerase. RT polymerizes a strand of DNA complementary to the original RNA template; the resulting strand of DNA is called cDNA. This cDNA can then be further amplified by PCR or isothermal methods, as outlined above. Custom DNA can also be obtained commercially from companies including, but not limited to, GenScript, GENEWIZ®, GeneArt® (Life Technologies), and Integrated DNA Technologies.

[0061] By "functional SMN1" is intended a gene encoding a native SMN protein, or a gene encoding another SMN protein that confers at least about 50%, at least about 75%, at least about 80%, at least about 90%, or about 100%, or more than 100%, the biological activity level of the native survival motor neuron protein, or a gene encoding a naturally occurring variant or polymorphism thereof that is not associated with disease. Furthermore, SMN2, an SMN1 homolog, also encodes an SMN protein, but is processed less efficiently to produce a functional protein. The degree of SMA exhibited by subjects who do not have a functional hSMN1 gene varies depending on the copy number of SMN2. Thus, in some subjects, the biological activity of the SMN protein may be less than 100% of that of the native SMN protein.

[0062] In some embodiments, such functional SMN has a sequence that is about 95% or more, or about 97% or more, or about 99% identical to a naturally occurring protein at the amino acid level. Such functional SMN proteins may also include naturally occurring polymorphisms. Identity can be determined by aligning 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)).

[0063] Percent identity can be readily determined for amino acid sequences spanning the entire length of a protein, the entire length of a polypeptide, about 32 amino acids, about 330 amino acids, or peptide fragments thereof, or the corresponding nucleic acid sequence coding sequence. Suitable amino acid fragments can be at least about 8 amino acids in length and can be up to about 700 amino acids in length. 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.

[0064] In some aspects, the modified SMN1 (e.g., hSMN1) gene described herein is engineered into a suitable genetic element (e.g., vector) (e.g., naked DNA, phage, transposon, cosmid, episome, etc.) useful for generating viral vectors and / or for delivery to host cells, which genetic element carries the SMN1 sequence. The selected vector can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-velocity ejection of DNA-coated pellets, viral infection, and protoplast fusion. Methods used to prepare such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook See, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0065] In some aspects, 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 may further comprise other regulatory sequences. In some aspects, the expression cassette is packaged within a viral vector capsid (e.g., a viral particle). Typically, such expression cassettes for generating viral vectors comprise the SMN1 (e.g., hSMN1) sequence described herein, which is flanked by a packaging signal and other expression control sequences (such as those described herein) of the viral genome. For example, for an AAV viral vector, the packaging signal is the 5' inverted terminal repeat (ITR) and the 3' ITR. The ITRs together with the expression cassette, once packaged into an AAV capsid, are referred to herein as the "recombinant AAV (rAAV) genome" or "vector genome" contained within the rAAV particle or capsid.

[0066] The term "expression" is used herein in its broadest sense and includes the production of RNA or the production of RNA and protein. With respect to RNA, the terms "expression" or "translation" specifically relate to the production of peptides or proteins. Expression can be transient or stable.

[0067] The term "translation" in the context of the present invention relates to the process in the ribosome whereby an mRNA chain controls the assembly of an amino acid sequence to produce a protein or peptide.

[0068] Promoter and regulatory elements: In some embodiments, the expression construct includes one or more regions containing sequences that promote expression of the coding sequence of the SMN1 gene (e.g., expression control sequences operably linked to the coding sequence). Examples of expression control sequences include, but are not limited to, promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences (e.g., promoters and enhancers) is contemplated herein.

[0069] In some embodiments, the expression cassette includes a promoter sequence as part of the expression control sequence, e.g., located between the 5' ITR sequence and the SMN1 coding sequence. Exemplary plasmids and vectors described herein use the ubiquitous chicken β-actin promoter (CB) in conjunction with the CMV immediate-early enhancer (CMV IE). Alternatively, other neuron-specific promoters can 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, which is incorporated herein by reference. Other neuron-specific promoters include the 67 kDa glutamic acid decarboxylase (GAD67) promoter, the homeobox Dlx5 / 6 promoter, the glutamate receptor 1 (GluRl) promoter, the preprotachykinin 1 (Tacl) promoter, the neuron-specific enolase (NSE) promoter, and the dopaminergic receptor 1 (Drdla) promoter. See, e.g., Delzor et al., Human Gene Therapy Methods. August 2012, 23(4):242-254. In another embodiment, the promoter is the GUSb promoter (http: / / www.jci.Org / articles / view / 41615#B30).

[0070] Other promoters (such as constitutive promoters and regulatable promoters) (e.g., WO2011 / 126808 and WO2013 / 04943) or promoters responsive to physiological cues may also be used. 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.

[0071] In addition to a promoter, the expression cassette and / or vector may include one or more other suitable transcription initiation sequences, termination sequences, enhancer sequences, effective RNA processing signals (such as splicing signals and polyadenylation (polyA) signals), sequences that stabilize cytoplasmic mRNA (e.g., WPRE), sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, optionally, 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. One example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include those suitable for CNS indications. In some embodiments, the expression cassette includes one or more expression enhancers. In some embodiments, the expression cassette includes two or more expression enhancers. These enhancers may be the same as each other or different from each other. For example, the enhancer can include a CMV immediate-early enhancer. This enhancer can be present in two copies located adjacent to each other. Alternatively, the dual copies of the enhancer can be separated by one or more sequences. In yet another embodiment, the expression cassette further includes an intron (e.g., a chicken beta-actin intron). Other suitable introns include those known in the art, such as those described in WO2011 / 126808. In some embodiments, introns incorporated upstream of the coding sequence improve 5'-capping and stability of the mRNA. Optionally, one or more other sequences can be selected to stabilize the mRNA. One example of such a sequence is a modified WPRE sequence, which can be engineered into a position upstream of the polyA sequence and downstream of the coding sequence (see, e.g., MA Zanta-Boussif, et al., Gene Therapy (2009) 16:605-619).

[0072] In some embodiments, such control sequences are "operably linked" to the SMN1 gene sequence. As used herein, the term "operably linked" refers to expression control sequences that are adjacent to the gene of interest, that act in trans to regulate the gene of interest, or that act at a distance to regulate the gene of interest.

[0073] Recombinant viral vectors: In some embodiments, an adeno-associated virus vector is provided comprising an AAV capsid and at least one expression cassette. In some embodiments, the at least one expression cassette comprises a nucleic acid sequence encoding SMN1 and an expression control sequence that directs expression of the SMN1 sequence in a host cell. The rAAV vector gene may also comprise 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 a deleted version thereof (AITR), which may be used for convenience and to facilitate 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, the resulting vector may be referred to as pseudotyped. Typically, the rAAV vector genome comprises 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 has been reported, termed the AITR, in which the D-sequence and terminal resolution site (trs) are deleted. In other embodiments, the full-length AAV The 5'ITR and the AAV 3'ITR are used.

[0074] The ITR sequences of the nucleic acids or nucleic acid vectors described herein can be from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or can be from multiple serotypes. 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 (Copyright) 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.

[0075] In some embodiments, the rAAV nucleic acid or rAAV genome can be single-stranded (ss). Meanwhile, in some embodiments, the rAAV nucleic acid or rAAV 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 comprises an SMN1 gene and one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the expression construct. In some embodiments, the nucleic acid is encapsidated by the viral capsid.

[0076] Thus, in some embodiments, an AAV particle comprises a viral capsid and a nucleic acid vector described herein, wherein the nucleic acid vector is encapsidated by the viral capsid. In some embodiments, the viral capsid comprises 60 capsid protein subunits, including VP1, VP2, and VP3. In some embodiments, the VP1, VP2, and VP3 subunits are present in the capsid in a ratio of about 1:1:10, respectively.

[0077] In some embodiments, recombinant adeno-associated viruses (rAAVs) are AAV DNase-resistant particles having an AAV protein capsid in which a nucleic acid sequence for delivery to a target cell is packaged. In some embodiments, the AAV capsid is composed of 60 capsid protein subunits (VP1, VP2, and VP3) arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. AAV capsids can be selected from those known to those skilled in the art, including variants thereof. In some embodiments, the AAV capsid is selected to efficiently transduce neural 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. See WO2018160585A2, WO2018160582A1, Royo, et al, Brain Res, 2008 Jan, 1190:15-22, Petrosyan et al, Gene Therapy, 2014 Dec, 21(12):991-1000, Holehonnur et al, BMC Neuroscience, 2014, 15:28, and Cearley et al, Mol Ther. 2008 Oct;16(10):1710-1718 (each of which is incorporated herein by reference). Other AAV capsids useful herein include AAVrh39, AAVrh20, AAVrh25, AAV10, AAVbb1, and AAVbb2, and variants thereof. Other AAV serotypes can also be selected as the source of capsids for AAV viral vectors (DNase-resistant viral particles), including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAVrhl64Rl, AAVrhl64R2, AAVrhl8, and any variant of AAV known or described herein, or yet to be discovered.See, for example, U.S. Published Patent Application No. 2007-0036760-A1, U.S. Published Patent Application No. 2009-0197338-A1, EP1310571. See also WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent No. 7790449 and U.S. Patent No. 7282199 (AAV8), WO2005 / 033321 and U.S. Patent No. 7,906,111 (AAV9), and WO2006 / 110689, and WO2003 / 042397 (rh10). Alternatively, recombinant AAV based on any of the AAVs described can be used as a source of AAV capsid. These documents also describe other AAVs that can be selected to generate AAV, and these documents are incorporated by reference. In some embodiments, AAV capsids for use in viral vectors can be generated by mutagenesis (e.g., by insertion, deletion, or substitution) of one of the aforementioned AAV capsids or its encoding nucleic acid. In some embodiments, the AAV capsid is a chimera containing domains derived from two, three, four, or more of the aforementioned AAV capsid proteins. In some embodiments, the AAV capsid is a mosaic of Vp1, Vp2, and Vp3 monomers derived from two or three different AAVs or recombinant AAVs. In some embodiments, a rAAV composition comprises one or more of the aforementioned capsids. The term "variant," as used herein with respect to AAV, 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 percent sequence identity across the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid can include variants that can vary by up to about 10% from any of the described or known AAV capsid sequences, i.e., 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 the 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.

[0078] In some embodiments, self-complementary AAVs are provided. The associated abbreviation "sc" 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. During infection, rather than waiting for the cell to mediate second-strand synthesis, the two complementary half-strands of scAAV join to form a single double-stranded DNA (dsDNA) unit ready for rapid replication and transcription. See, for example, D. M. McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis," Gene Therapy, (August 2001), Vol. 8, Number 16, Pages 1248-1254. Self-complementary 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.

[0079] Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, U.S. Published Patent Application No. 2007 / 0036760 (February 15, 2007), U.S. Patent No. 7,790,449, U.S. Patent No. 7,282,199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and U.S. Patent No. 7,588,772B2. In one system, a construct encoding a transgene flanked by ITRs and a construct(s) encoding rep and cap are transiently transfected into a producer cell line. In a second system, a construct encoding a transgene flanked by ITRs is transiently transfected into a packaging cell line that stably supplies rep and cap. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, necessitating the separation of rAAV from contaminating viruses. Systems have also been developed that do not require helper virus infection for AAV recovery; in these systems, the necessary helper functions (e.g., adenovirus E1, E2a, VA, and E4, or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied in trans by the system. In these systems, helper functions can be supplied by transiently transfecting cells with constructs encoding the necessary helper functions, or cells can be engineered to stably contain genes encoding helper functions whose expression 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 reviews of such production systems, see, for example, Zhang et al. et al, 2009, “Adenovirus-adeno-associated See, “AAV 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 entireties. Methods for 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 entireties: 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.

[0080] Optionally, the SMN1 gene described herein can be used to generate viral vectors other than rAAV, and these viral vectors 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. Suitably, when one of these other vectors is generated, it is generated as a replication-defective viral vector.

[0081] A "replication-defective virus" or "replication-defective viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged within a viral capsid or envelope, and any viral genomic sequences packaged within the viral capsid or envelope are replication-defective, i.e., they are unable to generate progeny virions but retain the ability to infect target cells. In some embodiments, the genome of the viral vector (which may be engineered to be "weakened" and contain only a transgene of interest flanked by signals necessary for amplification and packaging of the artificial genome) does not contain genes encoding enzymes required for replication, although such genes can be supplied during production. Thus, such viral vectors are considered safe for use in gene therapy because replication and infection by progeny virions cannot occur unless viral enzymes required for replication are present. Such replication-defective viruses may be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrating or non-integrating), or of another suitable viral source.

[0082] 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 such host cell 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.

[0083] Oligomeric compounds that increase production of full-length SMN2 mRNA In some embodiments, a combination therapy for treating SMA includes an ASO complementary to the pre-mRNA encoding SMN2 (also referred to in the present application as an SMN2 ASO). In some embodiments, the ASO increases full-length SMN2 mRNA. In some embodiments, the ASO alters splicing of the SMN2 pre-mRNA. In some embodiments, the ASO promotes the inclusion of exon 7 in the SMN2 mRNA. Some sequences and regions useful for altering SMN2 splicing can be found in PCT / US06 / 024469 (published as WO / 2007 / 002390) and WO2018014041A2, which are incorporated by reference in their entirety for all purposes.

[0084] In some embodiments, the SMN2 ASO effectively modulates SMN2 splicing, resulting in increased inclusion of exon 7 in SMN2 mRNA and ultimately increased SMN2 protein containing amino acids corresponding to exon 7. Such alternative SMN2 proteins are 100% identical to the wild-type SMN protein.

[0085] An ASO that effectively modulates SMN2 mRNA expression to produce functional SMN protein is considered an active ASO. Modulation of SMN2 expression can be measured in bodily fluids, which may or may not contain cells, tissues, or organs of an animal. Methods for obtaining samples for analysis (such as bodily fluids (e.g., sputum, serum, CSF), tissues (e.g., biopsy samples), or organs) and preparing the samples to enable analysis are well known to those skilled in the art. The effectiveness of treatment can be assessed by measuring biomarkers associated with target gene expression in one or more biological fluids, tissues, or organs obtained from an animal contacted with one or more of the compositions described herein.

[0086] In some embodiments, an increase in full-length SMN2 mRNA refers to a higher intracellular level of full-length SMN2 mRNA compared to a reference level (such as 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 the cell or organism (e.g., as described in the Examples below) or by an assay technique such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, monitoring gene expression on a microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), nucleic acid sequencing, Western blot, radioimmunoassay (RIA), other immunoassay, 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 in a sample obtained from a subject).

[0087] In some embodiments, the level of full-length SMN2 mRNA in the sample obtained from the subject receiving SMN2 ASO treatment can be compared with the level of full-length SMN2 mRNA in the subject not treated with SMN2 ASO, thereby determining the increase degree of full-length SMN2 mRNA caused by SMN2 ASO.In some embodiments, the reference level of full-length SMN2 mRNA is obtained from the same subject before SMN2 ASO is administered.In some embodiments, the reference level of full-length SMN2 mRNA is the range determined by the group of subjects not administered with SMN2 ASO.

[0088] In some embodiments, the increase in the level of full-length SMN2 mRNA is, for example, greater than 1-fold, greater than 1.5-5-fold, greater than 5-10-fold, greater than 10-50-fold, greater than 50-100-fold, greater than about 1.1-fold, greater than about 1.2-fold, greater than about 1.5-fold, greater than about 2-fold, greater than about 3-fold, greater than about 4-fold, greater than about 5-fold, greater than about 6-fold, greater than about 7-fold, greater than about 8-fold, greater than about 9-fold, greater than about 10-fold, greater than about 15-fold, greater than about 20-fold, greater than about 30-fold, greater than about 40-fold, greater than about 50-fold, greater than about 60-fold, greater than about 70-fold, greater than about 80-fold, greater than about 90-fold, greater than about 100-fold, or more than that, compared to a reference value.

[0089] In some embodiments, it can be determined whether an SMN2 ASO has increased full-length SMN2 mRNA in a subject to which the SMN2 ASO has been administered by comparing the ratio of full-length SMN2 mRNA to a shorter SMN2 mRNA (e.g., SMN2 mRNA that does not contain exon 7) 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 that does not contain 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 that does not contain exon 7) in a subject administered an SMN2 ASO is, for example, greater than 1x, greater than 1.5-5x, greater than 5-10x, greater than 10-50x, greater than 50-100x, greater than about 1.1x, greater than about 1.2x, greater than about 1.5x, greater than about 2x, greater than about 3x, greater than about 4x, greater than about 5x, greater than about 6x, greater than about 7x, greater than about 8x, greater than about 9x, greater than about 10x, greater than about 15x, greater than about 20x, greater than about 30x, greater than about 40x, greater than about 50x, greater than about 60x, greater than about 70x, greater than about 80x, greater than about 90x, greater than about 100x, or more, compared to a reference ratio.

[0090] 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 a subject at risk of having SMA before treatment. In some embodiments, production of exon 7-containing SMN protein is indicated by an increase in SMN2 The increase in the subject to which the ASO is administered is accompanied by an enhancement of exon 7-containing SMN protein levels, e.g., an enhancement of at least about 1-fold, at least about 1.5-5-fold, at least about 5-10-fold, at least about 10-50-fold, at least about 50-100-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 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. Contacting a bodily fluid, organ, or tissue with one or more compositions can result in expression of SMN1 and modulate expression of SMN2 in cells of the bodily fluid, organ, or tissue. Effective amounts of the compositions can be determined by monitoring the effect on expression of functional SMN protein by recombinant SMN1 gene and SMN2 ASO administered to a subject or contacted with cells.

[0091] 1. Antisense oligonucleotides (ASOs) In some embodiments, an ASO comprising a sequence complementary to a nucleic acid encoding human SMN2 is provided for use in treating a disease or condition associated with survival motor neuron protein (SMN), such as spinal muscular atrophy (SMA), where the treatment is carried out in conjunction with, for example, a recombinant SMN1 gene. In some embodiments, an ASO comprising a sequence complementary to a nucleic acid encoding human SMN2 is provided for use in treating a disease or condition associated with survival motor neuron protein (SMN), where the treatment is carried out in conjunction with, for example, a recombinant SMN1 gene, by administering the ASO directly to the central nervous system (CNS) or CSF.

[0092] 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 conjugated groups and / or terminal groups. In some embodiments, an oligomeric compound is an antisense oligonucleotide (ASO). As used herein, "antisense oligonucleotide" or "ASO" refers to an oligomeric compound that is at least partially complementary to a target nucleic acid to which it hybridizes and that produces at least one antisense activity as a result of such hybridization.

[0093] In some cases, the antisense oligonucleotide (ASO) increases full-length SMN protein in the subject. In some cases, the ASO increases full-length SMN 2 in the subject. mRNA. In some embodiments, the ASO that increases full-length SMN2 mRNA is an antisense oligonucleotide complementary to a nucleic acid encoding SMN2. In some embodiments, the ASO increases full-length SMN2 mRNA by altering the splicing pattern of SMN2 pre-mRNA. In some embodiments, the ASO promotes exon skipping during splicing of SMN2 pre-mRNA. In some embodiments, the ASO promotes the inclusion of exon 7 in SMN2 mRNA. In some embodiments, the ASO is designed to promote the inclusion of exon 7 in SMN2 mRNA by targeting intron 6, intron 7, or the boundary between exon 7 and an adjacent intron of SMN2 pre-mRNA. In some embodiments, the ASO comprises a nucleobase sequence complementary to intron 6 of SMN2 pre-mRNA. In some embodiments, the ASO comprises a nucleobase sequence complementary to exon 6 of SMN2 pre-mRNA. In some embodiments, the ASO comprises a nucleobase sequence complementary to intron 7 of SMN2 pre-mRNA. In some embodiments, SMN2 The ASO targeting intron 7 of 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. Examples of sequences and regions useful for altering SMN2 splicing, including but not limited to, can be found in PCT / US06 / 024469, which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the antisense oligonucleotide has a nucleobase sequence complementary to intron 7 of SMN2. Examples of such nucleobase sequences include, but are not limited to, those listed in the table below. [Table 1]

[0094] In some embodiments, the ASO targets intron 7 of 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 all nucleobases of SEQ ID NO:1. In some embodiments, the ASO has a nucleobase sequence consisting of all 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).

[0095] 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 example of the nucleotide sequence of nusinersen is UCACUUUCAUAAUGCUGG-3' (SEQ ID NO: 26). The active substance (nusinersen (also referred to as ISIS 396443)) is 5'- Me U Me CA Me C Me U Me U Me U Me CA Me UAA Me UG Me C Me It is a uniformly modified 2'-O-(2-methoxyethyl) phosphorothioate antisense oligonucleotide consisting of 18 nucleotide residues and having the sequence UGG-3' (SEQ ID NO: 25).

[0096] The chemical name nusinersen sodium corresponds to the molecular formula C234H323N61O128P17S17Na17, which 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-thio Cytidylyl-(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) ethyl)-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, with a relative molecular mass of 7501.0 g / mol and the structure shown in Figure 3.

[0097] Antisense is an effective means for regulating 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 regulating gene expression via antisense mechanisms, including target-occupancy-based antisense mechanisms. In one aspect, the antisense compounds provided herein regulate the splicing of target genes. Such regulation includes promoting or suppressing exon inclusion. Further provided herein are antisense compounds that target cis-splicing regulatory elements present in pre-mRNA molecules, including exonic splicing enhancers, exonic splicing silencers, intronic splicing enhancers, and intronic splicing silencers. Disrupting cis-splicing regulatory elements is believed to alter splice site selection, which can alter the composition of splice products.

[0098] Eukaryotic pre-mRNA processing is a complex process requiring multiple signals and protein factors to achieve proper mRNA splicing. Spliceosome-mediated exon specification requires more than just the standard 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 as repressing or enhancing the utilization of splice donor or splice acceptor sites, depending on their site and mode of action (Yeo et al. 2004, Proc. Natl. Acad. Sci. USA 101(44):15700-15705). The binding of specific proteins (trans-factors) to these regulatory sequences guides the splicing process, promoting or suppressing the use of specific splice sites, thereby regulating the ratio of spliced ​​products (Scamborova et al. 2004, Mol. Cell. Biol. 24(5):1855-1869; Hovhannisyan and Carstens, 2005, Mol. Cell. Biol. 25(1):250-263; Minovitsky et al. 2005, Nucleic Acids Res. 33(2):714-724).

[0099] In some embodiments, antisense oligonucleotides contain one or more modifications compared to naturally occurring oligomeric oligonucleotides (such as DNA or RNA). Such modified antisense oligonucleotides can have one or more desirable properties. In some embodiments, the modifications change the antisense activity of the antisense oligonucleotide, for example, by increasing the affinity of the antisense oligonucleotide for the target nucleic acid, increasing its resistance to one or more nucleases, and / or changing the pharmacokinetics or tissue distribution of the oligonucleotide. In some embodiments, the modified antisense oligonucleotide contains one or more modified nucleosides, and / or one or more modified nucleoside linkages, and / or one or more conjugated groups.

[0100] a. modified nucleosides In some embodiments, antisense oligonucleotides comprise one or more modified nucleosides. Such modified nucleosides may comprise modified sugars and / or modified nucleobases. In some embodiments, the inclusion of such modified nucleosides in oligonucleotides provides increased affinity for target nucleic acids and / or improved stability, including, but not limited to, increased resistance to degradation by nucleases and / or improved toxicity and / or uptake characteristics of the modified oligonucleotides.

[0101] i. Nucleobase The naturally occurring base moieties of nucleosides are heterocyclic bases, typically purines and pyrimidines. In addition to "unmodified" or "natural" nucleobases (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, the modified nucleobase is a nucleobase whose structure is substantially similar to that of the parent nucleobase, such as, for example, 7-deazapurine, 5-methylcytosine, or G-clamp. In some embodiments, the nucleobase mimic comprises a more complex structure (e.g., a tricyclic phenoxazine nucleobase mimic). Methods for preparing modified nucleobases are well known to those skilled in the art.

[0102] ii. Modified sugars and sugar substitutes The antisense oligonucleotides of the present application may optionally contain one or more nucleosides having a modified sugar moiety compared to the natural sugar. Oligonucleotides containing sugar-modified nucleosides may have enhanced stability against nucleases, increased binding affinity, or some other beneficial biological property. Such modifications include, but are not limited to, adding substituents, bridging non-geminal ring atoms to form bicyclic nucleic acids (BNAs), substituting ribosyl ring oxygen atoms for S, N(R), or C(R)(R) (R = H, C-C). 12alkyl, or protecting group), and combinations of such modifications, such as 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO2008 / 101157 published August 21, 2008 for other disclosed 5',2'-bissubstituted nucleosides), or ribosyl ring oxygen atom replaced with S with further substitution at the 2' position (see published U.S. patent application US20050130923 published June 16, 2005), or 5'-position substitution of BNAs (see PCT International Application WO2007 / 134181 published November 22, 2007, in which LNAs are substituted, for example, with a 5'-methyl group or a 5'-vinyl group).

[0103] Examples of nucleosides having modified sugar moieties include, but are not limited to, nucleosides containing a 5'-vinyl substituent, nucleosides containing a 5'-methyl (R or S) substituent, nucleosides containing a 4'-S substituent, nucleosides containing a 2'-F substituent, nucleosides containing a 2'-OCH substituent, and nucleosides containing a 2'-O(CH2)2OCH3 substituent. The 2'-position substituent can be allyl, amino, azido, thio, O-allyl, O-C1-C 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 C-C 10 It can also be selected from the group consisting of alkyl.

[0104] Examples of bicyclic nucleic acids (BNAs) include, but are not limited to, nucleosides comprising a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. 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' (see PCT / US2008 / 068922, published July 15, 2008). No. 7,399,845), 4'-CH2-N(OCH3)-2' (see PCT / US2008 / 064591), 4'-CH2-ON(CH3)-2' (see published U.S. patent application US2004-0171570 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).

[0105] In some embodiments, the modified sugar moiety comprising the modified nucleoside is not a bicyclic sugar moiety. In some embodiments, the sugar ring of the nucleoside can be modified at any position. Examples of useful sugar modifications include, but are not limited to, those compounds that contain a sugar substituent selected from OH, F, O-alkyl, S-alkyl, N-alkyl, or O-alkyl-O-alkyl, where such alkyl, alkenyl, and alkynyl can be 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 located at the 2' position of the sugar.

[0106] 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-C-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 C-C 10 alkyl).

[0107] In some embodiments, modified nucleosides suitable for use in the present invention are 2-methoxyethoxy, 2'-Omethyl (2'-OCH3), 2'-fluoro (2'-F).

[0108] In some embodiments, the modified nucleoside has a substituent at the 2' position, O[(CH2) 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 CH3]2, where n and m are from 1 to about 10. Other 2'-sugar substituents include C1-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.

[0109] In some embodiments, modified nucleosides contain a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitutions have been shown to improve 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).

[0110] In some embodiments, the 2'-sugar substituent is located at 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, specifically 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.

[0111] In some embodiments, suitable nucleosides have a sugar surrogate (such as a 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. 4,981,957, U.S. 5,118,800, U.S. 5,319,080, U.S. 5,359,044, U.S. 5,393,878, U.S. 5,446,137, U.S. 5,466,786, U.S. 5,514,785, U.S. 5,519,134, U.S. 5,567,811, U.S. 5,577,812, U.S. 5,578,813, U.S. 5,579,814, U.S. 5,579,815, U.S. 5,579,816, U.S. 5,579,817, U.S. 5,579,818, U.S. 5,579,819 ... No. 6,427, U.S. Pat. No. 5,591,722, U.S. Pat. No. 5,597,909, U.S. Pat. No. 5,610,300, U.S. Pat. No. 5,627,053, U.S. Pat. No. 5,639,873, U.S. Pat. No. 5,646,265, U.S. Pat. No. 5,658,873, U.S. Pat. No. 5,670,633, U.S. Pat. No. 5,792,747, and U.S. Pat. No. 5,700,920, each of which is incorporated herein by reference in its entirety.

[0112] 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' terminal position of the oligonucleotide.

[0113] B internucleoside bond Antisense oligonucleotides may optionally contain one or more modified internucleoside linkages. Two major 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 alter (typically improve) the nuclease resistance of oligonucleotides. In some embodiments, linkages with chiral atoms can be prepared as racemic mixtures (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.

[0114] The antisense oligonucleotides described herein may contain one or more asymmetric centers and may thus 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 include all such possible isomers as well as their racemic and optically pure forms.

[0115] 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.

[0116] c. Length In some embodiments, the present invention provides antisense oligonucleotides of any of a range of lengths. In some embodiments, the antisense compound or antisense oligonucleotide comprises or consists of X through Y linked nucleosides, where X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, with the proviso that X through Y are the numbers. For example, in some embodiments, the antisense compound or antisense oligonucleotide may be 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9-10 pieces, 9-11 pieces, 9-12 pieces, 9-13 pieces, 9-14 pieces, 9-15 pieces, 9-16 pieces, 9-17 pieces, 9-18 pieces, 9-19 pieces, 9-20 pieces, 9-21 pieces, 9-22 pieces, 9-23 pieces, 9-2 4 pieces, 9~25 pieces, 9~26 pieces, 9~27 pieces, 9~28 pieces, 9~29 pieces, 9~30 pieces, 10~11 pieces, 10~12 pieces, 10~13 pieces, 10~14 pieces, 10~15 pieces, 10~16 pieces, 10~17 pieces, 10~18 pieces, 10~19 pieces, 10~20 pieces, 10~21 pieces, 10~22 pieces, 10~23 pieces, 10~24 pieces, 10~25 pieces, 10~26 pieces, 10~27 pieces, 10~28 pieces, 10~29 pieces, 10~ 30 pieces, 11~12 pieces, 11~13 pieces, 11~14 pieces, 11~15 pieces, 11~16 pieces, 11~17 pieces, 11~18 pieces, 11~19 pieces, 11~20 pieces, 11~21 pieces, 11~22 pieces, 11~23 pieces, 11~24 pieces, 11~25 pieces, 11~26 pieces, 11~27 pieces, 11~28 pieces, 11~29 pieces, 11~30 pieces, 12~13 pieces, 12~14 pieces, 12~15 pieces, 12~16 pieces, 12~17 pieces, 12~ 18 pieces, 12~19 pieces, 12~20 pieces, 12~21 pieces, 12~22 pieces, 12~23 pieces, 12~24 pieces, 12~25 pieces, 12~26 pieces, 12~27 pieces, 12~28 pieces, 12~29 pieces, 12~30 pieces,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, 16-2 2, 16-23, 16-24, 16-25, 16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17~ 27, 17-28, 17-29, 17-30, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19 ~22, 19~23, 19~24, 19~25, 19~26, 19~29, 19~28, 19~29, 19~30, 20~21, 20~22, 20~23, 20~24, 20~25, 20~26, 20~27, 20~28, 20~29, 2 0~30, 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, 25-30It contains 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides, or is composed of these numbers of linked nucleosides.

[0117] In some embodiments, the antisense compound or antisense oligonucleotide is 15 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 16 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 17 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 18 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 19 nucleosides in length. In some embodiments, the antisense compound or antisense oligonucleotide is 20 nucleosides in length.

[0118] d. oligonucleotide motif In some embodiments, antisense oligonucleotides have chemically modified subunits arranged in a specific orientation 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, with each nucleoside containing a 2-MOE sugar moiety. In some embodiments, antisense oligonucleotides are uniformly modified, with each nucleoside containing a 2'-OMe sugar moiety. In some embodiments, antisense oligonucleotides are uniformly modified, with each nucleoside containing a morpholino sugar moiety.

[0119] 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.

[0120] 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.

[0121] In some embodiments, one or more alternating regions in an alternating motif include more than one nucleoside of one type. For example, an oligomeric compound may include one or more regions of any of the following nucleoside motifs: Nu1 Nu1 Nu2 Nu2 Nu1 Nu1, Nu1 Nu2 Nu2 Nu1 Nu2 Nu2, Nu1 Nu1 Nu2 Nu1 Nu1 Nu2, Nu1 Nu2 Nu2 Nu1 Nu2 Nu1 Nu1 Nu2 Nu2, Nu1 Nu2 Nu1 Nu2 Nu1 Nu1, Nu1 Nu1 Nu2 Nu1 NU2 Nu1 Nu2, Nu1 Nu2 Nu1 Nu2 Nu1 Nu1, Nu1 Nu2 Nu2 Nu1 Nu1 Nu2 Nu2 Nu1 Nu2 Nu1 Nu2 Nu1 Nu1, Nu2 Nu1 Nu2 Nu2 Nu1 Nu1 Nu2 Nu2 Nu1 Nu2 Nu1 Nu2 Nu1 Nu1, or Nu1 Nu2 Nu1 Nu2 Nu2 Nu1 Nu1 Nu2 Nu2 Nu1 Nu2 Nu1 Nu2 Nu1 Nu1, In the sequence, 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 nucleoside or an unmodified RNA nucleoside.

[0122] 2. Oligomeric compounds In some embodiments, an oligomeric compound is composed solely of an oligonucleotide. In some embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugated and / or terminal groups. Such conjugated and / or terminal groups can be added to an oligonucleotide having any of the chemical motifs described herein. Thus, for example, an oligomeric compound comprising an oligonucleotide having one or more regions of alternating nucleosides can include a terminal group.

[0123] a. Complex group In some embodiments, oligonucleotides are modified by the addition of one or more conjugated groups. Generally, conjugated groups modify one or more properties of the oligomeric compound to which they are attached, including, but not limited to, pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugated groups are routinely used in chemistry and are linked to a parent compound (e.g., an oligomeric compound, such as an oligonucleotide) either directly or via an optional conjugated linking moiety or group. Conjugated groups can 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 conjugated groups have been reported previously, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al. al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers (e.g., hexyl-S-tritylthiol) (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains (e.g., 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 (e.g., 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. al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0124] In some embodiments, the conjugate group comprises an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethicin, barbiturates, cephalosporins, sulfa drugs, antidiabetics, antibacterials, or antibiotics. Oligonucleotide-drug conjugates and their preparation are described in U.S. Patent Application Serial No. 09 / 334,130.

[0125] Representative U.S. patents that teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S. 4,828,979, U.S. 4,948,882, U.S. 5,218,105, U.S. 5,525,465, U.S. 5,541,313, U.S. 5,545,730, U.S. 5,552,538, U.S. 5,578,717, U.S. 5,580,731, U.S. 5,591,584, U.S. 5,109,124, U.S. 5 ,118,802, US 5,138,045, US 5,414,077, US 5,486,603, US 5,512.439, US 5,578,718, US 5,608,046, US 4,587,044, US 4,605,735, US 4,667,025, US 4,762,779, US 4,789,737, US 4,824,941, US 4,835,263, US 4,876,335, US 4,904,582, US 4,958,013, US 5,082,830, US 5,112,963, US 5,214,136, US 5,082,830, US 5,112,963, US 5,214,136, US 5,245,022, US 5,254,469, US 5,258,506, US 5,262,536, US 5,272,250, US 5,292,873, US 5,317,098, US 5,371,241, US 5,391,777 23, U.S. 5,416,203, U.S. 5,451,463, U.S. 5,510,475, U.S. 5,512,667, U.S. 5,514,785, U.S. 5,565,552, U.S. 5,567,810, U.S. 5,574,142, U.S. 5,585,481, U.S. 5,587,371, U.S. 5,595,726, U.S. 5,597,696, U.S. 5,599,923, U.S. 5,599,928, and U.S. 5,688,941. Conjugation groups can be attached to one or both ends of the oligonucleotide (terminal conjugation groups) and / or at any internal position.

[0126] b.Terminal group In some embodiments, the oligomeric compounds include terminal groups at one or both ends. In some embodiments, the terminal groups may include any of the composite groups described herein. In some embodiments, the terminal groups may include additional nucleosides and / or inverted abasic nucleosides. In some embodiments, the terminal groups are stabilizing groups.

[0127] In some embodiments, oligomeric compounds contain one or more terminal stabilizing groups that enhance properties (e.g., nuclease stability). Stabilizing groups include cap structures. As used herein, the term "cap structure" or "terminal cap moiety" refers to a chemical modification that can be added to both or one of the 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 detailed information, see, but are not limited to, Wincott et al., International PCT Publication No. WO 97 / 26270; Beaucage and Tyer, 1993, Tetrahedron 49, 1925; U.S. Patent Application Publication No. US 2005 / 0020525; and WO 03 / 004602).

[0128] 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 the target nucleic acid. In some embodiments, the terminal group is a non-nucleoside terminal group. Such non-terminal group can be any terminal group other than a nucleoside.

[0129] C. oligomeric compound motif In some embodiments, the oligomeric compound is T-(Nu1) n1 ,-(Nu2) n2 -(Nu1) n3 -(Nu2) n4 -(Nu1) n5 -Contains the motif T2, in which: Nu1 is the first type of nucleoside, Nu2 is a second type of nucleoside, n1 and n5 each independently represent 0 to 3; The sum of n2 and n4 is 10 to 25, n3 is 0 to 5, Each T1 and T2 is independently H, a hydroxyl protecting group, an optionally linked conjugated group, or a capping group.

[0130] 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. [Table A]

[0131] 3. Antisense In some embodiments, the oligomeric compounds are antisense compounds. Thus, in some embodiments, the oligomeric compounds exhibit antisense activity upon hybridization with a target nucleic acid (e.g., a target pre-mRNA or a target mRNA).

[0132] a. Hybridization In some embodiments, an antisense compound specifically hybridizes to a target nucleic acid when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired (e.g., physiological conditions in the case of in vivo assays or therapeutic treatments, or conditions under which the assay is performed in the case of in vitro assays).

[0133] Thus, "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 that undergo hybridization by the antisense compound. Stringent conditions are sequence-dependent and will vary under different circumstances; the "stringent conditions" under which an antisense oligonucleotide hybridizes to a target sequence are determined by the nature and composition of the antisense oligonucleotide and the assay in which the antisense oligonucleotide is tested.

[0134] It is known in the art that affinity modification of nucleotides can increase the number of tolerated mismatches compared to unmodified compounds.Similarly, certain nucleobase sequences may be more tolerant to mismatches than other nucleobase sequences.Those skilled in the art have the ability to determine the appropriate number of mismatches between oligonucleotides or between antisense oligonucleotides and target nucleic acids, and this determination is carried out by, for example, determining melting temperature (Tm).Tm or ATM can be calculated by methods known to those skilled in the art.For example, those skilled in the art can use the method described in Freier et al. (Nucleic Acids Research, 1997,25,22:4429-4443) to evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA duplexes.

[0135] b. pre-mRNA processing In some embodiments, the antisense compounds provided herein are complementary to pre-mRNA. In some embodiments, such antisense compounds alter the splicing of pre-mRNA. In some embodiments, the ratio of a variant of a mature mRNA corresponding to the target pre-mRNA to another variant of that mature mRNA is altered. In some embodiments, the ratio of a variant of a protein expressed from the target pre-mRNA to another variant of the protein is altered. Certain oligomeric compounds and nucleobase sequences that can be used to alter pre-mRNA splicing are described in, for example, 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, US2006 / 0172962, US2007 / 002390, US2005 / 0074801, US2007 / 0105807, US2005 / 0054836, WO2007 / 090073, WO2007 / 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. al., American J. of Human Genetics (April 2008) 82, 1-15, each of which is incorporated by reference in its entirety for all purposes. In some embodiments, the splicing-altering antisense sequences are modified according to the motifs described in this application.

[0136] In some embodiments, the ASO or oligomeric compound may include one or more modifications described in WO / 2018 / 014043 (PCT / US2017 / 042465), WO / 2018 / 014042 (PCT / US2017 / 042464), WO / 2018 / 014041 (PCT / US2017 / 042463), the contents of which are incorporated herein by reference in their entireties.

[0137] Combination Administration and Combination Treatment In some embodiments, a recombinant SMN1 gene (e.g., in a viral vector (e.g., rAAV)), an SMN2 ASO, or a combination thereof, is delivered to a subject described herein in a "therapeutically effective" amount (e.g., via simultaneous or sequential administration) to achieve a desired result (e.g., treatment of SMA or one or more symptoms thereof). In some embodiments, the desired result includes reducing muscle weakness, increasing muscle strength and tone, preventing or reducing scoliosis, or maintaining or improving respiratory health, or reducing tremors or twitching. Other desired endpoints may be determined by a physician.

[0138] In some embodiments, a combination of a recombinant SMN1 gene and an SMN2 ASO is delivered to a subject to increase weight. ASO combinations are delivered to a subject to prevent or reduce muscle weakness. In some embodiments, a recombinant SMN1 gene and SMN2 ASO combination are delivered to a subject to increase muscle strength. In some embodiments, a recombinant SMN1 gene and SMN2 ASO combination are delivered to a subject to increase muscle tone. In some embodiments, a recombinant SMN1 gene and SMN2 ASO combination are delivered to a subject to prevent or reduce scoliosis. In some embodiments, a recombinant SMN1 gene and SMN2 ASO combination are delivered to a subject to reduce tremors or twitching. In some embodiments, a recombinant SMN1 gene and SMN2 ASO combination are delivered to a subject to maintain or improve respiratory health. In some embodiments, a recombinant SMN1 gene and SMN2 ASO combination are delivered to a subject to prevent or reduce neuronal loss. In some embodiments, a recombinant SMN1 gene and SMN2 ASO combination are delivered to a subject to prevent or reduce motor neuron loss.

[0139] In some embodiments, a combination of a recombinant SMN1 gene and an SMN2 ASO is administered to achieve a synergistic effect. In some embodiments, the combination enhances the effect of the recombinant SMN1 gene, allowing for a reduced dose to be delivered to the subject (e.g., a reduced dose of rAAV encoding the recombinant SMN1 gene). In some embodiments, the combination enhances the effect of the SMN2 ASO, allowing for a reduced dose of ASO administered to the subject. In some embodiments, the reduced dose of rAAV encoding the recombinant SMN1 gene is 1x10 10 In some embodiments, the reduced dose of rAAV encoding the recombinant SMN1 gene is less than 1.0 x 10 8 ~1.0×10 10 In some embodiments, the reduced dose of rAAV encoding the recombinant SMN1 gene is 1.0 x 10 9 ~1.0×10 10 In some embodiments, the reduced dose of rAAV encoding the recombinant SMN1 gene is 1.0 x 10 10 ~1.0×10 13 In some embodiments, the reduced dose of rAAV encoding the recombinant SMN1 gene administered to a human subject is 3 x 10 13 In some embodiments, the reduced dose of rAAV encoding the recombinant SMN1 gene administered to a human subject is 1 x 10 14 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.

[0140] In some embodiments, the reduced dose of SMN2 ASO is 12 mg. A total of 5 mg to 60 mg of SMN2 ASO is administered to the subject per dose. In some embodiments, a total of 12 mg to 48 mg of SMN2 ASO is administered to the subject per dose. 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 12 mg of SMN2 ASO is administered to the subject per dose.

[0141] In some cases, SMA is detected in a fetus 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. Accordingly, methods of rescuing and / or treating a newborn subject with SMA are provided, comprising delivering a combination of a recombinant SNM1 gene and an SMN2 ASO to neural cells of the fetus and / or newborn subject (e.g., a human fetus and / or newborn). In some embodiments, methods of rescuing and / or treating a fetus with SMA are provided, comprising delivering a combination of a recombinant SNM1 gene and an SMN2 ASO to neural cells of the fetus in utero. In some embodiments, the combination is delivered in one or more of the compositions described herein via intrathecal injection. In some embodiments, in utero treatment is defined as administering a combination of a recombinant SNM1 gene and an SMN2 ASO described herein after detection of SMA in the fetus. See, e.g., David et al., Recombinant See 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.

[0142] In some embodiments, neonatal treatment involves delivery of at least one dose of the recombinant SNM1 gene and / or SMN2 ASO 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 occurs within a period of about 12 hours to about 1 week, about 2 weeks, about 3 weeks, or about 1 month, or about 24 hours to about 48 hours.

[0143] In some embodiments, for late-onset SMA, recombinant SNM1 and SMN2 genes One or both of the ASOs are delivered after the onset of symptoms. In some embodiments, treatment of the patient (e.g., the first injection) begins before the patient reaches 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.

[0144] In some embodiments, one or both of the recombinant SNM1 gene and SMN2 ASO are re-administered at a later date.

[0145] In some embodiments, multiple re-administrations are performed. Such re-administrations may involve re-administration of the recombinant SMN1 gene in the same type of viral vector, re-administration of the recombinant SMN1 gene in a different viral vector (e.g., using a different serotype of AAV capsid protein), or re-administration of the recombinant SMN1 gene via non-viral delivery. For example, if a patient is treated with a first rAAV (e.g., rAAV9) encoding SMN1, and the patient requires a second treatment with a recombinant SMN1 gene (e.g., in addition to administering an SMN2 ASO), a second, different rAAV (e.g., rAAVhu68) encoding the recombinant SMN1 gene can be subsequently administered, or vice versa. Similarly, if the patient has neutralizing antibodies against the first rAAV serotype, a second dose of the recombinant SMN1 gene can be delivered to the subject using a second, different rAAV serotype.

[0146] In some embodiments, treatment of SMA patients in conjunction with a recombinant SMN1 gene (e.g., in a viral vector, such as rAAV) may require additional therapy, such as transient co-treatment with an immunosuppressant before, during, and / or after treatment with the compositions described herein. Immunosuppressants for such co-treatment include, but are not limited to, steroids, antimetabolites, T-cell inhibitors, and alkylating agents, or procedures to remove circulating antibodies (e.g., plasmapheresis). For example, such transient treatment may include administration of a steroid (e.g., prednisone or prednisolone) once daily for seven days (with no administration on the seventh day) starting at a dose of about 60 mg, tapered by 10 mg / day. Other doses and immunosuppressants may also be selected.

[0147] 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., has 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 incapable of producing 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 eating. In some embodiments, the subject is identified by muscle electrophysiological measurements and / or muscle biopsy.

[0148] 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.

[0149] In some embodiments, the first dose of the pharmaceutical composition (e.g., a pharmaceutical composition of a recombinant SMN1 gene, an SMN2 ASO, or a combination of both) is administered in utero. In some such embodiments, the first dose is administered before the blood-brain barrier is fully developed. In some embodiments, the first dose is administered systemically to the subject in utero. In some embodiments, the first dose is administered in utero after the blood-brain barrier has formed. In some embodiments, the first dose is administered into the CSF.

[0150] In some embodiments, the first dose of the pharmaceutical composition (e.g., a pharmaceutical composition of a recombinant SMN1 gene, an SMN2 ASO, or a combination of 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 more than 15 years old.

[0151] In some embodiments, the SMN2 ASO is administered 1 to 6 times per year, and the recombinant SMN1 gene (e.g., in an rAAV) is administered once initially. In some embodiments, the SMN2 ASO and recombinant SMN1 gene are administered initially, followed by two or more subsequent doses of the SMN2 ASO alone. In some embodiments, the SMN2 ASO is administered twice per month. In some embodiments, such doses are administered monthly. In some embodiments, the SMN2 ASO is administered every two months. In some embodiments, the SMN2 ASO 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 to 5 years, 5 to 10 years, 10 to 15 years, 15 to 20 years, or more) after the initial administration.

[0152] In some embodiments, administration of at least one pharmaceutical composition (e.g., a pharmaceutical composition of a recombinant SMN1 gene, an SMN2 ASO, or a combination of both) results in a change in the subject's phenotype. 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 not containing exon 7, an increase in the absolute amount of SMN protein, improved muscle strength, improved electrical activity in at least one muscle, improved respiration, improved weight gain, reduced fatigue, and prolonged survival. In some embodiments, 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 upright, stand, and / or walk. In some embodiments, administration of at least one pharmaceutical composition enables the subject to eat, drink, and / or breathe without assistance. In some embodiments, the efficacy of treatment is assessed by electrophysiological assessment of muscles. In some embodiments, administration of the pharmaceutical composition improves at least one of the symptoms of SMA. In some embodiments, administration of the pharmaceutical composition improves at least one of the symptoms of SMA, and causes little or no inflammatory effects. In some embodiments, the absence of inflammatory effects is determined by the absence of significant increase in Aif1 levels during treatment.

[0153] In some embodiments, administration of at least one pharmaceutical composition delays the onset of at least one symptom of SMA. In some embodiments, administration of at least one pharmaceutical composition slows the progression of at least one symptom of SMA. In some embodiments, administration of at least one pharmaceutical composition reduces the severity of at least one symptom of SMA. In some embodiments, administration of at least one pharmaceutical composition causes undesirable side effects. In some embodiments, a treatment regimen is identified that provides desirable symptom relief while avoiding undesirable side effects.

[0154] Dosage and Formulation Thus, in some embodiments, a therapeutically effective amount of SMN2 ASO is administered to a subject with SMA. In some embodiments, the SMN2 ASO is administered alone to a subject. In some embodiments, the SMN2 ASO is administered to a subject together with other compounds and / or pharmaceutical compositions. In some embodiments, the SMN2 ASO and a recombinant nucleic acid (e.g., in an rAAV) are administered to a subject. In some embodiments, the SMN2 ASO and a recombinant nucleic acid encoding SMN1 (e.g., in an rAAV) are administered to a subject simultaneously (e.g., at the same time or during the same medical visit) or sequentially (e.g., during different medical visits). In some embodiments, the SMN2 ASO and the recombinant nucleic acid are administered to a subject together in a single composition. In some embodiments, the SMN2 ASO and the recombinant nucleic acid are administered to a subject separately.

[0155] In some embodiments, the SMN2 ASO and the recombinant nucleic acid encoding SMN1 are administered to the subject simultaneously (e.g., at the same time or at different times during a visit to a hospital, clinic, or other medical facility, e.g., at different times during the same medical visit). Thus, in some embodiments, simultaneous administration of the SMN2 ASO and the recombinant nucleic acid encoding SMN1 refers to administration during the same medical visit (e.g., during the same clinic day). In some embodiments, simultaneous administration of the SMN2 ASO and the recombinant nucleic acid encoding SMN1 refers to administration at different times during the same visit (e.g., during the same clinic day). In some embodiments, simultaneous administration of the SMN1 gene (e.g., an rAAV encoding SMN1) and the SMN2 ASO represents the initiation of a new treatment. In other embodiments, simultaneous administration of the SMN1 gene (e.g., an rAAV encoding SMN1) and the SMN2 ASO represents an additional treatment to a subject currently being treated with a different composition or a single composition (e.g., SMN1 gene therapy alone or SMN2 ASO therapy alone).

[0156] In some embodiments, the SMN2 ASO and the recombinant nucleic acid encoding SMN1 are administered to the subject sequentially during different visits (e.g., different clinic days). In some embodiments, sequential administration of the SMN2 ASO and the recombinant nucleic acid encoding SMN1 refers to administering the recombinant nucleic acid encoding SMN1 during an initial visit, followed by administration of the SMN2 ASO during a different visit (e.g., different clinic days). In some embodiments, sequential administration of the SMN2 ASO and the recombinant nucleic acid encoding SMN1 refers to administering the SMN2 ASO during an initial visit, followed by administration of the recombinant nucleic acid encoding SMN1 during a different visit (e.g., different clinic days). In some embodiments, the recombinant nucleic acid encoding SMN1 and the SMN2 ASO are administered at different frequencies. As used herein, sequential administration can include an administration protocol in which a second therapeutic agent (e.g., SMN2 ASO) can be administered one or more times during one or more different clinic visits, before or after administration of a first therapeutic agent (e.g., a recombinant nucleic acid encoding SMN1) during a clinic visit.

[0157] In some embodiments, the SMN2 ASO and recombinant nucleic acid are administered at different frequencies. In some embodiments, the SMN2 ASO is administered to the subject 1-6 times per year. In some embodiments, the recombinant nucleic acid is administered once. In some embodiments, an initial administration of the SMN2 ASO and recombinant nucleic acid is followed by two or more subsequent doses of the SMN2 ASO alone. In some embodiments, the SMN2 ASO is administered to the subject followed by a combination of the SMN2 ASO and recombinant nucleic acid in the same composition. In some embodiments, the SMN2 ASO is administered to the subject at a dose of 0.01-25 milligrams (e.g., 0.01-10 milligrams, 0.05-5 milligrams, 0.1-2 milligrams, or 0.5-1 milligrams) per kilogram of the subject's body weight, and the recombinant nucleic acid is administered at a dose of 2×10 10 ~2×10 14 GC (e.g., 1.0 × 10 13 ~1.0×10 14 For GC or IT dosing, for example, approximately 1.0 × 10 13 ~5.0×1014 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 body weight of the subject, and the recombinant nucleic acid is administered to a subject at a dose of 1 x 10 10 ~2×10 14 GC (e.g., 1.0 × 10 13 ~1.0×10 14 For GC or IT dosing, for example, approximately 1.0 × 10 13 ~5.0×10 14 For GC, or e.g., IV dosing, approximately 3 x 10 13 ~5×10 14 In some embodiments, the SMN2 ASO is administered in an rAAV at a dose of 0.01 to 10 milligrams per kilogram of subject body weight. In some embodiments, the SMN2 ASO is administered at a dose of 0.001 to 10 milligrams per kilogram of subject body weight. In some embodiments, the SMN2 ASO is administered at a dose of less than 0.001 milligrams per kilogram of subject body weight.

[0158] 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 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 SMN2 ASO and / or recombinant SMN1 gene is administered intravenously or intramuscularly to the subject. In some embodiments, the SMN2 ASO and / or recombinant SMN1 gene is administered intrathecally to the subject. In some embodiments, the SMN2 ASO and / or recombinant SMN1 gene is administered intracisternally to the subject. In some embodiments, the SMN2 ASO and / or recombinant SMN1 gene is administered intracisternally to the subject. In some embodiments, the administration of the SMN2 ASO and recombinant nucleic acid increases intracellular SMN protein levels in the subject. In some embodiments, the administration of the SMN2 ASO and recombinant nucleic acid increases intracellular SMN protein levels in the cervical, thoracic, and lumbar motor neuron regions of the subject.

[0159] In some embodiments, the dose of recombinant SMN1 gene (e.g., in rAAV) and the dose of SMN2 ASO are administered by bolus injection into 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, a bolus injection into CSF ​​and a bolus systemic injection are administered to the subject. In some embodiments, the CSF bolus dose and the systemic bolus dose can be the same or different from each other. In some embodiments, the CSF dose and the systemic dose are administered at different frequencies.

[0160] In some embodiments, a recombinant SMN1 gene (e.g., in an rAAV), SMN2 Pharmaceutical compositions containing ASOs, or combinations thereof are provided. The pharmaceutical compositions can be designed to be delivered to a subject in need thereof by any suitable route or combination of different routes. 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) delivery and / or intracisternal (ICM) delivery).

[0161] In some embodiments, direct delivery to the CNS is desired, and this delivery 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 ventricular or lumbar CSF, suboccipital puncture, or other suitable means. Meyer et al., Molecular Therapy (31 October 2014), demonstrated that direct CSF injection is effective in achieving widespread transgene expression throughout the spinal cord of mice and non-human primates, even at doses 10-fold lower than IV administration. 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, which is incorporated herein by reference. In some embodiments, the composition is delivered via lumbar injection.

[0162] In some aspects, the delivery vehicle and formulation are designed to avoid direct systemic delivery of a suspension comprising the AAV composition(s) described herein. Suitably, this may have the advantage of reducing systemic exposure compared to systemic administration, reducing toxicity, and / or reducing unwanted immune responses to the AAV and / or transgene product.

[0163] Compositions comprising recombinant SMN1 genes (e.g., in rAAV) and / or SMN2 ASOs can be formulated for any suitable route of administration (e.g., oral, inhaled, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, and other parenteral routes).

[0164] 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., WO2011 / 126808 and WO2013 / 049493). In some embodiments, such multiple viruses can include different replication-defective viruses (e.g., AAV, adenovirus, and / or lentivirus). Alternatively, delivery can be mediated by non-viral constructs (e.g., "naked DNA," "naked plasmid DNA," RNA, and mRNA) combined with various delivery compositions and nanoparticles (including, e.g., micelles, liposomes, cationic lipid-nucleic acid compositions, poly-glycan compositions, and other polymer-, lipid-, and / or cholesterol-based nucleic acid complexes), as well as other constructs (such as those described herein or known in the art). See, e.g., X. Su et al., Mol. Pharmaceutics, 2011, 8(3), pp. 774-787; web See publication: March 21, 2011, WO2013 / 182683, WO2010 / 053572, and WO2012 / 170930, both of which are incorporated herein by reference. Non-viral SMN1 delivery constructs may also be formulated for any suitable route of administration.

[0165] 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 purification methods suitable for separating empty capsids from vector particles have been described, such as the process described in International Patent Application No. PCT / US16 / 65976, entitled "Scalable Purification Method for AAV8," filed December 9, 2016, and its priority documents (U.S. Patent Application Nos. 62 / 322,098, filed April 13, 2016, and 62 / 266,341, filed 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 No. 62 / 322,083, filed April 13, 2016, and U.S. Patent Application No. 62 / 266,351, filed December 11, 2015) (AAV1), International Patent Application No. PCT / US16 / 66013, filed December 9, 2016, and its priority documents (U.S. Provisional Application No. 62 / 322,083, filed April 13, 2016, and U.S. Provisional Application No. 62 / 266,351, filed December 11, 2015) (AAV1). See also the purification methods described in International Patent Application Nos. PCT / US16 / 65970, filed December 9, 2016, and its priority applications (U.S. Provisional Application Nos. 62 / 266,357 and 62 / 266,357) (AAV9), all of which are incorporated herein by reference. 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 involves affinity capture at high salt concentrations, followed by an anion exchange resin process at high pH, ​​yielding rAAV vector particles substantially free of rAAV intermediates.

[0166] In the case of AAV viral vectors, quantification of genome copies ("GC") can be performed as a measure of the dosage 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 determining the AAV GC number is as follows: a purified AAV vector sample is first treated with DNase to remove 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 targeting a specific region of the viral genome (e.g., polyA signal). Another suitable method for determining genome copy number is quantitative PCR (qPCR), specifically 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 (Unedited version published online December 13, 2013)).

[0167] In some embodiments, the replication-defective virus composition can be formulated alone or in combination with an ASO in a dosage unit, the formulation or combination comprising about 1.0 x 10 9 GC~approx. 1.0×10 15 The amount of replication-defective virus is preferably in the range of GC (including all integers and fractions within the range) (which will treat an average subject weighing, for example, 70 kg), which for human patients is preferably 1.0 x 10 12 GC~1.0×10 14 The total dose administered to a subject may depend on the route of administration. In some embodiments, the composition contains at least 1 x 10 GC per dose. 9 GC, at least 2 × 10 9 GC, at least 3 × 10 9 GC, at least 4 × 10 9 GC, at least 5 × 10 9 GC, at least 6 × 109 GC, at least 7 × 10 9 GC, at least 8 × 10 9 GC, or at least 9 × 10 9 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. 10 GC, at least 2 × 10 10 GC, at least 3 × 10 10 GC, at least 4 × 10 10 GC, at least 5 × 10 10 GC, at least 6 × 10 10 GC, at least 7 × 10 10 GC, at least 8 × 10 10 GC, or at least 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 GC, at least 2 × 10 11 GC, at least 3 × 10 11 GC, at least 4 × 10 11 GC, at least 5 × 10 11 GC, at least 6 × 10 11 GC, at least 7 × 10 11 GC, at least 8 × 10 11 GC, or at least 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 GC, at least 2 × 10 12 GC, at least 3 × 10 12 GC, at least 4 × 10 12 GC, at least 5 × 10 12 GC, at least 6 × 10 12 GC, at least 7 × 10 12 GC, at least 8 × 10 12 GC, or at least 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. 13GC, at least 2 × 10 13 GC, at least 3 × 10 13 GC, at least 4 × 10 13 GC, at least 5 × 10 13 GC, at least 6 × 10 13 GC, at least 7 × 10 13 GC, at least 8 × 10 13 GC, or at least 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 GC, at least 2 × 10 14 GC, at least 3 × 10 14 GC, at least 4 × 10 14 GC, at least 5 × 10 14 GC, at least 6 × 10 14 GC, at least 7 × 10 14 GC, at least 8 × 10 14 GC, or at least 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 GC, at least 2 × 10 15 GC, at least 3 × 10 15 GC, at least 4 × 10 15 GC, at least 5 × 10 15 GC, at least 6 × 10 15 GC, at least 7 × 10 15 GC, at least 8 × 10 15 GC, or at least 9 × 10 15 In some embodiments, for human use, the dose range of the virus (e.g., rAAV) is 1×10 per dose. 10 ~About l×10 12 It can be a GC (including all integer or fractional quantities in the range).

[0168] These doses may be administered in various volumes of carrier, excipient, or buffer formulation, ranging from about 25 microliters to about 1,000 microliters, or about 10 milliliters, or up to 20 milliliters, inclusive, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In some embodiments, the volume of the 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 another embodiment, the volume is about 225 μl. In yet other embodiments, 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.

[0169] In other embodiments, a volume of about 1 μl to 150 mL can be selected, with larger volumes being selected for adults. Typically, for newborn infants, an appropriate volume is about 0.5 mL to about 10 mL. For slightly older infants, about 0.5 mL to about 15 mL can be selected. For infants just starting to walk, a volume of about 0.5 mL to about 20 mL can be selected. For children, a volume of up to about 30 mL can be selected. For children about 11 to 12 years old and adolescents about 13 to 19 years old, a volume of up to about 50 mL can be selected. In still other embodiments, a volume of about 5 mL to about 15 mL or about 7.5 mL to about 10 mL can be selected as the volume for intrathecal administration to a patient. Other appropriate volumes and doses can also be determined. The dose will be adjusted to balance the therapeutic effect against any side effects, and such doses can vary depending on the therapeutic application for which the recombinant vector is being used.

[0170] The recombinant SMN1 gene (e.g., in a viral vector (e.g., packaged within rAAV)) can be delivered to a host cell using a suitable method. The rAAV is preferably suspended in a physiologically compatible carrier and administered to a human or non-human mammalian patient. In some embodiments, the composition comprises a carrier, diluent, excipient, and / or adjuvant. An appropriate carrier can be selected to suit the route of administration. For example, one suitable carrier includes saline, which can be formulated with various buffers (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.

[0171] In some embodiments, the compositions may contain, in addition to the rAAV and / or ASO and carrier(s), other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Examples of suitable 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.

[0172] In some embodiments, compositions comprising rAAV and / or ASO may comprise a pharmaceutically acceptable carrier and / or be mixed with suitable excipients designed to be delivered to a subject via injection, osmotic pump, intrathecal catheter, or another device or route. In one example, the composition is formulated for intrathecal delivery. In some embodiments, intrathecal delivery comprises injection into the spinal canal (e.g., the subarachnoid space).

[0173] The viral vectors described in this application can be used in the preparation of a medicament for delivering SMN1 to a subject (e.g., a human patient) in need thereof, providing functional SMN to a subject, and / or treating spinal muscular atrophy in combination therapy with one or more SMN2 ASOs.

[0174] In some embodiments, the buffers, carriers, and / or other components of a pharmaceutical formulation containing rAAV are selected to include one or more components that prevent rAAV from adhering to the infusion tubing but do not interfere with the binding activity of the rAAV in vivo. When combined with ASOs, the buffers, carriers, and / or other components can also be selected to avoid undesirable interactions with the ASOs.

[0175] In some embodiments, the SMN2 ASO is formulated for delivery alone or co-formulated with a recombinant SMN1 gene (e.g., co-formulated with an rAAV comprising a recombinant nucleic acid encoding the SMN1 gene). In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated (alone or together with a recombinant SMN1 gene) for delivery (e.g., systemic administration) in an ASO amount ranging from 5 mg to 60 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated (alone or together with a recombinant SMN1 gene) for delivery (e.g., systemic administration) in an ASO amount ranging from 5 mg to 20 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated (alone or together with a recombinant SMN1 gene) for delivery (e.g., systemic administration) in an ASO amount ranging from 12 mg to 50 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated (alone or together with a recombinant SMN1 gene) for delivery (e.g., systemic administration) in an ASO amount ranging from 12 mg to 48 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated (alone or together with a recombinant SMN1 gene) for delivery (e.g., systemic administration) in an ASO amount ranging from 12 mg to 36 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated (alone or together with a recombinant SMN1 gene) for delivery (e.g., systemic administration) in an ASO amount of 28 mg per dose. In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated (alone or together with a recombinant SMN1 gene) for delivery (e.g., systemic administration) in an ASO amount of 12 mg per dose. In some such embodiments, the dose volume is 5 mL.

[0176] In some such embodiments, the ASO (e.g., SMN2 ASO) is formulated (alone or together with the recombinant SMN1 gene) for delivery (e.g., 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.

[0177] In some embodiments, the administration of the ASO to a subject is divided into an induction phase and a maintenance phase. In some such embodiments, the administered dose during the induction phase is higher than the administered dose during the maintenance phase. In some embodiments, the administered dose during the induction phase is lower than the administered dose during the maintenance phase. In some embodiments, the induction phase is achieved by bolus injection and the maintenance phase is achieved by continuous infusion. In some embodiments, a combination formulation is used during the induction phase.

[0178] In some embodiments, the pharmaceutical composition is administered as a bolus injection. In some such embodiments, the bolus injection dose comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises a total of 28 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose. In some such embodiments, the bolus injection dose comprises a total of 12 mg of antisense oligonucleotide (e.g., SMN2 ASO) per dose. In some such embodiments, the dose volume is 5 mL.

[0179] 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.

[0180] In some embodiments, such doses are administered twice a month. 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, a bolus injection into the CSF and a bolus systemic injection are administered to the subject. In such embodiments, the CSF bolus dose and the systemic bolus dose can be the same or different from each other. In some embodiments, the CSF dose and the systemic dose 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.

[0181] In some embodiments, the pharmaceutical composition is administered by continuous infusion (e.g., a dose can be administered over a period of time (e.g., 24 hours)). 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 dose volume is 5 mL.

[0182] In other embodiments, the administered dose is 0.05-25 milligrams of antisense compound per kilogram of subject body weight per day. In some embodiments, the administered dose is 0.1-10 milligrams of antisense compound per kilogram of subject body weight per day. In some embodiments, the administered dose is 0.5-10 milligrams of antisense compound per kilogram of subject body weight per day. In some embodiments, the administered dose is 0.5-5 milligrams of antisense compound per kilogram of subject body weight per day. In some embodiments, the administered dose is 1-5 milligrams of antisense compound per kilogram of subject body weight per day. In some embodiments, the invention provides dosing regimens comprising CNS infusion and at least one bolus systemic injection. In some embodiments, the invention provides dosing regimens comprising CNS infusion and at least one bolus subcutaneous injection. In some embodiments, the dose, whether bolus or infusion, is adjusted to achieve or maintain a concentration of antisense compound of 0.1-100 micrograms per gram of CNS tissue. In some embodiments, the dose, whether bolus or infusion, is adjusted to achieve or maintain a concentration of antisense compound between 1 and 10 micrograms per gram of CNS tissue, hi some embodiments, the dose, whether bolus or infusion, is adjusted to achieve or maintain a concentration of antisense compound between 0.1 and 1 micrograms per gram of CNS tissue.

[0183] In some embodiments, the present invention provides a dosing regimen comprising an infusion into the CNS and at least one bolus systemic injection. In some embodiments, the present invention provides a dosing regimen comprising an infusion into the CNS and at least one bolus subcutaneous injection. In some embodiments, the dose, whether bolus or infusion, is adjusted to achieve or maintain a concentration of antisense compound between 0.1 and 100 micrograms per gram of CNS tissue. In some embodiments, the dose, whether bolus or infusion, is adjusted to achieve or maintain a concentration of antisense compound between 1 and 10 micrograms per gram of CNS tissue. In some embodiments, the dose, whether bolus or infusion, is adjusted to achieve or maintain a concentration of antisense compound between 0.1 and 1 micrograms per gram of CNS tissue.

[0184] 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., in a viral vector (e.g., packaged within an rAAV)) and / or antisense compounds. In some embodiments, such pharmaceutical compositions comprise sterile saline and one or more therapeutic molecules. In some embodiments, such pharmaceutical compositions consist of sterile saline and one or more therapeutic molecules. In some embodiments, therapeutic molecules may be mixed with pharmaceutically acceptable active and / or inactive substances to prepare 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 degree of disease, or the dosage to be administered. In some embodiments, therapeutic molecules may be utilized in pharmaceutical compositions by combining such therapeutic molecules with a suitable pharmaceutically acceptable diluent or carrier. In some embodiments, a pharmaceutically acceptable diluent includes phosphate buffered saline (PBS). PBS is a suitable diluent for use in compositions to be delivered parenterally. Thus, in some embodiments, pharmaceutical compositions comprising one or more therapeutic molecules and a pharmaceutically acceptable diluent are used in the methods described herein. 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 capable of producing (directly or indirectly) their biologically active metabolites or residues upon administration to an animal, including a human. 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.

[0185] In some embodiments, prodrugs can include additional nucleosides at one or both ends of an oligomeric compound, which, when cleaved by endogenous nucleases in the body, form active antisense oligomeric compounds. Lipid-based vectors have been used in various nucleic acid therapy methods. For example, one method involves introducing nucleic acids into preformed liposomes or lipoplexes composed of a mixture of cationic and neutral lipids. Another method involves forming DNA complexes with mono-cationic or poly-cationic lipids in the absence of neutral lipids. Akinc et al., Nature Biotechnology 26, 561-569 (1 May 2008), which is incorporated herein by reference in its entirety, describes several preparations.

[0186] kit In some embodiments, kits are provided that include a recombinant SMN1 gene (e.g., in an rAAV) and / or an SMN2 ASO (e.g., included in a pharmaceutical composition). In some embodiments, such kits further include an additional therapeutic agent (such as one or more immunosuppressants). In some embodiments, such kits further include a delivery means (e.g., a syringe or infusion pump).

[0187] The following examples are illustrative only and are not intended to limit the invention. [Example]

[0188] Example 1: rAAV vector containing the hSMN1 gene A recombinant neurotropic AAV virus carrying codon-optimized human SMN1 cDNA was constructed.

[0189] Example 2: ASOs that increase full-length SMN2 mRNA (e.g., by promoting inclusion of exon 7 in hSMN2 mRNA) We prepared ASOs that increase full-length SMN2 mRNA (e.g., ASOs that promote the inclusion of exon 7 in SMN2 mRNA) (Fig. 3 ).

[0190] Example 3: Administration and biodistribution of an AAV vector containing the hSMN1 gene and an ASO that increases full-length SMN2 mRNA (e.g., an ASO that promotes inclusion of exon 7 in SMN2 mRNA). The rAAV described in Example 1 and the ASO described in Example 2 are administered to animal SMA disease models and control animals, including mouse, pig, and non-human primate (e.g., macaque) SMA disease and control animal models.

[0191] rAAV and ASO are administered via different routes, including intrathecal and systemic routes (e.g., via lumbar puncture delivery, intracisternal delivery, and intravenous delivery).

[0192] 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.

[0193] Figure 4. 3 x 10 M IgG4-dependent ... 13 The results of GC rAAV administration and 2 × 10 14 The results of intravenous administration of GC are shown.

[0194] Example 4: Combination of an rAAV vector containing the hSMN1 gene and an ASO that increases full-length SMN2 mRNA (e.g., an ASO that promotes the inclusion of exon 7 in SMN2 mRNA) Figure 5 shows a non-limiting example of the physical and biological characterization of a composition comprising both an rAAV vector (hSMN1 gene) and an ASO that increases full-length SMN2 mRNA (e.g., an ASO that promotes the inclusion of exon 7 in SMN2 mRNA).

[0195] 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 formulated together in the same formulation. The HPLC profiles of the rAAV vector and ASO are unchanged in Figure 5C, indicating that the combination of rAAV and ASO does not result in significant incompatibility.

[0196] Figure 5D provides data on the infectivity of rAAV in cells in vitro when delivered by rAAV vector alone or in combination with ASO, demonstrating that the presence of ASO in the combination does not significantly affect the infectivity of rAAV.

[0197] Figure 5E shows intracellular SMN protein expression levels and GEM formation in cells after treatment with rAAV, ASO, or both.

[0198] Example 5: Intracerebroventricular (ICV) Administration of Nusinersen and AAV-SMN1 Nusinersen and AAV-SMN1 were delivered via the right lateral ventricle into the cerebrospinal fluid (CSF) of 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 administered in combination with low- or high-dose AAV-SMN1 (1 × 10, respectively). 10 GC or 8 × 10 10 Mice are administered nusinersen or AAV-SMN1 along with nusinersen (GC) at birth (P0-P1). The mice's weight and righting reflex are measured and compared to those of control mice of the same genotype that receive either nusinersen or AAV-SMN1 alone.

[0199] Mice administered both nusinersen and AAV-SMN1 gained significantly more weight and had a significantly faster righting reflex compared to controls.

[0200] Studies will demonstrate that intracerebroventricular (ICV) administration of nusinersen and AAV-SMN1 increases the inclusion of SMN2 exon 7 in the spinal cord. Further studies will demonstrate an increase in the number of spinal motor neurons with increased SMN expression compared to controls.

[0201] Example 6: Administration of a composition of nusinersen and AAV-SMN1 Using a micro-osmotic pump (ALZET Osmotic Pumps, Cupertino, Calif., USA), a composition of nusinersen and AAV-SMN1 was delivered via the right lateral ventricle into the cerebrospinal fluid (CSF) of neonatal (P0-P1) SMA mice carrying the human SMN2 transgene. Low-dose nusinersen (1 μg) and low-dose AAV-SMN1 (1 × 10 10 GC), or a low-dose nusinersen (1 μg) and a high-dose AAV-SMN1 (8 × 10 10 GC), or a high-dose nusinersen (4 μg) and a low-dose AAV-SMN1 (1 × 10 10 GC), or a high-dose nusinersen (4 μg) and a high-dose AAV-SMN1 (8 × 10 10 The composition (Genetical Composition GC) is administered to mice at birth (P0-P1). The weight and righting reflex of the mice are measured and compared to the weight and righting reflex of control mice of the same genotype that are administered either nusinersen or AAV-SMN1 alone.

[0202] Mice administered the nusinersen and AAV-SMN1 composition gained significantly more weight and had a significantly faster righting reflex compared to controls.

[0203] Studies will demonstrate that intracerebroventricular (ICV) administration of a composition of nusinersen and AAV-SMN1 increases inclusion of SMN2 exon 7 in the spinal cord. Further studies will demonstrate an increase in the number of spinal motor neurons with increased SMN expression compared to controls.

[0204] Example 7: Administration and distribution analysis of nusinersen and AAV-SMN1 in non-human mammals The distribution of nusinersen and AAV-SMN1 compositions at different doses and administration routes is evaluated using SMA mice, SMA rhesus monkeys, and SMA cynomolgus monkeys. The nusinersen and AAV-SMN1 compositions are administered to some mice and some monkeys at a dose of approximately 1 mg / kg by intracerebroventricular (ICV) or intrathecal (IT) injection over 24 hours. 96 hours after the end of the injection period, the animals are sacrificed and tissues are collected. The concentrations of nusinersen and AAV-SMN1 are measured in samples from the cervical, thoracic, and lumbar regions of the spinal cord.

[0205] Additional mice, rhesus monkeys, and cynomolgus monkeys of the same genotype as above are administered the nusinersen and AAV-SMN1 composition at the same dose (approximately 1 mg / kg) by ICV or IT infusion. The animals are administered the nusinersen and AAV-SMN1 composition for 3, 7, or 14 days and then sacrificed 5 days after the end of the infusion period.

[0206] Example 8: Administration of nusinersen and AAV-SMN1 to human subjects Nusinersen and AAV-SMN1 will be administered to human subjects using routes including intracerebroventricular (ICV), intravenous (IV), and intrathecal (IP) (e.g., via lumbar puncture (LP) delivery and / or intracisternal (ICM) delivery). The compositions will be tested in both children and adults.

[0207] In some embodiments, the rAAV-SMN1 composition comprises about 1 x 10 14In some embodiments, the rAAV-SMN1 composition is administered to a child (e.g., a child with SMA) at a dose of about 1.5 x 10 GC, e.g., by lumbar puncture (LP) injection (e.g., over a 24-hour period). 14 The GC is administered to an adult (eg, an adult with SMA) in a dose of GC, for example, by intracisternal (ICM) infusion (eg, over a 24 hour period).

[0208] In some embodiments, other rAAV-SMN1 doses can be used, such as, for example, about 5-6×10 13 GC or higher, e.g., about 1.2 × 10 14 GC, or 1.5-1.8 x 10 14 Any suitable route of administration can be used, such as via IT delivery (e.g., infusion over 24 hours), via LP delivery, or via ICM delivery.

[0209] Example 9: Intracerebroventricular (ICV) Administration of Nusinersen and AAV-SMN1 Neonatal (P0-P1) SMA mice carrying the human SMN2 transgene were administered nusinersen and AAV-SMN1. Low or high doses of nusinersen (1 μg and 3 μg, respectively) were administered with low or high doses of AAV-SMN1 (1 × 10 10 GC or 3 × 10 10 Mice were administered nusinersen or AAV-SMN1 (GC) at birth (P0-P1). Body weight and righting reflex of the mice were measured and compared with those of control mice of the same genotype that received either nusinersen or AAV-SMN1 alone.

[0210] Mice that received both nusinersen and AAV-SMN1 were significantly heavier and had a significantly faster righting reflex compared to controls.

[0211] Figures 6A-6B show that using either the SMN1 gene (e.g., in an rAAV vector) or an ASO (such as nusinersen) (e.g., in a single dose), motor function was partially rescued at postnatal day (PND) 8 after dosing. ** , showing complete rescue of motor function at PND 16. Figure 6A shows the righting reflex (RR) of mice from four separate groups 8 and 16 days after administration of nusinersen. Figure 6B shows the body weight of mice from four separate groups 8 and 16 days after administration of nusinersen. Combination therapy may improve upon the partial rescue of RR (PND 7-16) and body weight seen with monotherapy.

[0212] Figures 7A-7C show the results of the first combination therapy study demonstrating the effect of combining SMN1 gene therapy with nusinersen on body weight and RR. Figure 7A shows the change in body weight over time. Figure 7B shows the change in RR over time. Figure 7C is a chart outlining the conditions for the three animal groups tested.

[0213] Figures 8A-8C show the results of a second combination therapy demonstrating the effect of combining SMN1 gene therapy with nusinersen on body weight and RR. Figure 8A is a chart outlining the conditions for the three animal groups tested. Figure 8B shows the change in body weight over time, and Figure 8C shows the change in RR (in days) over time.

[0214] Figures 9A-9B show a comparison of the % change in body weight from PND7 to PND13. Figure 9A shows a comparison of the % change in body weight from PND7 to PND13. 10 Figure 9B shows the % change in body weight at GC / ASO (nusinersen): 1 μg. Figure 9C shows the % change in body weight at a fixed dose of gene therapy (rAAV): 3 × 10 10 Figures 10A-10B show the % change in body weight at 3 μg of GC / ASO (nusinersen). Figure 10A shows the % change in RR at PND7-PND13. Figure 10A shows the % change in body weight at 3 μg of GC / ASO (nusinersen). Figure 10B shows the % change in RR at PND7-PND13. Figure 10A shows the % change in body weight at 3 μg of GC / ASO (nusinersen). 10GC / ASO (nusinersen): 1 μg. Figure 10B shows the % change in RR at a fixed dose of gene therapy (rAAV): 3 × 10 10 GC / ASO (nusinersen): % change in RR at 3 μg is shown.

[0215] Other Aspects All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0216] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to adapt it to various usages and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the appended claims.

[0217] equivalent While several inventive aspects have been described and exemplified herein, those skilled in the art will readily envision numerous other means and / or structures for performing the functions described herein and / or obtaining the results described herein and / or obtaining one or more of the advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive aspects described herein. More generally, those skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application(s) for which the teachings of the present invention are intended. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific inventive aspects described herein. Accordingly, it will be understood that the foregoing embodiments are exemplary only and that, within the scope of the appended claims and equivalents thereto, inventive aspects may be practiced otherwise than as specifically described and claimed. The inventive aspects of the present disclosure are directed individually to each feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not inconsistent with one another.

[0218] All definitions provided and used herein are understood to supersede dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0219] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each citation is made, which subject matter may, in some cases, include the entire document.

[0220] The indefinite articles "a" and "an," as used in the specification and claims, unless a different definition is expressly indicated, should be understood to mean "at least one."

[0221] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so coordinating, i.e., elements that may be present conjunctively or disjunctively. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so coordinating. Other elements may optionally be present beyond the elements specifically identified by the "and / or" clause, whether related or not to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language (such as "comprising"), may in one embodiment refer to A only (optionally including elements other than B); in another embodiment, refer to B only (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so forth.

[0222] As used in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when listed items are separated by "or" or "and / or," such "or" or "and / or" should be interpreted as inclusive, i.e., including not only at least one of an element or series of elements, but also including more than one of them, and optionally including additional unlisted items. Only terms clearly indicating otherwise (e.g., "only one of" or "exactly one of," or, when used in the claims, "consisting of") will refer to the inclusion of exactly one element of an element or series of elements. In general, the term "or" as used herein should only be interpreted as referring to exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity (e.g., "either," "one of," "only one of," or "exactly one of"). "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0223] It should be understood that the phrase "at least one," as used herein and in the claims with respect to a list of one or more elements, means that at least one element is selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, nor does it exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or not to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one A (optionally including multiple As) (and optionally including elements other than B) in the absence of any B; in another embodiment to at least one B (optionally including multiple Bs) (and optionally including elements other than A) in the absence of any A; in yet another embodiment to at least one A (optionally including multiple As) and at least one B (optionally including multiple Bs) (and optionally including other elements); and so on.

[0224] Unless a different definition is expressly indicated, it is also understood that, in any method claimed herein that includes multiple steps or actions, the order of such steps or actions of such method is not necessarily limited to the order in which such steps or actions of such method are described.

[0225] In the claims and the rest of this specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are understood to be open-ended, i.e., have the meaning of including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures. It should be understood that embodiments described herein using an open-ended transitional phrase (e.g., "comprising") also contemplate, in alternative embodiments, "consisting of" and "consisting essentially of" the feature(s) described by such open-ended transitional phrase. For example, if the disclosure recites "a composition comprising A and B," the disclosure also contemplates the alternative embodiments of "a composition consisting of A and B" and "a composition consisting essentially of A and B."

[0226] Although the sequence listing accompanying this application identifies each sequence as either "RNA" or "DNA" as appropriate, in practice, such sequences may be modified with any combination of chemical modifications. Those of skill in the art will readily understand that such designations as "RNA" or "DNA" used to describe modified oligonucleotides are, in some cases, arbitrary. For example, an oligonucleotide containing a nucleoside containing a 2'-OH sugar moiety and a thymine base may be described as a DNA with a modified sugar (in which the natural 2'-H in DNA is replaced with a 2'-OH) or an RNA with a modified base (in which the natural uracil in RNA is replaced with a thymine (the uracil is methylated)).

[0227] Thus, the nucleic acid sequences provided herein, including but not limited to those set forth in the Sequence Listing, are intended to encompass nucleic acids comprising naturally occurring RNA and / or DNA or modified RNA and / or DNA in any combination, including but not limited to such nucleic acids having modified nucleobases. As an additional non-limiting example, an oligomeric compound having the nucleobase sequence "ATCGATCG" encompasses any oligomeric compound having such a nucleobase sequence, with or without modification, including but not limited to such compounds comprising RNA bases (such as those having the sequence "AUCGAUCG"), as well as those having some DNA bases and some RNA bases (such as "AUCGATCG"), and oligomeric compounds having other modified nucleobases (such as "AT"CGAUCG) (where "C" represents a cytosine base containing a methyl group at the 5-position). The present invention provides, for example, the following items. (Item 1) 1. A method for treating spinal muscular atrophy (SMA) in a subject with SMA, said method comprising: a) a recombinant nucleic acid encoding survival motor neuron 1 (SMN1) protein; b) antisense oligonucleotides (ASOs) that increase full-length survival motor neuron 2 (SMN2) mRNA; to the subject. (Item 2) Item 10. The method of item 1, wherein the subject has one or more symptoms of SMA. (Item 3) 3. The method of claim 2, wherein the symptoms include limb muscle atrophy, difficulty or inability to walk, or difficulty breathing. (Item 4) 4. The method according to any one of items 1 to 3, wherein the subject is a human subject selected from a pediatric population and an adult population. (Item 5) Item 5. The method of item 4, wherein the subject is 18 years of age or older. (Item 6) Item 6. The method of item 5, wherein the subject is under 18 years of age. (Item 7) 7. The method of item 6, wherein the subject is about 2 weeks old, about 1 month old, about 3 months old, about 6 months old, about 1 year old, about 2 years old, about 3 years old, about 4 years old, or about 5 years old. (Item 8) 8. The method of any one of items 1 to 7, wherein the ASO alters the splicing pattern of survival motor neuron 2 (SMN2) pre-mRNA. (Item 9) The method of claim 8, wherein the ASO promotes the inclusion of exon 7 in survival motor neuron 2 (SMN2) mRNA. (Item 10) 10. The method of any one of items 1 to 9, wherein the ASO comprises a sequence complementary to intron 6 or intron 7 of a nucleic acid molecule encoding the SMN2 protein. (Item 11) 11. The method of claim 10, wherein the ASO comprises a sequence complementary to intron 6 of a nucleic acid molecule encoding the SMN2 protein. (Item 12) 11. The method of claim 10, wherein the ASO comprises a sequence complementary to intron 7 of a nucleic acid molecule encoding the SMN2 protein. (Item 13) 13. The method of any one of items 1 to 12, wherein the ASO comprises the nucleic acid sequence of SEQ ID NO: 1. (Item 14) Item 14. The method of item 13, wherein the ASO is nusinersen. (Item 15) 15. The method of any one of items 1 to 14, wherein the ASO comprises one or more nucleobase or backbone modifications. (Item 16) 16. The method of any one of items 1 to 15, wherein the recombinant nucleic acid comprises a promoter operably linked to the SMN1 gene. (Item 17) 17. The method of any one of items 1 to 16, wherein the recombinant nucleic acid is a recombinant AAV (rAAV) genome comprising flanking AAV inverted terminal repeats (ITRs). (Item 18) 18. The method of claim 17, wherein the recombinant nucleic acid is packaged within an rAAV particle and the rAAV particle is administered to the subject. (Item 19) 19. The method of claim 18, wherein the rAAV particles comprise AAV9 capsid proteins. (Item 20) 20. The method of any one of items 1 to 19, wherein the rAAV and the ASO are administered at the same time. (Item 21) 20. The method of any one of items 1 to 19, wherein the rAAV and the ASO are administered simultaneously. (Item 22) The method of item 20 or item 21, wherein the rAAV and the ASO are administered together in a single composition. (Item 23) The method of item 20 or item 21, wherein the rAAV and the ASO are administered in separate compositions. (Item 24) 20. The method of any one of items 1 to 19, wherein the rAAV and the ASO are administered at different frequencies. (Item 25) The method of any one of items 1 to 19 or 24, wherein the rAAV and the ASO are administered sequentially. (Item 26) 26. The method of any one of items 1 to 25, wherein the ASO is administered 1 to 6 times per year. (Item 27) 27. The method of any one of items 1 to 26, wherein the rAAV is administered once. (Item 28) 28. The method of any one of items 24 to 27, wherein an initial administration of the rAAV and the ASO is followed by two or more subsequent doses of the ASO alone. (Item 29) The SMN1 rAAV is 2 x 10 10 ~2×10 1429. The method of any one of items 1 to 28, wherein the ASO is administered at a dose of 0.01 to 10 milligrams per kilogram of body weight of the subject. (Item 30) 30. The method of item 29, wherein a total of 5 mg to 20 mg of ASO per dose is administered to the subject. (Item 31) 31. The method of claim 30, wherein 12 mg of ASO is administered to the subject per dose. (Item 32) 32. The method of any one of items 1 to 31, wherein the rAAV and the ASO are administered intrathecally to the subject. (Item 33) 32. The method of any one of items 1 to 31, wherein the rAAV and the ASO are administered to the cisternal space of the subject. (Item 34) 32. The method of any one of items 1 to 31, wherein the initial dose and / or subsequent doses of the ASO are administered intravenously or intramuscularly. (Item 35) 35. The method of any one of items 1 to 34, wherein administration of the rAAV and the ASO increases intracellular SMN protein levels in motor neurons of the subject. (Item 36) 36. The method of claim 35, wherein SMN protein levels are elevated in the subject's cervical, thoracic, and lumbar spinal cord. (Item 37) 37. The method of any one of items 1 to 36, wherein the subject has a deletion or loss-of-function point mutation in each SMN1 allele. (Item 38) Item 39. The method of Item 37, wherein the subject is homozygous for a mutation in the SMN1 gene. A method for treating spinal muscular atrophy (SMA) in a subject with SMA, the method comprising administering an effective amount of a composition comprising an rAAV encoding SMN1 to a subject who has previously been treated with an ASO that increases full-length SMN2 mRNA. (Item 40) A method for treating spinal muscular atrophy (SMA) in a subject with SMA, the method comprising administering an effective amount of a composition comprising an ASO that increases full-length SMN2 mRNA to a subject who has previously been administered an rAAV encoding SMN1. (Item 41) A composition comprising an rAAV encoding SMN1 and an ASO capable of increasing full-length SMN2 mRNA. (Item 42) 42. The composition of claim 41, wherein the rAAV comprises an AAV9 capsid protein. (Item 43) 43. The composition of claim 41 or 42, wherein the ASO is nusinersen. (Item 44) A pharmaceutical composition comprising the composition according to any one of items 41 to 43 and a pharmaceutically acceptable carrier.

Claims

[Claim 1] The invention as described in the drawings.

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