CN118284698A - Gene therapy for spinal muscular atrophy - Google Patents

Abstract

Aspects of the present disclosure relate to compositions and methods for treating spinal muscular atrophy (SMA). The present disclosure is based in part on isolated nucleic acids and vectors (e.g., viral vectors, such as rAAV vectors) encoding SMN1. In some embodiments, expression of SMN1 is driven by a native SMN 1 promoter or a variant thereof. In some embodiments, the isolated nucleic acids and vectors of the present disclosure have reduced toxicity and/or increased transgene expression relative to previously described SMN encoding vectors.

Description

Gene therapy for spinal muscular atrophy

Related applications

The present application claims the benefit of U.S. provisional application No. 63/341,650 entitled "GENE THERAPY FOR SPINAL MUSCULAR ATROPHY" filed on day 13 of 5 of 2022 and U.S. provisional application No. 63/282,246 entitled "GENE THERAPY FOR SPINAL MUSCULAR ATROPHY" filed on day 23 of 11 of 2021 in accordance with 35USC 119 (e), the contents of each of which are incorporated herein by reference in their entirety.

Reference to electronic sequence Listing

The contents of the electronic sequence Listing (U012070171 WO00-SEQ-LJG. Xml; size: 48,435 bytes; date of creation: 2022, 11, 18) are incorporated herein by reference in their entirety.

Background

Spinal muscular atrophy (spinal muscular atrophy, SMA) is a neuromuscular disease caused by loss of function of the motor neuron survival 1 (SMN 1) gene, characterized by motor neuron degeneration and progressive muscle weakness. About 1 out of every 11,000 newborns is affected by SMA and SMA remains the leading genetic cause of death in infants. In the last decade, two major breakthroughs have been made in the treatment of this devastating disease by increasing SMN protein in human patients. The first is an antisense oligonucleotide (ASO), norcinal (Nusinersen) and Li Sipu blue (risdiplam), which modify the splicing of existing SMN2 pre-mRNA. The second term is Zolgensma, which is a self-complementing adeno-associated virus 9 (scAAV 9) -mediated SMN1 gene replacement.

Disclosure of Invention

Aspects of the present disclosure relate to compositions and methods for treating Spinal Muscular Atrophy (SMA). The disclosure is based in part on isolated nucleic acids encoding SMN1 and vectors (e.g., viral vectors, such as rAAV vectors). In some embodiments, the expression of SMN1 is driven by a native SMN1 promoter or variant thereof. In some embodiments, the isolated nucleic acids and vectors of the present disclosure have reduced toxicity and/or increased transgene expression relative to the SMN encoding vectors previously described.

Thus, in some aspects, the disclosure provides recombinant adeno-associated virus (rAAV) vectors comprising a transgene comprising an endogenous SMN1 promoter operably linked to a codon-optimized nucleic acid sequence encoding human SMN1, flanked by adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs). In some embodiments, the codon optimized nucleic acid sequence comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical to the nucleic acid sequence set forth in SEQ ID NO. 1. In some embodiments, the codon optimized nucleic acid sequence comprises or consists of the sequence set forth in SEQ ID NO. 1. In some embodiments, the codon optimized nucleic acid sequence does not comprise the nucleic acid sequence shown in SEQ ID NO. 2. In some embodiments, human SMN1 comprises an amino acid sequence set forth in SEQ ID NO. 3.

In some embodiments, the endogenous SMN1 promoter is a human SMN1 promoter. In some embodiments, the endogenous SMN1 promoter comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical to a nucleic acid sequence set forth in SEQ ID NO. 4 or 5. In some embodiments, the endogenous SMN1 promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID NO. 4. In some embodiments, the endogenous SMN1 promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID NO. 5.

In some embodiments, a recombinant adeno-associated virus (rAAV) vector comprises a transgene comprising a promoter operably linked to a codon-optimized nucleic acid sequence encoding human SMN1, flanked by adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs), wherein the promoter is a constitutive promoter. In some embodiments, the promoter is a CB6 promoter. In some embodiments, the rAAV vector further comprises a CMV enhancer.

In some embodiments, the rAAV vector further comprises one or more miR-122 binding sites. In some embodiments, the one or more miR-122 binding sites are located between a codon-optimized nucleic acid sequence encoding human SMN1 and the 3' itr. In some embodiments, the rAAV vector comprises a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO.

In some embodiments, at least one AAV ITR is an AAV2 ITR. In some embodiments, at least one AAV ITR is a mutated ITR (mTR).

In some aspects, the disclosure provides vectors comprising the rAAV vectors described herein. In some embodiments, the vector is a plasmid or baculovirus vector.

In some aspects, the disclosure provides a cell comprising a rAAV vector described herein or a vector described herein. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a mammalian cell, a bacterial cell, or an insect cell (e.g., SF9 cell).

In some aspects, the disclosure provides isolated nucleic acids comprising a nucleic acid sequence set forth in any one of SEQ ID NO. 1, SEQ ID NO. 6, or SEQ ID NO. 7.

In some embodiments, the isolated nucleic acid further comprises an endogenous SMN1 promoter operably linked to the nucleic acid sequence. In some embodiments, the endogenous SMN1 promoter is a human SMN1 promoter. In some embodiments, the endogenous SMN1 promoter comprises or consists of a nucleic acid sequence set forth in SEQ ID NO. 4 or 5.

In some aspects, the disclosure provides recombinant adeno-associated virus (rAAV) comprising a recombinant adeno-associated virus (rAAV) vector comprising a transgene comprising an endogenous SMN1 promoter operably linked to a codon-optimized nucleic acid sequence encoding human SMN1, flanked by adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs); and at least one AAV capsid protein.

In some embodiments, the codon optimized nucleic acid sequence comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical to the nucleic acid sequence set forth in SEQ ID NO. 1. In some embodiments, the codon optimized nucleic acid sequence comprises or consists of the sequence set forth in SEQ ID NO. 1. In some embodiments, the codon optimized nucleic acid sequence does not comprise the nucleic acid sequence shown in SEQ ID NO. 2. In some embodiments, human SMN1 comprises SEQ ID NO:3, and a sequence of amino acids shown in 3.

In some embodiments, the endogenous SMN1 promoter is a human SMN1 promoter. In some embodiments, the endogenous SMN1 promoter comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical to a nucleic acid sequence set forth in SEQ ID NO. 4 or 5. In some embodiments, the endogenous SMN1 promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID NO. 4. In some embodiments, the endogenous SMN1 promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID NO. 5.

In some embodiments, a recombinant adeno-associated virus (rAAV) vector comprises a transgene comprising a promoter operably linked to a codon-optimized nucleic acid sequence encoding human SMN1, flanked by adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs), wherein the promoter is a constitutive promoter. In some embodiments, the promoter is a CB6 promoter. In some embodiments, the rAAV vector further comprises a CMV enhancer.

In some embodiments, the rAAV vector further comprises one or more miR-122 binding sites. In some embodiments, the one or more miR-122 binding sites are located between a codon-optimized nucleic acid sequence encoding human SMN1 and the 3' itr. In some embodiments, the rAAV vector comprises a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO. In some embodiments, at least one AAV ITR is an AAV2 ITR. In some embodiments, at least one AAV ITR is a mutated ITR (mTR). In some embodiments, the rAAV is a self-complementary AAV (scAAV).

In some embodiments, at least one AAV capsid protein is an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein, or variant thereof. In some embodiments, at least one AAV capsid protein is an AAV9 capsid protein.

In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising: (a) a self-complementary rAAV genome comprising: (i) a 5' itr; (ii) A human short SMN promoter comprising the nucleotide sequence of SEQ ID No. 5; (iii) A codon optimized nucleic acid sequence encoding SMN1 as shown in SEQ ID No. 1; (iv) a poly a tail; and (v) a 3' itr; and (b) AAV9 capsid protein. In some embodiments, the poly a tail is a rabbit globin poly a tail or a BGH poly a tail. In some embodiments, the rAAV further comprises one or more miR-122 binding sites.

In some aspects, the disclosure provides pharmaceutical compositions comprising a rAAV vector or rAAV described herein and a pharmaceutically acceptable excipient.

In some aspects, the disclosure provides methods of delivering a transgene to a cell, the methods comprising administering to the cell a rAAV vector, rAAV, or pharmaceutical composition described herein.

In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is located in the subject. In some embodiments, the subject has or is suspected of having Spinal Muscular Atrophy (SMA).

In some aspects, the disclosure provides methods for preventing or treating Spinal Muscular Atrophy (SMA) in a subject, the methods comprising administering to the subject a rAAV vector, rAAV, or pharmaceutical composition described herein.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has one or more mutations in the SMN1 gene.

In some embodiments, administering comprises systemic injection or local injection. In some embodiments, systemic injection comprises intravenous injection. In some embodiments, administering comprises injection into the Central Nervous System (CNS) of the subject.

In some embodiments, administration results in a reduced amount of Dorsal Root Ganglion (DRG) toxicity in the subject relative to administration of a rAAV encoding wild-type SMN1 to the subject. In some embodiments, the administration results in a reduced amount of Dorsal Root Ganglion (DRG) toxicity in the subject as compared to administration of a rAAV comprising a constitutive promoter operably linked to a wild-type SMN1 coding sequence to the subject.

In some embodiments, the administration results in reduced hepatotoxicity in a subject as compared to administration to the subject of an AAV vector comprising a constitutive promoter operably linked to a nucleic acid encoding human SMN 1.

In some embodiments, administration results in a reduction in complications associated with SMA. In some embodiments, complications of SMA include lung infection, spinal deformity (e.g., scoliosis, hip subluxation/dislocation), joint contracture, or respiratory failure.

In some embodiments, the administration results in an increase in survival of the subject as compared to administration of an AAV vector comprising a constitutive promoter operably linked to a nucleic acid encoding human SMN1 to the subject.

Brief description of the drawings

Fig. 1 shows a schematic diagram of an SMN1 encoding vector of the present disclosure. "scAAV-CB6-opt-SMN1" is a self-complementary AAV (scAAV) vector having a CB6 promoter and codon optimized SMN1 coding sequence. "SMNlp-SMN1" is a single stranded AAV genome having a 2.0kb endogenous human SMN1 promoter and codon optimized SMN1 coding sequence (CDS). "ssAAV-opt-SMN1 (MBL)" is a single stranded AAV genome having a 2.0kb endogenous human SMN1 promoter, synthetic MBL intron, and codon optimized SMN1 coding sequence (CDS). "SMNsp-SMN1" is a self-complementary AAV vector having a 0.9kb endogenous human SMN1 promoter and codon optimized SMN1 CDS. "Zolgensma" is a self-complementing AAV vector with the CMV enhancer/chicken beta-actin promoter and wild-type human SMN1 CDS.

FIGS. 2A-2F show representative data for ssaaV9.opt-SMN1 and Zolgensma in SMNΔ7 mice. FIG. 2A shows survival curves for SMNΔ7 mice treated with Zolgensma (2 xE14 Genomic Copy (GC)/kg) or three doses of SMNp-SMN1 rAAV (2 xE14 Genomic Copy (GC)/kg; 6.8xE13 GC/kg or 3.4xE13 GC/kg); ssaav9.Opt-SMN1 treated at the same or 1/3 dose had better survival than mice treated with Zolgensma. Figures 2B and 2C show representative data indicating that mice treated with ssaav9.Opt-SMN1 showed similar weight gain compared to mice treated with Zolgensma. FIG. 2D shows survival curves of scaAV9.opt-SMN1 and Zolgensma in SMNΔ7 mice; scaav9.Opt-SMN1 treated at the same or 1/3 dose had better survival than mice treated with Zolgensma. Figures 2E and 2F show representative data indicating that mice treated with ssaav9.Opt-SMN1 showed similar weight gain compared to mice treated with Zolgensma.

FIGS. 3A-3B show representative behavioral assay data for SMNΔ7 mice treated with scaAV9.opt-SMN 1. FIG. 3A shows that animals treated with scaAV9.Opt-SMN1 can self-righte from day 5 post-administration (RIGHT THEMSELVES). The representative data shown in fig. 3B demonstrate that all treated animals have functional muscles (functional muscle) as measured by the rotation test.

Fig. 4A-4B show representative toxicology data. The histological data shown in fig. 4A demonstrate that high levels of SMN1 overexpression are toxic to hepatocytes. Briefly, 5 E+11GC/mouse AAV vector was injected into animals by facial intravenous injection at P0. Liver was collected on day 8 post injection. Liver damage was observed in AAVsc-CB6-PI-SMN1 (6/6 animals) and Zolgensma (4/7 animals) vector treated groups, but not in control empty AAV9 (0/8 animals) or ssAAV-opt-SMN 1 (0/8) vector treated groups. FIG. 4B shows representative alanine Aminotransferase (ALT) assay test results.

FIG. 5 shows a schematic representation of the putative expression levels of the SMN1 encoding vector.

FIGS. 6A-6D show representative data for SMN.DELTA.7 mice treated with Zolgensma, SMNp-SMN 1rAAV or SMNsp-SMN1 rAAV. FIG. 6A shows survival curves for SMNΔ7 mice treated with Zolgensma (5 xE11 Genomic Copy (GC)) or two doses of SMNp-SMN 1rAAV (5 xE11GC or 1.67xE11 GC). Figure 6B shows the weight growth of smnΔ7 mice treated with Zolgensma (5 xe11 GC) or two doses of SMNp-SMN 1rAAV (5 xe11GC or 1.67xe11 GC). FIG. 6C shows survival curves for SMNΔ7 mice treated with Zolgensma (5 xE11 GC) or three doses of SMNsp-SMN1rAAV (5 xE11GC, 1.67xE11GC, or 0.8xE11 GC). Figure 6D shows body weight growth of smnΔ7 mice treated with Zolgensma (5 xe11 GC) or two doses of SMNsp-SMN1rAAV (5 xe11GC or 1.67xe11 GC). Untreated control animals are also shown in each figure.

FIGS. 7A-7D show that hepatotoxicity is induced by rAAV9sc-CMVen/CB-co-hSMN1 due to overexpression of hSMN1 in the liver. FIG. 7A shows that mice treated with rAAV9sc-CMVen/CB-co-hSMN1 by facial intravenous injection show yellow skin. FIG. 7B shows that hepatotoxicity was observed by liver histology in mice receiving rAAV9sc-CMVen/CB-co-hSMN 1. FIG. 7C shows the number of animals with hepatotoxicity following rAAV9sc-CMVen/CB-co-hSMN treatment, as compared to the reference vehicle and Untreated Control (UC). FIG. 7D shows SMN1 expression in liver from mice treated with rAAV9sc-CMVen/CB-co-hSMN, reference vector, or untreated control.

FIGS. 8A-8C show that the incorporation of miR-122 binding site into CMVen/CB-co-hSMN vector abrogates SMN1 expression in liver, thereby reducing hepatotoxicity. FIG. 8A shows AAV vectors with or without miR-122 binding sites. FIG. 8B shows that the incorporation of miR-122 binding site (CB 6-PI-opt hSMN1-miR-122 BS) in CMVen/CB-co-hSMN vector abrogates SMN1 expression in liver. FIG. 8C shows that mice treated with scaV 9-CB6-PI-opt hSMN1-miR-122BS were not hepatotoxic compared to mice treated with rAAV9sc-CMVen/CB-co-hSMN 1.

Figures 9A-9L show that second generation AAV (AAVsc-SMNp-co-hSMN 1) resulted in improved therapeutic results in SMA animal models. FIG. 9A shows that AAVsc-SMNp-co-hSMN1 produced higher expression efficiency in Neuro-2a cells compared to rAAV9sc-CMVen/CB6-co-hSMN 1. Fig. 9B shows the experimental design of mice treated by facial intravenous injection with AAVsc-SMNp-co-hSMN1, which mice were monitored daily after treatment. FIG. 9C shows that mice treated with AAVsc-SMNsp-co-hSMN1 increased more body weight than the reference vehicle group. Fig. 9D shows that administration of AAVsc-SMNsp-co-hSMN1 vector significantly improved the longevity of SMA mice. FIG. 9E shows that AAVsc-SMNsp-co-hSMN1 treated SMA mice were healthy at 5 months of age. FIG. 9F shows that SMA mice treated with AAVsc-SMNsp-co-hSMN1 were able to self-right earlier than the reference mice. Fig. 9G is a representative image from the transverse abdominal muscle (TRANSVERSE ABDOMINIS, TVA) showing the therapeutic effect on the restoration of innervation of neuromuscular junctions (NMJ). FIG. 9H shows that the neuromuscular junction structure in mice treated with AAVsc-SMNsp-co-hSMN1 recovered to a structure close to that of wild type mice, superior to that of reference treated SMA mice. Fig. 9I shows different stages of ear necrosis, which is one of the complications of SMA mice. FIG. 9J shows that treatment with AAVsc-SMNsp-co-hSMN1 reduces complications (e.g., ear necrosis) in SMA mice. Immunoblot analysis shown by 9K-9L indicates that AAVsc-SMNsp-co-hSMN1 is preferably expressed in the central nervous system and not in peripheral tissues. The expression pattern of the vector was close to the natural distribution of SMN in healthy control animals.

FIG. 10 shows representative survival curves for SMA mice untreated (NT), treated with baseline vector (BMK), or treated with scaAAV 9-SMNsp-hSMN 1; mice treated with scAAV9-SMNsp-hSMN1 had better survival than untreated mice or mice treated with BMK.

Detailed Description

Aspects of the present disclosure relate to compositions and methods for treating Spinal Muscular Atrophy (SMA). The disclosure is based in part on isolated nucleic acids encoding SMN1 and vectors (e.g., viral vectors, such as rAAV vectors). In some embodiments, the expression of SMN1 is driven by a native SMN1 promoter or variant thereof. In some embodiments, the isolated nucleic acids and vectors of the present disclosure have reduced toxicity and/or increased transgene expression and/or reduced off-target (off-target) tissue expression relative to vectors encoding SMN described previously.

Motor neuron survival 1 (SMN 1)

Aspects of the disclosure relate to compositions (e.g., isolated nucleic acids, vectors such as rAAV vectors, rAAV, etc.) encoding motor neuron survival 1 (SMN 1) proteins. SMN1 protein is a component of a complex that catalyzes the assembly of small nuclear ribonucleoprotein (snRNP) and plays an important role in cellular pre-mRNA splicing. SMN1 and SMN2 mutations are associated with Spinal Muscular Atrophy (SMA), a severe neuromuscular disease that results in loss of motor neurons and progressive muscular atrophy. Spinal Muscular Atrophy (SMA) is a rare neuromuscular disease that results in loss of motor neurons and progressive muscular atrophy. It is usually diagnosed during infancy or childhood and if untreated, becomes the most common genetic cause of death in infants. It may also appear later in life, with the course then being lighter. A common feature is the progressive weakness of the voluntary muscles (voluntary muscles), the arms, legs and respiratory muscles being affected first. SMA patients may also develop complications, and related problems may include poor head control, dysphagia, pulmonary infection, spinal deformities (e.g., scoliosis, hip subluxation/dislocation), joint contractures, and respiratory failure.

The age of onset and the severity of symptoms form the basis for the traditional classification of spinal muscular atrophy into several categories.

Spinal muscular atrophy is caused by abnormalities (mutations) in the SMN1 gene that encodes SMN, a protein necessary for motor neuron survival. Loss of these neurons in the spinal cord can block signaling between the brain and skeletal muscle. Another gene, SMN2, is considered a disease modifying gene because generally the more copies of SMN2, the lighter the disease progression. The diagnosis of SMA is based on symptoms and confirmed by genetic testing.

The natural course of the disease varies in outcome, with the most severe cases dying within weeks after birth, while the long-term SMA form has a normal life expectancy.

Drugs targeting the genetic cause of the disease include norcinnabar (nusinersen), li Sipu blue (risdiplam) and gene therapy drug onasemnogene abeparvovec. Support care includes physical therapy, job therapy (occupational therapy), respiratory support, nutritional support, orthopedic intervention, and mobility support.

In humans, SMN1 is encoded by the SMN1 gene, as shown in NCBI reference sequence numbers nm_000344, nm_001297715, and nm_ 022874. In some embodiments, the SMN1 protein comprises an amino acid sequence set forth in any one of NCBI reference sequence numbers np_059107, np_075013, np_075014, np_075015, np_000335. In some embodiments, the SMN1 protein comprises the amino acid sequence set forth in SEQ ID NO. 3. In some embodiments, the SMN1 protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 3.

In some aspects, the disclosure relates to isolated nucleic acids comprising a codon-optimized (e.g., optimized to increase expression, reduce toxicity, reduce immunogenicity, etc.) nucleic acid sequence encoding an SMN1 protein. In some embodiments, the isolated nucleic acid encoding the SMN1 protein comprises a sequence identical to SEQ ID NO:1, at least 70%, 75%, 80%, 90%, 95% or 99% identical. In some embodiments, the isolated nucleic acid encoding an SMN1 protein comprises the nucleic acid sequence set forth in SEQ ID NO. 1. In some embodiments, the isolated nucleic acid encoding the SMN1 protein consists of the nucleic acid sequence set forth in SEQ ID NO. 1.

In some embodiments, the isolated nucleic acid encoding the SMN1 protein comprises at least one (e.g., 1, 2, 3, 4, 5, 10,15, 20, 25, 50, 100, 150, 200, 250, or more) nucleotide substitution, insertion, deletion, or any combination thereof, relative to a wild-type SMN1 encoding nucleic acid sequence, such as the nucleic acid sequence set forth in SEQ ID No. 2.

"Nucleic acid" sequence refers to a DNA or RNA sequence. In some embodiments, the proteins and nucleic acids of the present disclosure are isolated. As used herein, the term "isolated" refers to an artificial generation. As used herein, with respect to nucleic acids, the term "isolated" refers to: (i) Amplification in vitro by, for example, polymerase Chain Reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, such as by cleavage and gel separation; or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is one that can be readily manipulated by recombinant DNA techniques well known in the art. Thus, the nucleotide sequences contained in the vector, wherein the 5 'and 3' restriction sites are known or for which the Polymerase Chain Reaction (PCR) primer sequences have been disclosed, are considered isolated, but the nucleic acid sequences present in their natural state in their natural hosts are not isolated. The isolated nucleic acid may be substantially purified, but is not required. For example, a nucleic acid isolated within a cloning or expression vector is impure in that it may be only a small percentage of the material in the cell in which it is located. However, as the term is used herein, such a nucleic acid is isolated in that it can be readily manipulated by standard techniques known to those of ordinary skill in the art. As used herein, the term "isolated" with respect to a protein or peptide refers to a protein or peptide that has been isolated from its natural environment or that has been artificially produced (e.g., by chemical synthesis, by recombinant DNA techniques, etc.).

The isolated nucleic acid of the disclosure may be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, an isolated nucleic acid as described in the present disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) Inverted Terminal Repeat (ITR) or variant thereof. The isolated nucleic acid (e.g., recombinant AAV vector) can be packaged into a capsid protein and administered to a subject and/or delivered to a selected target cell. "recombinant AAV (rAAV) vectors" typically consist of at least a transgene and its regulatory sequences, and 5 'and 3' AAV Inverted Terminal Repeats (ITRs). The transgene may comprise a region encoding, for example, a protein and/or an expression control sequence (e.g., a poly-a tail), as described elsewhere in this disclosure.

In general, ITR sequences are about 145bp in length. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, although some minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (see, e.g., Sambrook et al.,"Molecular Cloning.A Laboratory Manual",2d ed.,Cold Spring Harbor Laboratory,New York(1989); and K.Fisher et al, J Virol. 70:520:532 (1996)). Examples of such molecules employed in the present disclosure are "cis-acting" plasmids containing transgenes, wherein the selected transgene sequences and associated regulatory elements are flanked by 5 'and 3' aav ITR sequences. AAV ITR sequences can be obtained from any known AAV, including the currently identified mammalian AAV types. In some embodiments, the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR. In some embodiments, an isolated nucleic acid encoding a transgene is flanked by AAV ITRs (e.g., in the 5 '-ITR-transgene-ITR-3' direction). In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, at least one AAV ITR is a truncated AAV ITR (e.g., a mutated ITR, also known as mTR), such as the ΔITR described in McCarty (2008) Molecular Therapy (10): 1648-1656.

In addition to the major elements defined above for recombinant AAV vectors, the vectors also comprise conventional control elements operably linked to the transgenic elements in a manner that allows them to be transcribed, translated, and/or expressed in cells transfected with the vectors produced by the present disclosure or infected with the viruses produced by the present disclosure. As used herein, "operably linked" sequences include expression control sequences adjacent to a gene of interest and expression control sequences that function in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly a) signals; stabilizing the sequence of cytoplasmic mRNA; sequences that increase translation efficiency (e.g., kozak consensus sequences); a sequence that enhances protein stability; and, when desired, sequences that enhance secretion of the encoded product. Many expression control sequences, including natural, constitutive, inducible and/or tissue specific promoters, are known in the art and may be used.

As used herein, a nucleic acid sequence (e.g., coding sequence) and a regulatory sequence are said to be operably linked when they are covalently linked in a manner that places the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequence. If it is desired to translate a nucleic acid sequence into a functional protein, two DNA sequences are said to be operably linked if the induction of a promoter in the 5' regulatory sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frame shift mutation, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, if a promoter region is capable of affecting transcription of a DNA sequence such that the resulting transcript can be translated into a protein or polypeptide of interest, the promoter region will be operably linked to the nucleic acid sequence. Similarly, two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins that have been translated in-frame. In some embodiments, the operably linked coding sequences produce a fusion protein.

The region comprising the transgene (e.g., a transgene encoding an SMN1 protein, etc.) may be located at any suitable location in the isolated nucleic acid that is capable of expressing at least one transgene, selectable marker protein, or reporter protein.

It will be appreciated that where the transgene encodes more than one gene product (e.g., SMN1 protein and another protein or interfering nucleic acid), each gene product may be located at any suitable location within the transgene. For example, a nucleic acid encoding a first polypeptide may be located in an intron of a transgene, and a nucleic acid sequence encoding a second polypeptide may be located in another untranslated region (e.g., between the last codon of the protein coding sequence and the first base of the poly-a signal of the transgene).

"Promoter" refers to a DNA sequence recognized by a cell's synthetic machinery or an introduced synthetic machinery that is required to initiate specific transcription of a gene. The phrase "operably linked," "operably positioned," "under control," or "under transcriptional control" refers to a promoter that is in the correct position and orientation relative to a nucleic acid to control RNA polymerase initiation and gene expression.

For nucleic acids encoding proteins, polyadenylation sequences are typically inserted after the transgene sequence and before the 3' aav ITR sequences. The rAAV constructs used in the present disclosure may also comprise introns, ideally located between promoter/enhancer sequences and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40T intron sequence. In some embodiments, the intron is a non-natural intron or a synthetic intron (e.g., an MBL intron). Another vector element that may be used is an Internal Ribosome Entry Site (IRES). IRES sequences are used to produce more than one polypeptide from a single gene transcript. IRES sequences can be used to produce proteins containing more than one polypeptide chain. These and other common vector element choices are conventional, and many such sequences are available [ see, e.g., sambrook et al, and documents cited therein, e.g., pages 3.18.3.26 and 16.17.27 and Ausubel et al, current Protocols in Molecular Biology, john Wiley & Sons, new York,1989]. In some embodiments, the polyprotein (polyprotein) comprises foot-and-mouth disease virus 2A sequences; this is a small peptide (about 18 amino acids in length) that has been shown to mediate cleavage (Ryan,M D et al.,EMBO,1994;4:928-933;Mattion,N M et al.,J Virology,November 1996;p.8124-8127;Furler,S et al.,Gene Therapy,2001;8:864-873; of polyprotein and Halpin, C et al, the Plant Journal,1999; 4:453-459). Cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retrovirus) (Ryan,M D et al.,EMBO,1994;4:928-933;Mattion,N M et al.,J Virology,November 1996;p.8124-8127;Furler,S et al.,Gene Therapy,2001;8:864-873; and Halpin,C et al.,The Plant Journal,1999;4:453-459;de Felipe,P et al.,Gene Therapy,1999;6:198-208;de Felipe,P et al.,Human Gene Therapy,2000;11:1921-1931.; and Klump, H et al GENE THERAPY,2001; 8:811-817).

Examples of constitutive promoters include, but are not limited to, the retrovirus Rous Sarcoma Virus (RSV) LTR promoter (optionally with an RSV enhancer), the Cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [ see, e.g., boshart et al, cell,41:521-530 (1985) ], the SV40 promoter, the dihydrofolate reductase promoter, the β -actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF 1a promoter [ Invitrogen ]. In some embodiments, the promoter is an RNA pol II promoter. In some embodiments, the promoter is an RNA pol III promoter, such as U6 or H1. In some embodiments, the promoter is an RNA pol II promoter. In some embodiments, the promoter is a chicken β -actin (CBA) promoter. In some embodiments, the promoter comprises a U1a promoter.

Inducible promoters allow for regulation of gene expression and may be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of specific physiological states such as the acute phase, specific differentiation states of cells, or in replicating cells alone. Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to Invitrogen, clontech and Ariad. Many other systems have been described and can be readily selected by one skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep Metallothionein (MT) promoter, dexamethasone (Dex) inducible Mouse Mammary Tumor Virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); ecdysone insect promoters (No. et al, proc.Natl. Acad.Sci.USA,93:3346-3351 (1996)), tetracycline inhibition systems (Gossen et al, proc.Natl. Acad.Sci.USA,89:5547-5551 (1992)), tetracycline induction systems (Gossen et al, science,268:1766-1769 (1995), see also Harvey et al, curr.Opin.chem.biol.,2:512-518 (1998)), RU486 induction systems (Wang et al, nat.Biotech.,15:239-243 (1997) and Wang et al, gene Ther, 4:432-441 (1997)), and rapamycin induction systems (Magari et al, J.Clin. Invest.,100:2865-2872 (1997)). Other types of inducible promoters useful herein are those regulated by specific physiological states such as temperature, acute phase, specific differentiation state of cells, or in replicating cells only.

Aspects of the disclosure relate to isolated nucleic acids and rAAV vectors comprising a nucleic acid sequence encoding SMN1 (e.g., a codon optimized nucleic acid sequence encoding SMN 1) operably linked to a native promoter. In some embodiments, the native promoter comprises a human SMN1 promoter or variant thereof. In some embodiments, the human SMN1 promoter comprises a nucleic acid sequence set forth in SEQ ID NO. 4. In some embodiments, the promoter comprises a portion of a human SMN1 promoter comprising the nucleic acid sequence set forth in SEQ ID NO. 5 (i.e., a short hSMN1 promoter). In some embodiments, the human SMN1 promoter or variant thereof comprises a sequence identical to SEQ ID NO:4 or SEQ ID NO:5, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical. Human SMN1 promoters are well known in the art, for example as described in Echaniz-Lagura et al, am.J. hum.Genet.64:1365-1370, 1999. Where expression of the transgene is desired to mimic natural expression (e.g., expression of physiological levels of hSMN1 in an appropriate cell type), a natural promoter may be preferred. A natural promoter may be used when expression of the transgene must be regulated in time or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. Without wishing to be bound by any theory, the use of the human SMN1 promoter in the isolated nucleic acids and rAAV vectors described herein is capable of modulating expression of human SMN1 protein by the vector and reducing toxicity, e.g., dorsal Root Ganglion (DRG) toxicity or hepatotoxicity, in a subject relative to expression of human SMN1 protein from isolated nucleic acids and rAAV vectors comprising other promoters, e.g., CMV promoter, chicken-beta actin (CBA) promoter, CB6 promoter, etc. In further embodiments, other natural expression control elements such as enhancer elements, polyadenylation sites, and/or Kozak consensus sequences may also be used to mimic natural expression.

In some embodiments, the regulatory sequences confer tissue specific gene expression capacity. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: a proximal retinal cleavage protein promoter (retinoschisin proximal promoter), an internotoreceptor retinol-binding protein enhancer (RS/IRBPa), rhodopsin Kinase (RK), a liver-specific thyroxine-binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a Pancreatic Polypeptide (PPY) promoter, a synaptosin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian junction protein (DES) promoter, an alpha-myosin heavy chain (alpha-MHC) promoter, or a cardiac troponin T (cTnT) promoter. Other exemplary promoters include the beta-actin promoter, the hepatitis b virus core promoter (Sandig et al., gene ter., 3:1002-9 (1996)); alpha Fetoprotein (AFP) promoter (Arbuthnot et al., hum. Gene Ther.,7:1503-14 (1996)), osteocalcin promoter (Stein et al., mol. Biol. Rep.,24:185-96 (1997)); bone sialoprotein promoter (Chen et al, j.bone miner, res.,11:654-64 (1996)), CD2 promoter (Hansal et al, j.immunol.,161:1063-8 (1998)); an immunoglobulin heavy chain promoter; t cell receptor alpha chain promoters, neuronal such as Neuronal Specific Enolase (NSE) promoters (ANDERSEN ET al., cell. Mol. Neurobiol.,13:503-15 (1993)), neurofilament light chain gene promoters (Piccioli et al., proc. Natl. Acad. Sci. USA,88:5611-5 (1991)), and neuronal specific vgf gene promoters (Piccioli al., neuron,15:373-84 (1995)), and the like, as will be apparent to those skilled in the art.

In some embodiments, the promoter preferably drives transgene expression in certain tissues. In some embodiments, the disclosure provides nucleic acids comprising a tissue-specific promoter operably linked to a transgene. As used herein, a "tissue-specific promoter" refers to a promoter that preferentially modulates (e.g., drives or upregulates) gene expression in a particular cell type relative to other cell types. The cell type specific promoter may be specific for any cell type, such as Central Nervous System (CNS) cells, liver cells (e.g., hepatocytes), cardiac cells, muscle cells, and the like. In some embodiments, the tissue-specific promoter is a muscle tissue or cell-specific promoter. Examples of CNS-specific promoters include, but are not limited to, synaptotagmin (Syn), GFAP, ca 2 ⁺/calmodulin-dependent protein kinase II (hCAMKII), and the like.

In some aspects, the disclosure relates to isolated nucleic acids comprising a transgene encoding one or more miRNA binding sites. Without wishing to be bound by any particular theory, the incorporation of miRNA binding sites into gene expression constructs allows for modulation of transgene expression (e.g., inhibition of transgene expression) in cells and tissues expressing the corresponding miRNA. In some embodiments, the incorporation of one or more miRNA binding sites into the transgene allows for off-targeting of transgene expression in a cell type specific manner (de-targeting). In some embodiments, one or more miRNA binding sites are located in the 3 'untranslated region (3' utr) of the transgene, e.g., between the last codon of a nucleic acid sequence encoding one or more complement control proteins (complement control protein) described herein and the poly a sequence.

In some embodiments, a rAAV vector described herein comprises a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO.

In some embodiments, transgene expression results in overexpression of the transgene in the liver, resulting in hepatotoxicity (see, e.g., ,Hinderer et al.,Severe Toxicity in Nonhuman Primates and Piglets Following High-Dose Intravenous Administration of an Adeno-Associated Virus Vector Expressing Human SMN,Volume:29Issue 3,285-298:2018, 3, 1). In some embodiments, to reduce hepatotoxicity, an AAV vector comprises a transgene comprising one or more (e.g., 1,2,3, 4, 5, or more) miRNA binding sites that off-target transgene expression by hepatocytes. For example, in some embodiments, the transgene comprises one or more miR-122 binding sites. In some embodiments, a rAAV vector described herein comprises one or more miR-122 binding sites. In some embodiments, a rAAV vector comprising a miR-122 binding site comprises a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO.

In some embodiments, the transgene comprises one or more (e.g., 1, 2, 3, 4, 5, or more) miRNA binding sites that off-target transgene expression by immune cells (e.g., antigen Presenting Cells (APCs), such as macrophages, dendritic cells, etc.). The incorporation of miRNA binding sites for immune-related mirnas can off-target transgene (e.g., one or more inhibitory nucleic acids) expression by antigen presenting cells, thereby reducing or eliminating immune responses (cellular and/or humoral immune responses) by the subject to the products of the transgenes, e.g., as described in US2018/0066279, the entire contents of which are incorporated herein by reference.

As used herein, an "immune-related miRNA" is a miRNA that is preferably expressed in a cell of the immune system, such as an Antigen Presenting Cell (APC). In some embodiments, the immune-related miRNA is a miRNA expressed in an immune cell at a level that appears to be at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold higher than a non-immune cell (e.g., a control cell such as a HeLa cell, HEK293 cell, mesenchymal cell, etc.). In some embodiments, the immune system cells (immune cells) expressing the immune-related mirnas are B cells, T cells, killer T cells, helper T cells, γδ T cells, dendritic cells, macrophages, monocytes, vascular endothelial cells, or other immune cells. In some embodiments, the cells of the immune system are B cells ：B220、BLAST-2(EBVCS)、Bu-1、CD19、CD20(L26)、CD22、CD24、CD27、CD57、CD72、CD79a、CD79b、CD86、chB6、D8/17、FMC7、L26、M17、MUM-1、Pax-5(BSAP) and PC47H that express one or more of the following markers. In some embodiments, the cells of the immune system are T cells that express one or more of the following markers: ART2, CD1a, CD1d, CD11b (Mac-1), CD134 (OX 40), CD150, CD2, CD25 (interleukin 2 receptor α)、CD3、CD38、CD4、CD45RO、CD5、CD7、CD72、CD8、CRTAM、FOXP3、FT2、GPCA、HLA-DR、HML-1、HT23A、Leu-22、Ly-2、Ly-m22、MICG、MRC OX 8、MRC OX-22、OX40、PD-1( programmed death-1), RT6, TCR (T cell receptor), thy-1 (CD 90) and TSA-2 (thymus sharing Ag-2). In some embodiments, the immune-related miRNA is selected from ：miR-15a、miR-16-1、miR-17、miR-18a、miR-19a、miR-19b-1、miR-20a、miR-21、miR-29a/b/c、miR-30b、miR-31、miR-34a、miR-92a-1、miR-106a、miR-125a/b、miR-142-3p、miR-146a、miR-150、miR-155、miR-181a、miR-223 and miR-424, miR-221, miR-222, let-7i, miR-148 and miR-152. In some embodiments, the transgenes described herein comprise one or more binding sites for miR-142.

Recombinant adeno-associated virus (rAAV)

In some aspects, the disclosure provides isolated adeno-associated viruses (AAV). As used herein, the term "isolated" with respect to AAV refers to an AAV that is artificially produced or obtained. Isolated AAV may be produced using recombinant methods. Such AAV is referred to herein as a "recombinant AAV". Recombinant AAV (rAAV) preferably has tissue-specific targeting capabilities such that the transgene of the rAAV is specifically delivered to one or more predetermined tissues (e.g., muscle tissue, ocular tissue, neurons, etc.). AAV capsids are important elements that determine these tissue-specific targeting capabilities (e.g., tissue tropism). Thus, rAAV having a capsid appropriate for the targeted tissue may be selected.

In some embodiments, the rAAV of the disclosure comprises a nucleotide sequence as set forth in SEQ ID NO. 1, 6, or 7, or encodes a protein having an amino acid sequence as set forth in SEQ ID NO. 3. In some embodiments, a rAAV of the disclosure comprises a nucleotide sequence that hybridizes to SEQ ID NO: 1. the nucleotide sequences shown in 6-11 are 99% identical, 95% identical, 90% identical, 85% identical, 80% identical, 75% identical, 70% identical, 65% identical, 60% identical, 55% identical or 50% identical nucleotide sequences.

In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising: (a) a self-complementary rAAV genome comprising: (i) a 5' itr; (ii) A human short SMN promoter comprising the nucleotide sequence of SEQ ID No. 5; (iii) A codon optimized nucleic acid sequence encoding SMN1 as shown in SEQ ID No. 1; (iv) a poly a tail; and (v) a 3' itr; and (b) AAV9 capsid protein. In some embodiments, the poly a tail is a rabbit globin poly a or BGH poly a tail. In some embodiments, the rAAV further comprises one or more miR-122 binding sites.

Methods for obtaining recombinant AAV having a desired capsid protein are well known in the art. (see, e.g., US 2003/013872, the contents of which are incorporated herein by reference in their entirety). Generally, the methods involve culturing a host cell comprising: a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector comprising an AAV Inverted Terminal Repeat (ITR) and a transgene; and an ancillary function sufficient to allow packaging of the recombinant AAV vector into an AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the cap gene of an AAV. AAV comprises three capsid proteins, namely virosome proteins 1 to 3 (referred to as VP1, VP2 and VP 3), all of which are transcribed from a single cap gene by alternative splicing. In some embodiments, VP1, VP2, and VP3 have molecular weights of about 87kDa, about 72kDa, and about 62kDa, respectively. In some embodiments, the capsid protein forms a globular 60-mer protein shell around the viral genome after translation. In some embodiments, the function of the capsid protein is to protect the viral genome, deliver the genome, and interact with the host. In some aspects, the capsid proteins deliver the viral genome to a host in a tissue-specific manner.

In some embodiments, the AAV capsid protein has tropism for Central Nervous System (CNS) tissues. In some embodiments, the AAV capsid protein targets a neuronal cell type, astrocyte, oligodendrocyte, glial cell, or the like. In some embodiments, the AAV capsid protein has an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, aav.php-eB, AAVrh39, AAVrh43, and variants of any of the foregoing. In some embodiments, the rAAV comprises AAV9 capsid proteins.

In some embodiments, the rAAV vector or rAAV particle comprises a mutated ITR lacking a functional terminal dissociation site (TRS). The term "lack of a terminal dissociation site" may refer to an AAV ITR comprising a mutation (e.g., a sense mutation, such as a nonsensical mutation or a missense mutation) that eliminates the function of the terminal dissociation site (TRS) of the ITR, or to a truncated AAV ITR (e.g., Δtrs ITR) lacking a nucleic acid sequence encoding a functional TRS. Without wishing to be bound by any particular theory, rAAV vectors comprising ITRs lacking a functional TRS produce self-complementary rAAV vectors (scAAV or scrav vectors), e.g., as described in McCarthy (2008) Molecular Therapy (10): 1648-1656.

The components cultured in the host cell to encapsulate the rAAV vector in the AAV capsid may be provided to the host cell in trans. Or any one or more of the desired components (e.g., recombinant AAV vectors, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell that has been engineered to contain one or more of the desired components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the desired components under the control of an inducible promoter. However, the desired components may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein when discussing regulatory elements suitable for use with a transgene. In yet another alternative, the selected stable host cell may contain the selected component under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, stable host cells can be produced that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter), but which contain rep and/or cap proteins under the control of an inducible promoter. Other stable host cells can also be produced by those skilled in the art.

In some embodiments, the disclosure relates to host cells containing nucleic acids comprising a codon optimized coding sequence encoding a transgene (e.g., SMN 1). "host cell" refers to any cell that contains or is capable of containing a substance of interest. Typically, the host cell is a mammalian cell. In some embodiments, the host cell is a neuron. The host cell may be used as a recipient for an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAV. The term includes progeny of the original cell that has been transfected. Thus, as used herein, a "host cell" may refer to a cell that has been transfected with an exogenous DNA sequence. It will be appreciated that the progeny of a single parent cell need not be identical, in morphology or in genomic or total DNA complement, to the original parent, due to natural, accidental, or deliberate mutation. In some embodiments, the host cell is a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. In some embodiments, the host cell is a central nervous system cell, such as a neuron or glial cell.

Any suitable genetic element (vector) may be used to deliver the recombinant AAV vectors, rep sequences, cap sequences, and helper functions required to produce the rAAV of the present disclosure to packaging host cells. The selected genetic element may be delivered by any suitable method, including those described herein. Methods for constructing any of the embodiments of the present disclosure are known to the nucleic acid operator and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., ,Sambrook et al.,Molecular Cloning:ALaboratory Manual,Cold Spring Harbor Press,Cold Spring Harbor,N.Y. similarly, methods of producing rAAV virions are well known and selection of suitable methods does not constitute a limitation of the present disclosure. See, for example, K.Fisher et al, J.Virol.,70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAV can be produced using a triple transfection method (described in detail in U.S. patent No. 6,001,650). Typically, recombinant AAV is produced by transfecting a host cell with an AAV vector (comprising a transgene flanked by ITR elements), an AAV helper function vector, and an accessory function vector to be packaged into an AAV particle. AAV helper function vectors encode "AAV helper function (AAV HELPER functions)" sequences (e.g., rep and cap) that function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the disclosure include pHLP19 described in U.S. Pat. No. 6,001,650 and pRep6cap6 vectors described in U.S. Pat. No. 6,156,303, the disclosures of which are incorporated herein by reference in their entirety. Accessory function vectors encode nucleotide sequences that have viral and/or cellular functions that are not AAV-derived, upon which AAV depends for replication (e.g., "accessory function (accessory function)"). Accessory functions include those required for AAV replication, including but not limited to those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly. The viral-based accessory function may be derived from any known helper virus, such as adenovirus, herpes virus (except herpes simplex virus type 1) and vaccinia virus.

In some aspects, the disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, which has been "transfected" when the foreign DNA has been introduced into the cell membrane. Numerous transfection techniques are known in the art. See, e.g., ,Graham et al.(1973)Virology,52:456,Sambrook et al.(1989)Molecular Cloning,a laboratory manual,Cold Spring Harbor Laboratories,New York,Davis et al.(1986)Basic Methods in Molecular Biology,Elsevier, and Chu et al (1981) Gene 13:197. Such techniques may be used to introduce one or more exogenous nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, into a suitable host cell.

As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA fragment has been introduced, e.g., a DNA fragment that results in transcription of a biologically active polypeptide or production of a biologically active nucleic acid (e.g., RNA).

As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when combined with appropriate control elements and which can transfer gene sequences between cells. In some embodiments, the vector is a viral vector, e.g., a rAAV vector, a lentiviral vector, an adenoviral vector, a retroviral vector, and the like. Thus, the term includes cloning and expression vehicles and viral vectors. In some embodiments, the vector comprises a baculovirus vector, which can be used to produce viral particles in certain insect cells (e.g., SF9 cells). In some embodiments, useful vectors are considered to be those in which the nucleic acid fragment to be transcribed is under the transcriptional control of a promoter.

Delivery of transgenes to tissue

The isolated nucleic acids, rAAV, and compositions of the present disclosure can be delivered to a subject in the form of a composition according to any suitable method known in the art. For example, the rAAV, preferably suspended in a physiologically compatible carrier (i.e., in a composition), can be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque). In some embodiments, the host animal does not include a human. In some embodiments, the subject is a human.

Delivery of the rAAV may be by, for example, intramuscular injection or infusion into muscle tissue or cells of the subject. As used herein, "muscle tissue" refers to any tissue derived from or contained in skeletal, smooth, or cardiac muscle of a subject. Non-limiting examples of muscle tissue include skeletal muscle, smooth muscle, cardiac muscle, myocytes, sarcomere (sarcomere), myofibrils, and the like.

May be administered into the blood stream by injection into a vein, artery or any other blood vessel. In some embodiments, the rAAV is administered into the blood stream by a technique well known in the surgical arts, i.e., isolated limb perfusion (isolated limb perfusion), which essentially enables the technician to isolate the limb from the systemic circulation prior to administration of the rAAV virion. The skilled artisan can also use a variant of the isolated limb perfusion technique described in U.S. patent No. 6,177,403 to administer virions into the vasculature of the isolated limb to potentially enhance transduction to muscle cells or tissue.

Aspects of the disclosure relate to compositions comprising a recombinant AAV comprising a capsid protein and a nucleic acid encoding a transgene, wherein the transgene comprises a codon optimized nucleic acid sequence encoding an SMN1 protein. In some embodiments, the nucleic acid further comprises an AAV ITR. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

The compositions of the disclosure may comprise a rAAV alone, or in combination with one or more other viruses (e.g., encoding a second rAAV having one or more different transgenes). In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAV, each having one or more different transgenes.

The skilled artisan can readily select an appropriate carrier in view of the indication for which the rAAV is intended. For example, one suitable carrier includes saline, which may be formulated with a variety of buffer solutions (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. The choice of carrier does not constitute a limitation of the present disclosure.

Optionally, the compositions of the present disclosure may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and carrier(s). Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, parachlorophenol, and poloxamers (nonionic surfactants) such asF-68. Suitable chemical stabilizers include gelatin and albumin.

The rAAV is administered in a sufficient amount to transfect cells of the tissue of interest and provide sufficient levels of gene transfer and expression without undue side effects. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to selected organs (e.g., portal intravenous delivery to the liver), intraocular injection, subretinal injection, oral administration, inhalation (including intranasal and intratracheal delivery), intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. The routes of administration may be combined if desired.

The dose of rAAV virions required to achieve a particular "therapeutic effect", e.g., in genomic copies per kilogram body weight (GC/kg), will vary depending on a number of factors, including, but not limited to: the route of administration of the rAAV virions, the level of gene or RNA expression required to achieve a therapeutic effect, the particular disease or disorder being treated, and the stability of the gene or RNA product. The skilled artisan can readily determine the range of rAAV virion doses to be used to treat patients with a particular disease or disorder based on the factors described above, as well as other factors well known in the art. In some embodiments, a rAAV described herein is administered to a subject at a dose of about 1ml to about 100ml of a solution containing about 10 ⁹ to 10 ¹⁶ copies of the genome. In some cases, the dose administered is about 10 ¹¹ to 10 ¹³ rAAV genome copies.

An effective amount of rAAV is an amount sufficient to target an infected animal, target a target tissue. In some embodiments, an effective amount of rAAV is administered to the subject at a pre-symptomatic stage of the degenerative disease. In some embodiments, the rAAV or composition is administered to the subject after exhibiting one or more signs or symptoms of the degenerative disease.

The effective amount of rAAV may also depend on the mode of administration. For example, in some cases, targeting muscle tissue (e.g., muscle cells) by intramuscular administration or subcutaneous injection may require a different (e.g., higher or lower) dose than targeting muscle tissue by another method (e.g., systemic administration, topical administration, etc.). In some embodiments, intramuscular Injection (IM) of rAAV with certain serotypes (e.g., AAV2, AAV6, AAV9, etc.) mediates efficient transduction of muscle cells. Thus, in some embodiments, the injection is intramuscular Injection (IM). In some embodiments, the injection is systemic administration (e.g., intravenous injection). In certain instances, multiple doses of rAAV are administered. In some embodiments, the administration is systemic administration. In some embodiments, systemic administration includes intravenous administration. In some embodiments, the administration is topical to the central nervous system, e.g., by an intra-brain injection, intrathecal injection, intracranial injection, and the like.

In some embodiments, the rAAV composition is formulated to reduce aggregation of AAV particles in the composition, particularly in the presence of high rAAV concentrations (e.g., -10 ¹³ GC/mL or higher). Methods for reducing rAAV aggregation are well known in the art and include, for example, surfactant addition, pH adjustment, salt concentration adjustment, and the like (see, e.g., wright FR, et al Molecular Therapy (2005) 12,171-178, the contents of which are incorporated herein by reference).

The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, and the development of suitable dosing and treatment regimens for using the specific compositions described herein in various treatment regimens is also well known to those skilled in the art.

Typically, these formulations may contain at least about 0.1% or more of the active compound, although the percentage of active ingredient(s) may of course vary and may conveniently be between about 1 or 2% to about 70% or 80% or more of the total formulation weight or volume. Reasonably, the amount of active compound in each therapeutically useful composition can be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations, and thus, various dosages and treatment regimens may be desirable.

In certain instances, rAAV-based therapeutic constructs suitably formulated in the pharmaceutical compositions disclosed herein by intraocular, subretinal, subcutaneous, intracardiac, intranasal, parenteral, intravenous, intramuscular, intrathecal, oral, intraperitoneal, or by inhalation delivery would be desirable. In some embodiments, modes of administration as described in U.S. Pat. nos. 5,543,158, 5,641,515, and 5,399,363 (each incorporated herein by reference in their entirety) can be used to deliver rAAV. In some embodiments, the preferred mode of administration is by portal intravenous injection.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal conditions of storage and use, these formulations contain preservatives to prevent microbial growth. In many cases, the pharmaceutical forms are sterile and fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and/or vegetable oils. For example, proper fluidity can be maintained by the use of a coating agent, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like, can be used to prevent the action of microorganisms. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of delayed absorption agents, for example, aluminum monostearate and gelatin.

For administration of injectable aqueous solutions, for example, the solution may be suitably buffered if desired and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Sterile aqueous media that can be used are well known in the art. For example, a dose may be dissolved in 1mL of isotonic NaCl solution and added to 1000mL of subcutaneous perfusate (hypodermoclysis fluid) or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences", 15 th edition, pages 1035-1038 and 1570-1580). Depending on the host, the dosage will necessarily vary somewhat. In any event, the person responsible for administration will determine the appropriate dosage for the individual host.

Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The rAAV compositions disclosed herein can also be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and salts with inorganic acids such as hydrochloric or phosphoric acids or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts with the free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. When formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergies or similar adverse reactions when administered to a host.

The compositions of the present disclosure may be introduced into suitable host cells using delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like. In particular, the transgene delivered by the rAAV vector may be formulated for delivery encapsulated in a lipid particle, liposome, vesicle, nanosphere, nanoparticle, or the like.

Such formulations may be preferred for pharmaceutically acceptable formulations for introducing the nucleic acid or rAAV constructs disclosed herein. The formation and use of liposomes is generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulation half-life have been developed (U.S. patent No. 5,741,516). In addition, various methods of liposome and liposome-like formulations have been described as potential drug carriers (U.S. Pat. nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868 and 5,795,587).

Liposomes have been successfully used in many cell types that are generally resistant to transfection by other procedures. Furthermore, liposomes have no DNA length limitations typically present in viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiation therapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, a number of successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also known as multilamellar vesicles (MLVs)). MLVs typically have diameters of 25nm to 4 μm. Sonication of MLV results in formation of a diameter of 200 toSmall Unilamellar Vesicles (SUVs) within the scope, which contain an aqueous solution in the core.

Alternatively, nanocapsule formulations of rAAV may be used. Nanocapsules can generally capture substances in a stable and reproducible manner. To avoid the side effects caused by overload of intracellular polymers, such ultrafine particles (about 0.1 μm in size) should be designed with polymers that can degrade in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles meeting these requirements are contemplated.

In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods of delivering the rAAV compositions to a host. Ultrasonic introduction (i.e., ultrasound) has been used and is described in U.S. patent No. 5,656,016 as a means for increasing the rate and efficacy of drug penetration into and through the circulatory system. Other drug delivery alternatives contemplated are intra-osseous injection (U.S. patent No. 5,779,708), microchip devices (U.S. patent No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. patent nos. 5,770,219 and 5,783,208), and feedback controlled delivery (U.S. patent No. 5,697,899).

Therapeutic method

Aspects of the present disclosure relate to compositions and methods for preventing or treating certain neurological and/or neuromuscular diseases, such as Spinal Muscular Atrophy (SMA). The present disclosure is based in part on isolated nucleic acids, rAAV, etc. comprising a codon optimized nucleic acid sequence encoding an SMN1 protein. In some embodiments, the rAAV vector comprises a native (e.g., human) SMN1 promoter, e.g., as set forth in SEQ ID NO. 4 or 5.

The disclosure is based in part on increasing SMN1 protein expression using the rAAV and vectors described herein, which also results in reduced toxicity in a subject (e.g., relative to a subject to which a previously used rAAV-SMN1 vector, e.g., zolgensma, has been administered). In some embodiments, the reduced toxicity is reduced liver toxicity. In some embodiments, the reduced toxicity is reduced Dorsal Root Ganglion (DRG) toxicity. The amount of toxicity reduction in the subject may vary. In some embodiments, toxicity is reduced by a factor of 2-5, 3-10, 5-20, 10-50, 30-80, or 50-100 (e.g., relative to a subject to whom the previously used rAAV-SMN1 vector has been administered). In some embodiments, toxicity is reduced by more than 100-fold (e.g., relative to a subject to whom a previously used rAAV-SMN1 vector, e.g., zolgensma, has been administered). Methods of measuring toxicity in a subject are known and include measuring cytotoxicity (e.g., cell death), measuring liver toxicity (e.g., by ALT assay), and the like.

In some embodiments, the administration results in a reduction in complications associated with SMA. In some embodiments, complications of SMA include lung infection, spinal deformity (e.g., scoliosis, hip subluxation/dislocation), joint contracture, or respiratory failure.

In some embodiments, the administration results in increased survival of the subject relative to administration to the subject of an AAV vector comprising a constitutive promoter operably linked to a nucleic acid encoding human SMN 1. In some embodiments, the survival of a subject is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% relative to a subject that has been administered a previously used rAAV-SMN1 vector, e.g., zolgensma. The present disclosure may use any suitable known method of measuring survival.

In some embodiments, the subject is a mammalian subject, e.g., a human subject. In some embodiments, the subject is characterized by having one or more mutations in the SMN1 or SMN2 gene, e.g., one or more mutations that result in a reduction (or deletion) of a functional SMN1 protein in a cell of the subject. Examples of SMN1 mutations are described, for example, in Wirth, hum mutat.2000;15 228-37. In some embodiments, the subject has reduced (or no) functional SMN1 protein in cells. In some embodiments, the subject is a non-human mammal, such as a mouse, rat, goat, sheep, pig, cow, camel, llama, or non-human primate.

In some embodiments, administration of an isolated nucleic acid, rAAV, or composition described herein to a cell or subject increases SMN1 expression in the cell or subject by 2-fold to 100-fold (e.g., 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, 100-fold, etc.) as compared to a control subject. As used herein, a "control" subject refers to a subject that has not been administered an isolated nucleic acid, rAAV, or composition described herein. In some embodiments, the control subject is the same subject to which the isolated nucleic acids, rAAV, or compositions described herein were administered (e.g., prior to administration).

In some aspects, the disclosure relates to a method for treating SMA in a subject, the method comprising: administering to a subject an isolated nucleic acid, rAAV, or pharmaceutical composition as described herein.

As used herein, the term "preventing" refers to administering or administering a composition comprising a transgene encoding an SMN1 protein to a subject who has not yet developed symptoms or diseases associated with aberrant SMN1 activity but who has a predisposition to develop a disease associated with aberrant SMN1 activity in order to stop or slow the progression of one or more symptoms of the disease associated with aberrant SMN1 activity. In some embodiments, the disease is SMA. In some embodiments, "preventing" also refers to stopping or slowing the progression of complications (e.g., SMA) associated with aberrant SMN1 activity as described herein.

As used herein, the term "treating" refers to administering or administering a composition comprising a transgene encoding a SMN1 protein to a subject having symptoms or diseases associated with aberrant SMN1 activity or having a predisposition to a disease associated with aberrant SMN1 activity, with the purpose of curing, treating, alleviating, altering, rescuing, ameliorating, improving or affecting a disorder, disease symptom or predisposition associated with aberrant SMN1 activity. In some embodiments, the disease is SMA. In some embodiments, "treating" also refers to curing, treating, alleviating, altering, saving, ameliorating, improving, or affecting a complication associated with aberrant SMN1 activity (e.g., SMA) as described herein.

Alleviating the disease associated with aberrant SMN1 activity includes delaying the progression or progression of the disease, or reducing the severity of the disease. Remission of a disease does not necessarily require a healing outcome. As used herein, "delaying" the progression of a disease (e.g., a disease associated with aberrant SMN1 activity, such as SMA) refers to delaying, impeding, slowing, stabilizing, and/or delaying the progression of the disease. The length of time for such delay may vary depending on the disease history and/or the individual receiving the treatment. A method of "delaying" or alleviating the progression of a disease, or delaying the onset of a disease, is a method that reduces the likelihood of developing one or more symptoms of a disease within a given time frame and/or reduces the extent of symptoms within a given time frame, as compared to the absence of the method. Such comparisons are typically based on clinical studies using a number of subjects sufficient to give a statistically significant result.

"Progression" or "progression" of a disease refers to the initial manifestation and/or subsequent progression of the disease. Standard clinical techniques well known in the art may be used to detect and assess the progression of the disease. However, development also refers to progress that may not be detectable. For the purposes of this disclosure, development or progression refers to the biological process of symptoms. "progression" includes occurrence, recurrence and onset. As used herein, a "episode" or "occurrence" of a disease associated with aberrant SMN1 activity or angiogenesis includes an initial episode and/or recurrence.

The compositions (or expressed transgenes) described herein can be administered in one or more target cells of a subject (e.g., a mammalian subject, such as a human). In some embodiments, the composition (e.g., isolated nucleic acid, rAAV, etc.) is administered to cells of the Central Nervous System (CNS) of the subject. Examples of CNS cells include, but are not limited to, neurons, astrocytes, glial cells, and the like.

Kit and related compositions

In some embodiments, the agents described herein may be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. The kit may include one or more containers containing the components of the present disclosure and instructions for use. In particular, such kits may include one or more of the agents described herein, as well as instructions describing the intended use and proper use of such agents. In certain embodiments, the agents in the kit may be pharmaceutical formulations and dosages suitable for the particular application and method of administration of the agents. Kits for research purposes may contain the components in the appropriate concentrations or amounts to perform various experiments.

The kit may be designed to facilitate use of the methods described herein by researchers and may take a variety of forms. Where applicable, each composition of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In certain instances, some compositions may be configurable (constitutable) or processable (e.g., processed into an active form), e.g., by adding a suitable solvent or other substance (e.g., water or cell culture medium), which may or may not be provided with the kit. As used herein, "instructions" may define the components of the instructions and/or generalizations, and generally include written instructions on or with the packaging of the present disclosure. The instructions may also include any verbal or electronic instructions provided in any manner that will clearly recognize that the instructions will follow the kit, such as audiovisual (e.g., video tape, DVD, etc.), internet and/or web-based communications, etc. The written instructions may be in a form prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals or biological products, and the instructions may also reflect approval by the agency of manufacture, use or sale for administration to animals.

The kit may comprise any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and/or separating and mixing the sample and administering to the subject. The kit may comprise a container containing the medicament described herein. The medicament may be in the form of a liquid, gel or solid (powder). The medicament may be prepared aseptically, packaged in syringes and transported refrigerated. Or it may be contained in a vial or other container for storage. The second container may contain other medicaments that are prepared aseptically. Or the kit may include an active agent pre-mixed and transported in a syringe, vial, tube or other container. The kit may have one or more or all of the components required to administer the agent to an animal, such as a syringe, a topical applicator, or an intravenous needle cannula (intravenous needle tubing) and a bag, particularly where the kit is used to create a specific somatic animal model.

The kit may take a variety of forms, such as a blister pouch, shrink-wrap pouch, vacuum-sealed pouch, sealable thermoformed tray, or similar pouch or tray form, wherein the accessory is loosely packaged within the pouch (pouch), one or more tubes, containers, boxes, or bags (bag). The kit may be sterilized after the addition of the accessories, allowing the individual accessories in the container to be opened in other ways. The kit may be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kit may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, fabrics (e.g., gauze) for applying or removing disinfectant, disposable gloves, supports for the medicament prior to application, and the like, depending on the particular application.

The instructions included in the kit may relate to methods for constructing an AAV vector described herein. Furthermore, the kits of the present disclosure may include instructions, negative and/or positive controls, containers, diluents and buffers for the sample, sample preparation tubes, and printed or electronic forms of reference AAV sequences for sequence comparison.

Examples

Example 1

Intravenous administration Zolgensma to individuals under 2 years of age has shown significant clinical benefit to SMA type 1 infants, but a steep rise in liver transaminase is observed in most patients. Systemic administration of AAV 9-like SMN vectors was observed to cause toxicity in large animals, including Dorsal Root Ganglion (DRG) neurodegeneration and inflammation. AAV 9-mediated long-term overexpression of SMN in a mouse model has been observed to induce dose-dependent tardive dyskinesia. In summary, expression of SMN1 beyond physiological levels delivered by AAV may be a safety issue for AAV-based SMA gene therapy.

This example describes the engineering of AAV-SMN1 vectors to address potential safety issues. First, the SMN1 coding sequence (CDS) has been codon optimized to maximize SMN1 expression, thereby reducing the vector dose delivered to the patient. Second, endogenous SMN1 promoters are used to drive AAV-based SMN1 expression in patients to improve safety. Fig. 1 shows a schematic diagram of an SMN1 encoding vector of the present disclosure. "scAAV-CB6-opt-SMN1" is a self-complementary AAV (scAAV) vector having a CB6 promoter and codon optimized SMN1 coding sequence. "SMNp-SMN1" is a single stranded AAV genome having a 2.0kb endogenous human SMN1 promoter and codon optimized SMN1 coding sequence (CDS). "ssAAV-opt-SMN1 (MBL)" is a single stranded AAV genome having a 2.0kb endogenous human SMN1 promoter, synthetic MBL intron, and codon optimized SMN1 coding sequence (CDS). "SMNsp-SMN1" is a self-complementary AAV vector having a 0.9kb endogenous human SMN1 promoter and codon optimized SMN1 CDS. "Zolgensma" is a self-complementing AAV vector with the CMV enhancer/chicken beta-actin promoter and wild-type human SMN1 CDS.

In vivo expression of rAAV vectors encoding SMN1 was studied. FIGS. 2A-2F show representative data for ssaaV9.opt-SMN1 and Zolgensma in SMNΔ7 mice. FIG. 2A shows survival curves for SMNΔ7 mice treated with Zolgensma (2 xE14 Genomic Copy (GC)/kg) or three doses of SMNp-SMN1rAAV (2 xE14 Genomic Copy (GC)/kg, 6.8xE13 GC/kg, or 3.4xE13 GC/kg); ssaav9.Opt-SMN1 treated at the same or 1/3 dose had better survival than mice treated with Zolgensma. Representative data shown in fig. 2B and 2C indicate that mice treated with ssaav9.Opt-SMN1 showed similar weight gain compared to mice treated with Zolgensma. FIG. 2D shows survival curves of scaAV9.opt-SMN1 and Zolgensma in SMNΔ7 mice; scaav9.Opt-SMN1 treated at the same or 1/3 dose had better survival than mice treated with Zolgensma. Representative data shown in fig. 2E and 2F indicate that mice treated with ssaav9.Opt-SMN1 showed similar weight gain compared to mice treated with Zolgensma.

FIGS. 3A-3B show representative behavioral assay data for SMNΔ7 mice treated with scaAV9.opt-SMN 1. FIG. 3A shows that animals treated with scaAV9.opt-SMN1 can self-righte from day 5 post-administration. The representative data shown in fig. 3B demonstrate that all treated animals have functional muscles as measured by the rotation test.

Fig. 4A-4B show representative toxicology data. The histological data shown in fig. 4A demonstrate that high levels of SMN1 overexpression are toxic to hepatocytes. Briefly, 5 E+11GC/mouse AAV vector was injected into animals by facial intravenous injection at P0. Liver was collected on day 8 post injection. Liver damage was observed in AAVsc-CB6-PI-SMN1 (6/6 animals) and Zolgensma (4/7 animals) vector treated groups, but not in control empty AAV9 (0/8 animals) or ssAAV-opt-SMN 1 (0/8) vector treated groups. FIG. 4B shows representative alanine Aminotransferase (ALT) assay test results.

FIG. 5 shows a graph illustrating the putative expression levels of SMN1 encoding vectors. Briefly, SMN1 encoding vectors described in the present disclosure may allow for high levels of SMN1 expression in Central Nervous System (CNS) tissues without over-expression in muscle or other non-target organs, which may lead to hepatotoxicity in the subject.

Three modified SMN1 encoding rAAV vectors depicted in fig. 1 were packaged within AAV9 capsids. rAAV was then injected into smnΔ7 mice at P1 and the mice were monitored for body weight and survival. Both engineered AAV-SMN1 vectors (e.g., SMNp-SMN1 and SMNsp-SMN 1) outperform Zolgensma in terms of viability. One-third of the dose (relative to Zolgensma) of the engineered vector (e.g., 1.67xe11 Genome Copy (GC) per mouse) was observed to be more effective in extending longevity than Zolgensma of 5xe11 GC per mouse (fig. 6A and 6C). Fig. 6B shows that smnΔ7 mice treated with SMNp-SMN1 of 5xe11 GC increased more body weight than mice treated with Zolgensma after day 17, and that a dose of SMNp-SMN1 of 1.67xe11 GC reached a similar body weight compared to Zolgensma groups on day 55. Mice treated with SMNsp-SMN1 vector at two different doses (5 xe11 GC and 1.67xe11 GC) also showed growth advantage from day-23 compared to Zolgensma groups at 5xe11 GC dose (fig. 6D).

Example 2

To test whether AAV vectors having a codon optimized hSMN1 sequence (co-hSMN 1) at CMVen/CB promoter (CMVen/CB-co-hSMN 1; SEQ ID NO: 8) induced hepatotoxicity, AAV vectors were packaged into AAV9 capsids (rAAV 9sc-CMVen/CB-co-hSMN 1) and rAAV was administered by facial intravenous injection at 3.3x10e14 GC/kg to SMNdelta7 mice at postnatal day 1. All treated animals (n=6) died earlier than untreated SMA mice. Skin yellowing and hepatocyte damage were found in animals treated with vehicle on day 8 post injection (fig. 7A-7C). SMN1 overexpression was confirmed in the liver (fig. 7D).

In addition, it was tested whether elimination of SMN expression in the liver would alleviate liver damage. To off-target the rAAV from the liver, the miR-122 binding site was engineered into CMVen/CB-co-hSMN1 (CB 6-PI-opt hSMN1-miR-122BS;SEQ ID NO:9) and packaged into the AAV9 capsid (scaV 9-CB6-PI-opt hSMN1-miR-122 BS) (FIG. 8A). rAAV9sc-CMVen/CB-co-hSMN1 and scAAV9-CB6-PI-opt hSMN1-miR-122BS were injected into SMNdelta mice on postnatal day 1, respectively. Liver expression and hepatotoxicity of SMN1 were assessed. The results showed that by using the miR-122 binding site in AAV constructs, SMN expression was eliminated from the liver (fig. 8B), and no hepatotoxicity was observed in mice treated with scAAV9-CB6-PI-opt hSMN1-miR-122BS (fig. 8C).

Furthermore, restoring physiological levels of hSMN1 expression in appropriate cell types with low effective doses of rAAV may reduce hepatotoxicity. To achieve physiologically regulated hSMN1 expression, a second generation (2 nd gen) scaVV 9 (SMN 1sp-co-SMN1 (referred to as SMN1p-co-SMN1 in FIGS. 9D, 9E and 9J) was designed to express co-hSMN1 from an endogenous short hSMN1 promoter (SEQ ID NO: 5); SEQ ID NO: 7). scAAV9-CMVen/CB-opt-hSMN1 was used as positive control. Neuro-2a cells were transfected with SMN1p-co-SMN1 or CMVen/CB-opt-hSMN1 and SMN1 expression was assessed. The results show that the second generation construct with the endogenous SMN promoter shows higher expression efficiency in Neuro-2a cells (fig. 9A).

Next, SMN1p-co-hSMN1 (SEQ ID NO: 7) was packaged into AAV9 capsids (scAAV 9-SMNp-co-hSMN 1) and injected into neonatal SMA mice by facial intravenous injection (FIG. 9B). Another group of SMA mice used scaVV 9-CMVen/CB-hSMN1, which had a similar profile to that ofThe same expression cassette as used in (a) was used as a reference vector. AAVsc-SMNsp-co-hSMN1 vector or reference vector was injected into SMA mice in P0 by facial intravenous injection to establish a parallel animal experiment. Mice were monitored daily for body weight (fig. 9C) and survival (fig. 9D). Mice treated with AAVsc-SMNsp-co-hSMN1 increased more body weight than the reference vehicle group. The administration of AAVsc-SMNsp-co-hSMN1 vector significantly improved the longevity of SMA mice. Animals treated with AAVsc-SMNsp-co-hSMN1 were healthy at 5 months of age (fig. 9E). The muscle function of the mice was also assessed. AAVsc-SMNsp-co-hSMN1 treatment achieved better recovery of muscle function than the reference group. The grid test data indicated that SMA mice treated with AAVsc-SMNsp-co-hSMN1 were able to self-right earlier than the reference mice (fig. 9F). The effect of treatment at P0 on neuromuscular junction (NMJ) in young (P12) animals was also assessed. Representative images from the transverse abdominal muscle (TVA) showed therapeutic effects on restoration of innervation in NMJ (fig. 9G). Neuromuscular junction structure in AAVsc-SMNsp-co-hSMN1 treated mice recovered to a structure close to that of wild type mice, superior to that of SMA mice treated with the reference (fig. 9H).

In addition, AAVsc-SMNsp-co-hSMN1 treatment reduced complications in SMA mice. Ear necrosis is one of the common complications in this animal model. The severity of the complications was divided into different disease levels (fig. 9I). Animals treated with AAVsc-SMNsp-co-hSMN1 exhibited less severity than the reference (fig. 9J). The older the animal, the more severe the symptoms. For AAVsc-SMNp-co-hSMN1 treated animals, 90 days of data were collected; for reference treated animals, 60 days of data were collected.

The administration of AAVsc-SMNsp-co-hSMN1 in SMA mice achieved an organ/tissue expression pattern similar to that of healthy vehicle animals. Immunoblotting showed that AAVsc-SMNp-co-hSMN1 was preferably expressed in the central nervous system and not in peripheral tissues. The expression pattern of this vector was close to the natural distribution of SMN in healthy control animals (fig. 9K-9L).

In addition, SMA mice that received AAVsc-SMNsp-co-hSMN1 or baseline rAAV (BMK) at P5 were monitored for survival. Administration of scAAV9-SMNsp-hSMN1 (7e+11vg/mouse) vector resulted in 50% survival of SMA mice 90 days post injection, whereas SMA mice receiving baseline rAAV had survived none 30 days post injection (fig. 10).

Representative sequence

Codon-optimized human SMN1 nucleic acid sequence (SEQ ID NO: 1)

ATGGCCATGAGCAGCGGCGGCAGTGGCGGCGGCGTGCCCGAGCAGGAGGATTCTGTGCTGTTCCGGAGAGGAACAGGCCAGAGCGATGACTCCGATATCTGGGACGACACAGCCCTTATCAAGGCCTACGACAAGGCCGTGGCCAGCTTTAAGCACGCCCTGAAGAATGGCGATATCTGCGAGACAAGCGGAAAGCCTAAGACCACCCCTAAAAGAAAGCCCGCCAAGAAAAACAAGTCCCAGAAAAAAAACACCGCCGCTAGCCTGCAGCAGTGGAAGGTGGGCGACAAATGCAGCGCCATCTGGTCCGAGGACGGCTGCATCTACCCTGCTACCATCGCCAGCATCGACTTCAAGCGGGAAACCTGCGTGGTGGTCTACACAGGCTATGGCAATAGGGAGGAACAAAATCTCTCTGATCTGCTGTCTCCTATTTGTGAAGTGGCTAACAACATCGAGCAGAACGCCCAGGAAAATGAGAACGAAAGCCAAGTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCTGGAAACAAGTCTGACAACATCAAGCCCAAGTCTGCCCCTTGGAACAGCTTCCTGCCCCCTCCTCCTCCAATGCCTGGCCCCAGACTGGGCCCCGGCAAGCCTGGCCTGAAGTTCAACGGCCCTCCTCCACCCCCTCCTCCTCCACCTCCCCATCTGCTGAGCTGCTGGCTGCCTCCTTTTCCCAGCGGCCCCCCTATCATCCCCCCACCACCTCCTATCTGTCCCGACAGCCTGGACGACGCCGATGCTCTGGGATCCATGCTGATCAGCTGGTACATGTCTGGCTACCACACCGGCTACTACATGGGCTTCCGGCAGAACCAGAAGGAAGGAAGATGCAGCCACAGCCTGAACTGA

Wild type SMN1 nucleic acid sequence (SEQ ID NO: 2)

ATGGCGATGAGCAGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAGGAGGATTCCGTGCTGTTCCGGCGCGGCACAGGCCAGAGCGATGATTCTGACATTTGGGATGATACAGCACTGATAAAAGCATATGATAAAGCTGTGGCTTCATTTAAGCATGCTCTAAAGAATGGTGACATTTGTGAAACTTCGGGTAAACCAAAAACCACACCTAAAAGAAAACCTGCTAAGAAGAATAAAAGCCAAAAGAAGAATACTGCAGCTTCCTTACAACAGTGGAAAGTTGGGGACAAATGTTCTGCCATTTGGTCAGAAGACGGTTGCATTTACCCAGCTACCATTGCTTCAATTGATTTTAAGAGAGAAACCTGTGTTGTGGTTTACACTGGATATGGAAATAGAGAGGAGCAAAATCTGTCCGATCTACTTTCCCCAATCTGTGAAGTAGCTAATAATATAGAACAGAATGCTCAAGAGAATGAAAATGAAAGCCAAGTTTCAACAGATGAAAGTGAGAACTCCAGGTCTCCTGGAAATAAATCAGATAACATCAAGCCCAAATCTGCTCCATGGAACTCTTTTCTCCCTCCACCACCCCCCATGCCAGGGCCAAGACTGGGACCAGGAAAGCCAGGTCTAAAATTCAATGGCCCACCACCGCCACCGCCACCACCACCACCCCACTTACTATCATGCTGGCTGCCTCCATTTCCTTCTGGACCACCAATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGAAGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATGGGTTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATTAA

SMN1 amino acid sequence (SEQ ID NO: 3)

MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN

SMN1 Long promoter nucleic acid sequence (SEQ ID NO: 4)

tcgaagctttataaaaacatacttttttttttacttttttttttttttctgagacacagcctcactctgtcgcccaggctggagtgcaggttttcatgtttatctgtgagatgtacctttggcacattactttcctgacatgagatttaaatttttttttttatcttgtgacaatttaacttttttgacacataaaaattgtacatatttatttgtttgagatggagtcgcactctgtcactcaggctggagtgcagtggcgtgatcttggctcactgcaacctccgcctcccgagttcaagtgattctcctggctcagcctcccaagcagctgtcattacaggcctgcaccaccacacccggctgattttgtatttttaggagaaacagggtttcaccatgttgggccaggctggtcttgaagtcctgacctcaagtgatccacccaccttggcctcccaaagtgctgggattataggcatgagccaccgtaccagacccctaaaaattgtatatatttaaggtgtaccatttgatgtttagatatacattgtgaaatgattacattccacatattacctctacagagttaccatttttgtacacttggtcaacatcatcccattctccccttcctccacagatatttcttgtatactatatagaagccaagggtattttgggggaagagctcaaagttcctttcgtggagttaaaaatatatatatactatgtacatataagccatttagcaaccctagatgcttaataaagaatactggaggcccggtgtggtggctcacacctgtaatcccagcactttgggaggccgaggcggtcggattacgaggtcaggagttcaagaccagcctggccaacatggtgaaaccccatctttactaaaaatacaaaaattagccgggtgtggtggtgggcgcctgtaatcccagctactcggggggctgaggcagaattgcttgaacctgggaggcagaggttgcagtgagctgagatcacgccactgcattccagcctgggtgacagagcaatattctgtcgcaaaaaaaaaaagaatactggaggctgggcgaggtggctcacacctgtaatcccagcattttgggatgccagaggcgggcggaatntcttgagctcaggagttcgagaccagcctacacaatatgctccaaacgccgcttntacaaaacatacagaaactacccgggtgtggtggcgnncccctgtggtcctagatacttgggaggttgaggcgggaggatcgcttgagctcgggaggtcgaggctgcaatgagccgagatggtgccactgcattctgacgacagagcgagattccgtttcaaaacaaacaacaaataaggttgggggatcaaatatcttctagtgtttaaggatctgccttccttcctgcccccatgtttgtctttccttgtttgtctttatatagatcaagcaggttttaaattcctagtaggagcttacatttacttttccaagggggagggggaataaatatctacacacacacacacacacacacacacacacacacacacacacacacacacacaccacactggagttcgagacgaggcctaagcaacatgccgaaaccccgtctctactaaatacaaaaaatagctgagcttggtggcgcacgcctatagtcctagctactggggaggctgaggtgggaggatcgcttgagcccaagaagtcgaggctgcagtgagccgagatcgcgccgctgcactccagcctgagcgacagggcgaggctctgtctcaaaacaaacaaacaaaaaaaaaaaggaaaggaaatataacacagtgaaatgaaaggattgagagaaatgaaaaatatacacgccacaaatgtgggagggcgataaccactcgtagaaagcgtgagaagttactacaagcggtcctcccgggcaccgtactgttccgctcccagaagccccgggcgccggaagtcgtcactcttaagaagggacggggccccacgctgcgcacccgcgggtttgct

SMN1 short promoter nucleic acid sequence (SEQ ID NO: 5)

ggatgccagaggcgggcggaatntcttgagctcaggagttcgagaccagcctacacaatatgctccaaacgccgcttntacaaaacatacagaaactacccgggtgtggtggcgnncccctgtggtcctagatacttgggaggttgaggcgggaggatcgcttgagctcgggaggtcgaggctgcaatgagccgagatggtgccactgcattctgacgacagagcgagattccgtttcaaaacaaacaacaaataaggttgggggatcaaatatcttctagtgtttaaggatctgccttccttcctgcccccatgtttgtctttccttgtttgtctttatatagatcaagcaggttttaaattcctagtaggagcttacatttacttttccaagggggagggggaataaatatctacacacacacacacacacacacacacacacacacacacacacacacacacacaccacactggagttcgagacgaggcctaagcaacatgccgaaaccccgtctctactaaatacaaaaaatagctgagcttggtggcgcacgcctatagtcctagctactggggaggctgaggtgggaggatcgcttgagcccaagaagtcgaggctgcagtgagccgagatcgcgccgctgcactccagcctgagcgacagggcgaggctctgtctcaaaacaaacaaacaaaaaaaaaaaggaaaggaaatataacacagtgaaatgaaaggattgagagaaatgaaaaatatacacgccacaaatgtgggagggcgataaccactcgtagaaagcgtgagaagttactacaagcggtcctcccgggcaccgtactgttccgctcccagaagccccgggcgccggaagtcgtcactcttaagaagggacggggccccacgctgcgcacccgcgggtttgct

SMNlp-co-SMN1 rAAV vector nucleic acid sequence (SEQ ID NO: 6)

gctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctaccagggtaatggggatcctctagaactatagctagtcgacattgattattgactagttcgaagctttataaaaacatacttttttttttacttttttttttttttctgagacacagcctcactctgtcgcccaggctggagtgcaggttttcatgtttatctgtgagatgtacctttggcacattactttcctgacatgagatttaaatttttttttttatcttgtgacaatttaacttttttgacacataaaaattgtacatatttatttgtttgagatggagtcgcactctgtcactcaggctggagtgcagtggcgtgatcttggctcactgcaacctccgcctcccgagttcaagtgattctcctggctcagcctcccaagcagctgtcattacaggcctgcaccaccacacccggctgattttgtatttttaggagaaacagggtttcaccatgttgggccaggctggtcttgaagtcctgacctcaagtgatccacccaccttggcctcccaaagtgctgggattataggcatgagccaccgtaccagacccctaaaaattgtatatatttaaggtgtaccatttgatgtttagatatacattgtgaaatgattacattccacatattacctctacagagttaccatttttgtacacttggtcaacatcatcccattctccccttcctccacagatatttcttgtatactatatagaagccaagggtattttgggggaagagctcaaagttcctttcgtggagttaaaaatatatatatactatgtacatataagccatttagcaaccctagatgcttaataaagaatactggaggcccggtgtggtggctcacacctgtaatcccagcactttgggaggccgaggcggtcggattacgaggtcaggagttcaagaccagcctggccaacatggtgaaaccccatctttactaaaaatacaaaaattagccgggtgtggtggtgggcgcctgtaatcccagctactcggggggctgaggcagaattgcttgaacctgggaggcagaggttgcagtgagctgagatcacgccactgcattccagcctgggtgacagagcaatattctgtcgcaaaaaaaaaaagaatactggaggctgggcgaggtggctcacacctgtaatcccagcattttgggatgccagaggcgggcggaatatcttgagctcaggagttcgagaccagcctacacaatatgctccaaacgccgcttCtacaaaacatacagaaactacccgggtgtggtggcgtgcccctgtggtcctagatacttgggaggttgaggcgggaggatcgcttgagctcgggaggtcgaggctgcaatgagccgagatggtgccactgcattctgacgacagagcgagattccgtttcaaaacaaacaacaaataaggttgggggatcaaatatcttctagtgtttaaggatctgccttccttcctgcccccatgtttgtctttccttgtttgtctttatatagatcaagcaggttttaaattcctagtaggagcttacatttacttttccaagggggagggggaataaatatctacacacacacacacacacacacacacacacacacacacacacacacacacacaccacactggagttcgagacgaggcctaagcaacatgccgaaaccccgtctctactaaatacaaaaaatagctgagcttggtggcgcacgcctatagtcctagctactggggaggctgaggtgggaggatcgcttgagcccaagaagtcgaggctgcagtgagccgagatcgcgccgctgcactccagcctgagcgacagggcgaggctctgtctcaaaacaaacaaacaaaaaaaaaaaggaaaggaaatataacacagtgaaatgaaaggattgagagaaatgaaaaatatacacgccacaaatgtgggagggcgataaccactcgtagaaagcgtgagaagttactacaagcggtcctcccgggcaccgtactgttccgctcccagaagccccgggcgccggaagtcgtcactcttaagaagggacggggccccacgctgcgcacccgcgggtttgctATGGCCATGAGCAGCGGCGGCAGTGGCGGCGGCGTGCCCGAGCAGGAGGATTCTGTGCTGTTCCGGAGAGGAACAGGCCAGAGCGATGACTCCGATATCTGGGACGACACAGCCCTTATCAAGGCCTACGACAAGGCCGTGGCCAGCTTTAAGCACGCCCTGAAGAATGGCGATATCTGCGAGACAAGCGGAAAGCCTAAGACCACCCCTAAAAGAAAGCCCGCCAAGAAAAACAAGTCCCAGAAAAAAAACACCGCCGCTAGCCTGCAGCAGTGGAAGGTGGGCGACAAATGCAGCGCCATCTGGTCCGAGGACGGCTGCATCTACCCTGCTACCATCGCCAGCATCGACTTCAAGCGGGAAACCTGCGTGGTGGTCTACACAGGCTATGGCAATAGGGAGGAACAAAATCTCTCTGATCTGCTGTCTCCTATTTGTGAAGTGGCTAACAACATCGAGCAGAACGCCCAGGAAAATGAGAACGAAAGCCAAGTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCTGGAAACAAGTCTGACAACATCAAGCCCAAGTCTGCCCCTTGGAACAGCTTCCTGCCCCCTCCTCCTCCAATGCCTGGCCCCAGACTGGGCCCCGGCAAGCCTGGCCTGAAGTTCAACGGCCCTCCTCCACCCCCTCCTCCTCCACCTCCCCATCTGCTGAGCTGCTGGCTGCCTCCTTTTCCCAGCGGCCCCCCTATCATCCCCCCACCACCTCCTATCTGTCCCGACAGCCTGGACGACGCCGATGCTCTGGGATCCATGCTGATCAGCTGGTACATGTCTGGCTACCACACCGGCTACTACATGGGCTTCCGGCAGAACCAGAAGGAAGGAAGATGCAGCCACAGCCTGAACTGAgcggccgcaagcttcctgaggatccgatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaagcaattcgttgatctgaatttcgaccacccataatacccattaccctggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgataactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattag

SMN1sp-co-SMN1 rAAV vector nucleic acid sequence (SEQ ID NO: 7)

ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtgtagccatgctctaggaagatcaattcggtacaattcacgcgtggatgccagaggcgggcggaatAtcttgagctcaggagttcgagaccagcctacacaatatgctccaaacgccgcttCtacaaaacatacagaaactacccgggtgtggtggcgTGcccctgtggtcctagatacttgggaggttgaggcgggaggatcgcttgagctcgggaggtcgaggctgcaatgagccgagatggtgccactgcattctgacgacagagcgagattccgtttcaaaacaaacaacaaataaggttgggggatcaaatatcttctagtgtttaaggatctgccttccttcctgcccccatgtttgtctttccttgtttgtctttatatagatcaagcaggttttaaattcctagtaggagcttacatttacttttccaagggggagggggaataaatatctacacacacacacacacacacacacacacacacacacacacacacacacacacaccacactggagttcgagacgaggcctaagcaacatgccgaaaccccgtctctactaaatacaaaaaatagctgagcttggtggcgcacgcctatagtcctagctactggggaggctgaggtgggaggatcgcttgagcccaagaagtcgaggctgcagtgagccgagatcgcgccgctgcactccagcctgagcgacagggcgaggctctgtctcaaaacaaacaaacaaaaaaaaaaaggaaaggaaatataacacagtgaaatgaaaggattgagagaaatgaaaaatatacacgccacaaatgtgggagggcgataaccactcgtagaaagcgtgagaagttactacaagcggtcctcccgggcaccgtactgttccgctcccagaagccccgggcgccggaagtcgtcactcttaagaagggacggggccccacgctgcgcacccgcgggtttgctATGGCCATGAGCAGCGGCGGCAGTGGCGGCGGCGTGCCCGAGCAGGAGGATTCTGTGCTGTTCCGGAGAGGAACAGGCCAGAGCGATGACTCCGATATCTGGGACGACACAGCCCTTATCAAGGCCTACGACAAGGCCGTGGCCAGCTTTAAGCACGCCCTGAAGAATGGCGATATCTGCGAGACAAGCGGAAAGCCTAAGACCACCCCTAAAAGAAAGCCCGCCAAGAAAAACAAGTCCCAGAAAAAAAACACCGCCGCTAGCCTGCAGCAGTGGAAGGTGGGCGACAAATGCAGCGCCATCTGGTCCGAGGACGGCTGCATCTACCCTGCTACCATCGCCAGCATCGACTTCAAGCGGGAAACCTGCGTGGTGGTCTACACAGGCTATGGCAATAGGGAGGAACAAAATCTCTCTGATCTGCTGTCTCCTATTTGTGAAGTGGCTAACAACATCGAGCAGAACGCCCAGGAAAATGAGAACGAAAGCCAAGTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCTGGAAACAAGTCTGACAACATCAAGCCCAAGTCTGCCCCTTGGAACAGCTTCCTGCCCCCTCCTCCTCCAATGCCTGGCCCCAGACTGGGCCCCGGCAAGCCTGGCCTGAAGTTCAACGGCCCTCCTCCACCCCCTCCTCCTCCACCTCCCCATCTGCTGAGCTGCTGGCTGCCTCCTTTTCCCAGCGGCCCCCCTATCATCCCCCCACCACCTCCTATCTGTCCCGACAGCCTGGACGACGCCGATGCTCTGGGATCCATGCTGATCAGCTGGTACATGTCTGGCTACCACACCGGCTACTACATGGGCTTCCGGCAGAACCAGAAGGAAGGAAGATGCAGCCACAGCCTGAACTGAgcggccgcaagcttatcgataccgtcgactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcctaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattagg

CMVen/CB-co-hSMN1 rAAV vector nucleic acid sequence (SEQ ID NO: 8)

ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtgtagccatgctctaggaagatcaattcaattcacgcgtcgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggaGtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgtcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagcaagctctagcctcgagaattcaccggtgccaccATGGCCATGAGCAGCGGCGGCAGTGGCGGCGGCGTGCCCGAGCAGGAGGATTCTGTGCTGTTCCGGAGAGGAACAGGCCAGAGCGATGACTCCGATATCTGGGACGACACAGCCCTTATCAAGGCCTACGACAAGGCCGTGGCCAGCTTTAAGCACGCCCTGAAGAATGGCGATATCTGCGAGACAAGCGGAAAGCCTAAGACCACCCCTAAAAGAAAGCCCGCCAAGAAAAACAAGTCCCAGAAAAAAAACACCGCCGCTAGCCTGCAGCAGTGGAAGGTGGGCGACAAATGCAGCGCCATCTGGTCCGAGGACGGCTGCATCTACCCTGCTACCATCGCCAGCATCGACTTCAAGCGGGAAACCTGCGTGGTGGTCTACACAGGCTATGGCAATAGGGAGGAACAAAATCTCTCTGATCTGCTGTCTCCTATTTGTGAAGTGGCTAACAACATCGAGCAGAACGCCCAGGAAAATGAGAACGAAAGCCAAGTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCTGGAAACAAGTCTGACAACATCAAGCCCAAGTCTGCCCCTTGGAACAGCTTCCTGCCCCCTCCTCCTCCAATGCCTGGCCCCAGACTGGGCCCCGGCAAGCCTGGCCTGAAGTTCAACGGCCCTCCTCCACCCCCTCCTCCTCCACCTCCCCATCTGCTGAGCTGCTGGCTGCCTCCTTTTCCCAGCGGCCCCCCTATCATCCCCCCACCACCTCCTATCTGTCCCGACAGCCTGGACGACGCCGATGCTCTGGGATCCATGCTGATCAGCTGGTACATGTCTGGCTACCACACCGGCTACTACATGGGCTTCCGGCAGAACCAGAAGGAAGGAAGATGCAGCCACAGCCTGAACTGAgccaagcttcctgaggatccgatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggcctaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattagg

CMVen/CB-co-hSMN1_miR122_BS rAAV vector nucleic acid sequence (SEQ ID NO: 9)

ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtgtagccatgctctaggaagatcaattcaattcacgcgtcgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggaGtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgtcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagcaagctctagcctcgagaattcaccggtgccaccATGGCCATGAGCAGCGGCGGCAGTGGCGGCGGCGTGCCCGAGCAGGAGGATTCTGTGCTGTTCCGGAGAGGAACAGGCCAGAGCGATGACTCCGATATCTGGGACGACACAGCCCTTATCAAGGCCTACGACAAGGCCGTGGCCAGCTTTAAGCACGCCCTGAAGAATGGCGATATCTGCGAGACAAGCGGAAAGCCTAAGACCACCCCTAAAAGAAAGCCCGCCAAGAAAAACAAGTCCCAGAAAAAAAACACCGCCGCTAGCCTGCAGCAGTGGAAGGTGGGCGACAAATGCAGCGCCATCTGGTCCGAGGACGGCTGCATCTACCCTGCTACCATCGCCAGCATCGACTTCAAGCGGGAAACCTGCGTGGTGGTCTACACAGGCTATGGCAATAGGGAGGAACAAAATCTCTCTGATCTGCTGTCTCCTATTTGTGAAGTGGCTAACAACATCGAGCAGAACGCCCAGGAAAATGAGAACGAAAGCCAAGTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCTGGAAACAAGTCTGACAACATCAAGCCCAAGTCTGCCCCTTGGAACAGCTTCCTGCCCCCTCCTCCTCCAATGCCTGGCCCCAGACTGGGCCCCGGCAAGCCTGGCCTGAAGTTCAACGGCCCTCCTCCACCCCCTCCTCCTCCACCTCCCCATCTGCTGAGCTGCTGGCTGCCTCCTTTTCCCAGCGGCCCCCCTATCATCCCCCCACCACCTCCTATCTGTCCCGACAGCCTGGACGACGCCGATGCTCTGGGATCCATGCTGATCAGCTGGTACATGTCTGGCTACCACACCGGCTACTACATGGGCTTCCGGCAGAACCAGAAGGAAGGAAGATGCAGCCACAGCCTGAACTGAgcGGCCacaaacaccattgtcacactccaacaaacaccattgtcacactccaacaaacaccattgtcacactccaagcttcctgaggatccgatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggcctaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattagg

SMNlp-co-SMN1_miR122_BS rAAV vector nucleic acid sequence (SEQ ID NO: 10)

gctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctaccagggtaatggggatcctctagaactatagctagtcgacattgattattgactagttcgaagctttataaaaacatacttttttttttacttttttttttttttctgagacacagcctcactctgtcgcccaggctggagtgcaggttttcatgtttatctgtgagatgtacctttggcacattactttcctgacatgagatttaaatttttttttttatcttgtgacaatttaacttttttgacacataaaaattgtacatatttatttgtttgagatggagtcgcactctgtcactcaggctggagtgcagtggcgtgatcttggctcactgcaacctccgcctcccgagttcaagtgattctcctggctcagcctcccaagcagctgtcattacaggcctgcaccaccacacccggctgattttgtatttttaggagaaacagggtttcaccatgttgggccaggctggtcttgaagtcctgacctcaagtgatccacccaccttggcctcccaaagtgctgggattataggcatgagccaccgtaccagacccctaaaaattgtatatatttaaggtgtaccatttgatgtttagatatacattgtgaaatgattacattccacatattacctctacagagttaccatttttgtacacttggtcaacatcatcccattctccccttcctccacagatatttcttgtatactatatagaagccaagggtattttgggggaagagctcaaagttcctttcgtggagttaaaaatatatatatactatgtacatataagccatttagcaaccctagatgcttaataaagaatactggaggcccggtgtggtggctcacacctgtaatcccagcactttgggaggccgaggcggtcggattacgaggtcaggagttcaagaccagcctggccaacatggtgaaaccccatctttactaaaaatacaaaaattagccgggtgtggtggtgggcgcctgtaatcccagctactcggggggctgaggcagaattgcttgaacctgggaggcagaggttgcagtgagctgagatcacgccactgcattccagcctgggtgacagagcaatattctgtcgcaaaaaaaaaaagaatactggaggctgggcgaggtggctcacacctgtaatcccagcattttgggatgccagaggcgggcggaatAtcttgagctcaggagttcgagaccagcctacacaatatgctccaaacgccgcttCtacaaaacatacagaaactacccgggtgtggtggcgTGcccctgtggtcctagatacttgggaggttgaggcgggaggatcgcttgagctcgggaggtcgaggctgcaatgagccgagatggtgccactgcattctgacgacagagcgagattccgtttcaaaacaaacaacaaataaggttgggggatcaaatatcttctagtgtttaaggatctgccttccttcctgcccccatgtttgtctttccttgtttgtctttatatagatcaagcaggttttaaattcctagtaggagcttacatttacttttccaagggggagggggaataaatatctacacacacacacacacacacacacacacacacacacacacacacacacacacaccacactggagttcgagacgaggcctaagcaacatgccgaaaccccgtctctactaaatacaaaaaatagctgagcttggtggcgcacgcctatagtcctagctactggggaggctgaggtgggaggatcgcttgagcccaagaagtcgaggctgcagtgagccgagatcgcgccgctgcactccagcctgagcgacagggcgaggctctgtctcaaaacaaacaaacaaaaaaaaaaaggaaaggaaatataacacagtgaaatgaaaggattgagagaaatgaaaaatatacacgccacaaatgtgggagggcgataaccactcgtagaaagcgtgagaagttactacaagcggtcctcccgggcaccgtactgttccgctcccagaagccccgggcgccggaagtcgtcactcttaagaagggacggggccccacgctgcgcacccgcgggtttgctATGGCCATGAGCAGCGGCGGCAGTGGCGGCGGCGTGCCCGAGCAGGAGGATTCTGTGCTGTTCCGGAGAGGAACAGGCCAGAGCGATGACTCCGATATCTGGGACGACACAGCCCTTATCAAGGCCTACGACAAGGCCGTGGCCAGCTTTAAGCACGCCCTGAAGAATGGCGATATCTGCGAGACAAGCGGAAAGCCTAAGACCACCCCTAAAAGAAAGCCCGCCAAGAAAAACAAGTCCCAGAAAAAAAACACCGCCGCTAGCCTGCAGCAGTGGAAGGTGGGCGACAAATGCAGCGCCATCTGGTCCGAGGACGGCTGCATCTACCCTGCTACCATCGCCAGCATCGACTTCAAGCGGGAAACCTGCGTGGTGGTCTACACAGGCTATGGCAATAGGGAGGAACAAAATCTCTCTGATCTGCTGTCTCCTATTTGTGAAGTGGCTAACAACATCGAGCAGAACGCCCAGGAAAATGAGAACGAAAGCCAAGTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCTGGAAACAAGTCTGACAACATCAAGCCCAAGTCTGCCCCTTGGAACAGCTTCCTGCCCCCTCCTCCTCCAATGCCTGGCCCCAGACTGGGCCCCGGCAAGCCTGGCCTGAAGTTCAACGGCCCTCCTCCACCCCCTCCTCCTCCACCTCCCCATCTGCTGAGCTGCTGGCTGCCTCCTTTTCCCAGCGGCCCCCCTATCATCCCCCCACCACCTCCTATCTGTCCCGACAGCCTGGACGACGCCGATGCTCTGGGATCCATGCTGATCAGCTGGTACATGTCTGGCTACCACACCGGCTACTACATGGGCTTCCGGCAGAACCAGAAGGAAGGAAGATGCAGCCACAGCCTGAACTGAgcGGCCacaaacaccattgtcacactccaacaaacaccattgtcacactccaacaaacaccattgtcacactccaAGCTTCCtgaggatccgatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaagcaattcgttgatctgaatttcgaccacccataatacccattaccctggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgataactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattag

SMNsp-co-SMN1_miR122_BS rAAV vector nucleic acid sequence (SEQ ID NO: 11)

ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtgtagccatgctctaggaagatcaattcggtacaattcacgcgtggatgccagaggcgggcggaatAtcttgagctcaggagttcgagaccagcctacacaatatgctccaaacgccgcttCtacaaaacatacagaaactacccgggtgtggtggcgTGcccctgtggtcctagatacttgggaggttgaggcgggaggatcgcttgagctcgggaggtcgaggctgcaatgagccgagatggtgccactgcattctgacgacagagcgagattccgtttcaaaacaaacaacaaataaggttgggggatcaaatatcttctagtgtttaaggatctgccttccttcctgcccccatgtttgtctttccttgtttgtctttatatagatcaagcaggttttaaattcctagtaggagcttacatttacttttccaagggggagggggaataaatatctacacacacacacacacacacacacacacacacacacacacacacacacacacaccacactggagttcgagacgaggcctaagcaacatgccgaaaccccgtctctactaaatacaaaaaatagctgagcttggtggcgcacgcctatagtcctagctactggggaggctgaggtgggaggatcgcttgagcccaagaagtcgaggctgcagtgagccgagatcgcgccgctgcactccagcctgagcgacagggcgaggctctgtctcaaaacaaacaaacaaaaaaaaaaaggaaaggaaatataacacagtgaaatgaaaggattgagagaaatgaaaaatatacacgccacaaatgtgggagggcgataaccactcgtagaaagcgtgagaagttactacaagcggtcctcccgggcaccgtactgttccgctcccagaagccccgggcgccggaagtcgtcactcttaagaagggacggggccccacgctgcgcacccgcgggtttgctATGGCCATGAGCAGCGGCGGCAGTGGCGGCGGCGTGCCCGAGCAGGAGGATTCTGTGCTGTTCCGGAGAGGAACAGGCCAGAGCGATGACTCCGATATCTGGGACGACACAGCCCTTATCAAGGCCTACGACAAGGCCGTGGCCAGCTTTAAGCACGCCCTGAAGAATGGCGATATCTGCGAGACAAGCGGAAAGCCTAAGACCACCCCTAAAAGAAAGCCCGCCAAGAAAAACAAGTCCCAGAAAAAAAACACCGCCGCTAGCCTGCAGCAGTGGAAGGTGGGCGACAAATGCAGCGCCATCTGGTCCGAGGACGGCTGCATCTACCCTGCTACCATCGCCAGCATCGACTTCAAGCGGGAAACCTGCGTGGTGGTCTACACAGGCTATGGCAATAGGGAGGAACAAAATCTCTCTGATCTGCTGTCTCCTATTTGTGAAGTGGCTAACAACATCGAGCAGAACGCCCAGGAAAATGAGAACGAAAGCCAAGTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCTGGAAACAAGTCTGACAACATCAAGCCCAAGTCTGCCCCTTGGAACAGCTTCCTGCCCCCTCCTCCTCCAATGCCTGGCCCCAGACTGGGCCCCGGCAAGCCTGGCCTGAAGTTCAACGGCCCTCCTCCACCCCCTCCTCCTCCACCTCCCCATCTGCTGAGCTGCTGGCTGCCTCCTTTTCCCAGCGGCCCCCCTATCATCCCCCCACCACCTCCTATCTGTCCCGACAGCCTGGACGACGCCGATGCTCTGGGATCCATGCTGATCAGCTGGTACATGTCTGGCTACCACACCGGCTACTACATGGGCTTCCGGCAGAACCAGAAGGAAGGAAGATGCAGCCACAGCCTGAACTGAgcGGCCacaaacaccattgtcacactccaacaaacaccattgtcacactccaacaaacaccattgtcacactccaagcttatcgataccgtcgactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatAGCCtaggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattagg

Claims (86)

1. A recombinant adeno-associated virus (rAAV) vector comprising a transgene comprising an endogenous SMN1 promoter operably linked to a codon-optimized nucleic acid sequence encoding human SMN1, flanked by adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs).

2. The rAAV vector of claim 1, wherein the codon optimized nucleic acid sequence comprises a nucleotide sequence that hybridizes to SEQ ID NO:1, at least 70%, 80%, 90%, 95% or 99% identical.

3. The rAAV vector of claim 1 or 2, wherein the codon optimized nucleic acid sequence comprises SEQ ID NO:1 or a sequence set forth in SEQ ID NO:1, and a sequence set forth in seq id no.

4. The rAAV vector according to any one of claims 1 to 3, wherein the codon optimized nucleic acid sequence does not comprise the nucleic acid sequence set forth in SEQ ID No. 2.

5. The rAAV vector according to any one of claims 1 to 3, wherein the human SMN1 comprises the amino acid sequence set forth in SEQ ID No. 3.

6. The rAAV vector of any one of claims 1-5, wherein the endogenous SMN1 promoter is a human SMN1 promoter.

7. The rAAV vector of any one of claims 1-6, wherein the endogenous SMN1 promoter comprises a sequence identical to SEQ ID NO:4 or 5, at least 70%, 80%, 90%, 95% or 99% identical.

8. The rAAV vector according to any one of claims 1 to 7, wherein the endogenous SMN1 promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID No. 4.

9. The rAAV vector according to any one of claims 1 to 7, wherein the endogenous SMN1 promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID No. 5.

10. The rAAV vector of any one of claims 1-9, further comprising one or more miR-122 binding sites.

11. The rAAV vector of claim 10, wherein the one or more miR-122 binding sites are located between a codon-optimized nucleic acid sequence encoding human SMN1 and the 3' itr.

12. The rAAV vector according to any one of claims 1 to 11, wherein at least one AAV ITR is an AAV2 ITR.

13. The rAAV vector according to any one of claims 1 to 12, wherein at least one AAV ITR is a mutated ITR (mTR).

14. A vector comprising the rAAV vector of any one of claims 1 to 13.

15. The vector of claim 14, wherein the vector is a plasmid or a baculovirus vector.

16. A cell comprising the rAAV vector of any one of claims 1 to 13 or the vector of claim 14 or 15.

17. An isolated nucleic acid comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 6-11.

18. The isolated nucleic acid of claim 17, further comprising an endogenous SMN1 promoter operably linked to the nucleic acid sequence.

19. The isolated nucleic acid of claim 18, wherein the endogenous SMN1 promoter is a human SMN1 promoter, optionally wherein the SMN1 promoter comprises SEQ ID NO:4 or 5 or a nucleic acid sequence represented by SEQ ID NO:4 or 5.

20. A recombinant adeno-associated virus (rAAV), comprising:

(i) A recombinant adeno-associated virus (rAAV) vector comprising a transgene comprising an endogenous SMN1 promoter operably linked to a codon-optimized nucleic acid sequence encoding human SMN1, flanked by adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs); and

(Ii) At least one AAV capsid protein.

21. The rAAV of claim 20, wherein the codon-optimized nucleic acid sequence comprises a nucleotide sequence that hybridizes to SEQ ID NO:1, at least 70%, 80%, 90%, 95% or 99% identical.

22. The rAAV of claim 20 or 21, wherein the codon optimized nucleic acid sequence comprises or consists of the sequence set forth in SEQ ID No. 1.

23. The rAAV of any one of claims 20-22, wherein the codon-optimized nucleic acid sequence does not comprise the nucleic acid sequence set forth in SEQ ID No. 2.

24. The rAAV of any one of claims 20-23, wherein the human SMN1 comprises the amino acid sequence set forth in SEQ ID No. 3.

25. The rAAV of any one of claims 20-24, wherein the endogenous SMN1 promoter is a human SMN1 promoter.

26. The rAAV of any one of claims 20-25, wherein the endogenous SMN1 promoter comprises a sequence identical to SEQ ID NO:4 or 5, at least 70%, 80%, 90%, 95% or 99% identical.

27. The rAAV of any one of claims 20-26, wherein the endogenous SMN1 promoter comprises or consists of a nucleic acid sequence set forth in SEQ ID No. 4.

28. The rAAV of any one of claims 20-26, wherein the endogenous SMN1 promoter comprises or consists of a nucleic acid sequence set forth in SEQ ID No. 5.

29. The rAAV of any one of claims 20-28, further comprising one or more miR-122 binding sites.

30. The rAAV of claim 29, wherein the one or more miR-122 binding sites are located between a codon-optimized nucleic acid sequence encoding human SMN1 and the 3' itr.

31. The rAAV of any one of claims 20-30, wherein at least one AAV ITR is an AAV2 ITR.

32. The rAAV of any one of claims 20-31, wherein at least one AAV ITR is a mutated ITR (mTR).

33. The rAAV of any one of claims 20-32, wherein the rAAV is a self-complementary AAV (scAAV).

34. The rAAV of any one of claims 20-33, wherein the at least one AAV capsid protein is an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein, or variant thereof.

35. The rAAV of any one of claims 20-34, wherein the at least one AAV capsid protein is an AAV9 capsid protein.

36. A pharmaceutical composition comprising the rAAV vector or rAAV of any one of the preceding claims and a pharmaceutically acceptable excipient.

37. A method of delivering a transgene to a cell, the method comprising administering the rAAV vector of any one of claims 1 to 13, the rAAV of any one of claims 20 to 35, or the pharmaceutical composition of claim 36 to the cell.

38. The method of claim 37, wherein the cell is a mammalian cell.

39. The method of claim 37 or 38, wherein the cell is a human cell.

40. The method of any one of claims 37-39, wherein the cell is located in a subject.

41. The method of claim 40, wherein the subject has or is suspected of having Spinal Muscular Atrophy (SMA).

42. A method of preventing or treating Spinal Muscular Atrophy (SMA) in a subject, the method comprising administering the rAAV vector of any one of claims 1 to 13, the rAAV of any one of claims 20-35, or the pharmaceutical composition of claim 36 to the subject.

43. The method of claim 42, wherein the subject is a mammal.

44. The method of claim 42 or 43, wherein the subject is a human.

45. The method of any one of claims 42-44, wherein the subject has one or more mutations in the SMN1 gene.

46. The method of any one of claims 42-45, wherein the administering comprises systemic injection or local injection.

47. The method of claim 46, wherein the systemic injection comprises intravenous injection.

48. The method of any one of claims 42-47, wherein the administering comprises injection into the Central Nervous System (CNS) of the subject.

49. The method of any one of claims 42-48, wherein the administration results in a reduced amount of Dorsal Root Ganglion (DRG) toxicity in the subject relative to administration of a rAAV comprising a constitutive promoter operably linked to a wild-type SMN1 coding sequence to the subject.

50. The method of any one of claims 42-49, wherein the administration results in reduced hepatotoxicity in the subject relative to administration to the subject of an AAV vector comprising a constitutive promoter operably linked to a nucleic acid encoding human SMN 1.

51. The method of any one of claims 42-50, wherein the administration results in a reduction of complications associated with SMA.

52. The method of any one of claims 42-51, wherein the administration results in an increase in survival rate of the subject relative to administration to the subject of an AAV vector comprising a constitutive promoter operably linked to a nucleic acid encoding human SMN 1.

53. The method of claim 51, wherein the SMA complication comprises a lung infection, spinal deformity (e.g., scoliosis, hip subluxation/dislocation), joint contracture, or respiratory failure.

54. A recombinant adeno-associated virus (rAAV) vector comprising a transgene comprising a promoter operably linked to a codon-optimized nucleic acid sequence encoding human SMN1, flanked by adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs).

55. The rAAV vector according to claim 54, wherein the codon-optimized nucleic acid sequence comprises a nucleotide sequence that hybridizes to SEQ ID NO:1, at least 70%, 80%, 90%, 95% or 99% identical.

56. The rAAV vector of claim 54 or 55, wherein the codon-optimized nucleic acid sequence comprises SEQ ID NO:1 or a sequence set forth in SEQ ID NO:1, and a sequence set forth in seq id no.

57. The rAAV vector according to any one of claims 54-56, wherein the codon-optimized nucleic acid sequence does not comprise the nucleic acid sequence set forth in SEQ ID No. 2.

58. The rAAV vector according to any one of claims 54-57, wherein the human SMN1 comprises the amino acid sequence of SEQ ID No. 3.

59. The rAAV vector according to any one of claims 54-58, wherein the promoter is a CB6 promoter.

60. The rAAV vector of any one of claims 54-59, wherein the rAAV vector further comprises a CMV enhancer.

61. The rAAV vector of any one of claims 54-60, further comprising one or more miR-122 binding sites.

62. The rAAV vector of claim 61, wherein the one or more miR-122 binding sites are located between a codon-optimized nucleic acid sequence encoding human SMN1 and the 3' itr.

63. The rAAV vector of any one of claims 54-62, wherein at least one AAV ITR is an AAV2 ITR.

64. The rAAV vector according to any one of claims 54-63, wherein at least one AAV ITR is a mutated ITR (mTR).

65. A vector comprising the rAAV vector of any one of claims 54-64.

66. The vector of claim 65, wherein the vector is a plasmid or a baculovirus vector.

67. A cell comprising the rAAV vector of any one of claims 54-64 or the vector of claim 65 or 66.

68. A recombinant adeno-associated virus (rAAV), the rAAV comprising:

(a) A self-complementary rAAV genome comprising:

(i)5'ITR

(ii) A human short SMN promoter comprising the nucleotide sequence of SEQ ID No. 5;

(iii) A codon optimized nucleic acid sequence encoding SMN1 as shown in SEQ ID No. 1;

(iv) poly a tail; and

(V) 3' ITR; and

(B) AAV9 capsid protein.

69. The rAAV of claim 68, wherein the poly a tail is a rabbit globin poly a tail or a BGH poly a tail.

70. The rAAV of claim 68 or 69, further comprising one or more miR-122 binding sites.

71. A recombinant adeno-associated virus (rAAV), comprising:

(i) The recombinant adeno-associated virus (rAAV) vector of any one of claims 54-65, and

(Ii) At least one AAV capsid protein.

72. The rAAV of claim 71, wherein the rAAV is a self-complementary AAV (scAAV).

73. The rAAV of claim 71 or 72, wherein the at least one AAV capsid protein is an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein, or variant thereof.

74. The rAAV of any one of claims 71-73, wherein the at least one AAV capsid protein is an AAV9 capsid protein.

75. A method for preventing or treating Spinal Muscular Atrophy (SMA) in a subject, the method comprising administering the rAAV vector of any one of claims 54-64 or the rAAV of any one of claims 71-74 to the subject.

76. The method of claim 75, wherein the subject is a mammal.

77. The method of claim 75 or 76, wherein the subject is a human.

78. The method of any one of claims 75-77, wherein the subject has one or more mutations in the SMN1 gene.

79. The method of any one of claims 75-78, wherein the administering comprises systemic injection or local injection.

80. The method of claim 79, wherein the systemic injection comprises intravenous injection.

81. The method of any one of claims 75-80, wherein the administering comprises injection to the Central Nervous System (CNS) of the subject.

82. The method of any one of claims 75-81, wherein the administration results in a reduced amount of Dorsal Root Ganglion (DRG) toxicity in the subject relative to administration of a rAAV comprising a constitutive promoter operably linked to a wild-type SMN1 coding sequence to the subject.

83. The method of any one of claims 75-82, wherein the administration results in reduced hepatotoxicity in the subject relative to administration of an AAV vector comprising a constitutive promoter operably linked to a nucleic acid encoding human SMN 1.

84. The method of any one of claims 75-83, wherein the administration results in a reduction in complications associated with SMA.

85. The method of any one of claims 74-83, wherein the administration results in an increase in survival of the subject relative to administration to the subject of an AAV vector comprising a constitutive promoter operably linked to a nucleic acid encoding human SMN 1.

86. The method of claim 84, wherein the complication of SMA comprises a lung infection, spinal deformity (e.g., scoliosis, hip subluxation/dislocation), joint contracture, or respiratory failure.