Recombinant AAV for gene therapy of SMA disease

Abstract

The present invention provides a polynucleotide encoding hSMN1 and a recombinant AAV. The invention also provides a method of treating SMA disease comprising administering the recombinant AAV to a subject in need thereof.

Description

Technical Field

[0001] The present invention relates to gene therapy. In particular, the present invention relates to a recombinant adeno-associated virus (AAV) for gene therapy of SMA disease. Background Art

[0002] Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by mutations in the SMN1 gene on chromosome 5, which leads to the death of neurons in the anterior horn of the spinal cord and subsequent atrophy. The prevalence of type I SMA is reported to be approximately 1-2 cases per 100,000 people. The prevalence of type I SMA in China is estimated to be 1,200-1,500 cases, and the prevalence of other subtypes is 20,000 cases.

[0003] Type I SMA is a devastating disease. Patients develop the disease at ages 0-6 months and are never able to sit up. Life expectancy is usually less than 2 years.

[0004] Several therapies have been approved for SMA, including: i) gene therapy (Zolgensma), which was approved by the USFDA on May 24, 2019 for the treatment of pediatric patients under 2 years of age and approved by the EMA in May 2020; ii) Nusinersen (antisense oligonucleotide, ASO), which was approved by the FDA in December 2016 and approved by the EMA in May 2017; and iii) Evrysdi, which was approved by the FDA in August 2020. However, the approved therapies have several drawbacks that limit their widespread use.

[0005] Zolgensma is indeed very expensive (about 2 million USD / IV injection). High-dose injections may cause high levels of liver enzymes, which may trigger serious liver damage (https: / / www.medicalnewstoday.com / articles / drugs-zolgensma).

[0006] Nusinersen is delivered directly into the central nervous system (CNS) via intrathecal injection (i.e., delivered into the cerebrospinal fluid via lumbar puncture to reach the target within the CNS where motor neuron loss begins). After the initial 4 loading doses, Nusinersen is administered 3 times per year (https: / / www.spinraza.com and https: / / medlineplus.gov / druginfo / meds / a617010.html). Nusinersen requires continuous administration in an invasive and complex manner, carries a high risk, and requires a trained physician / nurse to perform.

[0007] Evrysdi is supplied as an oral solution but is to be taken once daily after meals and must be prepared by a pharmacist before dispensing. Evrysdi can be administered orally with the oral syringe provided. If the patient is unable to swallow and has a nasogastric or gastrostomy tube, it can be administered through the tube. After delivering Evrysdi, the tube should be flushed with water (https: / / www.centerwatch.com / directories / 1067-fda-approved-drugs / listing / 4644-evrysdi-risdiplam). Evrysdi is not convenient to administer. Young patients (2 years or younger) or those with disabilities require assistance from parents / nurses as they are obviously unable to take the medication orally on their own.

[0008] Therefore, there is a need for a new treatment for SMA, especially type I SMA, such as a gene therapy that can provide sustained expression of functional SMN1 at desired levels. Summary of the invention

[0009] To meet this need, the inventors have developed a gene therapy for SMA disease that provides sustained endogenous production of SMN1 following transfer of a functional copy of an SMN1 gene (eg, an SMN1 gene) encoding a wild-type SMN1 polypeptide to a patient.

[0010] In a first aspect, the present invention provides a polynucleotide of interest comprising a nucleotide sequence selected from SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0011] In a second aspect, the present invention provides an expression construct comprising a polynucleotide of interest comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 and operably linked to a promoter. In some embodiments, the nucleotide sequence is SEQ ID NO: 11.

[0012] In some embodiments, the construct further comprises an intron. In some embodiments, the intron is between the promoter and the polynucleotide of interest. In some embodiments, the intron comprises a nucleotide sequence selected from SEQ ID NO:24 and 29. In some embodiments, the construct further comprises an enhancer. In some embodiments, the enhancer is upstream of the promoter. In some embodiments, the enhancer comprises the nucleotide sequence shown in SEQ ID NO:22. In some embodiments, the promoter comprises a nucleotide sequence selected from SEQ ID NO:23, 28 and 31.

[0013] In a third aspect, the present invention provides a recombinant adeno-associated virus (rAAV) comprising a genome comprising an expression construct of the present invention. In some embodiments, the rAAV is prepared by a system comprising a transgenic plasmid comprising the rAAV genome, a packaging plasmid encoding REP and / or CAP proteins, and a helper plasmid. Therefore, the present invention also provides a vector comprising an expression construct of the present invention.

[0014] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the rAAV of the present invention.

[0015] In a fifth aspect, the present invention provides a host cell comprising the polynucleotide, expression construct or rAAV of the present invention.

[0016] In a sixth aspect, the present invention provides a method for treating a disease associated with SMN1 deficiency, comprising administering the rAAV or pharmaceutical composition of the present invention to a subject in need thereof.

[0017] The present invention also provides the use of the polynucleotides, expression constructs, rAAV, pharmaceutical compositions and / or host cells of the present invention in the preparation of a medicament for treating a disease associated with SMN1 deficiency in a subject.

[0018] In some embodiments, the disease is SMA, such as type I SMA. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown are the structures of the construct of the invention (A), GFP control construct (B), and Zolgensma construct (C) and Zolgensma GFP construct (D).

[0020] Figure 2 Maps of the transgenic plasmid (A), helper plasmid (B), packaging plasmid (C), and fLuc2 plasmid (D) are shown.

[0021] Figure 3Western blots for detecting hSMN1 expression of transgenic plasmids in 293T cells are shown, with α-tubulin as a reference. Lanes 2-20 represent samples from cells transfected with transgenic plasmids containing CoSMN2-20, respectively; Lane C represents samples from untransfected cells; Lane Zol represents samples from cells transfected with transgenic plasmids containing Zolgensma constructs; and Lane GFP represents samples from cells transfected with transgenic plasmids encoding GFP.

[0022] Figure 4 Luciferase activity in transfected cells is shown, columns 2-20 represent cell samples co-transfected with transgenic plasmids containing CoSMN2-20, respectively; column "cells" represents samples from untransfected cells; column Zol represents samples from cells co-transfected with a transgenic plasmid containing a Zolgensma construct; and column GFP represents samples from cells co-transfected with a transgenic plasmid encoding GFP.

[0023] Figure 5 Relative expression of hSMN1 in 293T cells is shown, and the results of Western blots were corrected by luciferase activity and normalized to Zol samples.

[0024] Figure 6 The relative expression of hSMN1 by the rAAV of the present invention in 293T cells is shown.

[0025] Figure 7 The relative expression of hSMN1 by the rAAV of the present invention in wild-type mice is shown.

[0026] Figure 8 The relative expression of hSMN1 by the rAAV of the present invention in the SMA model is shown.

[0027] Fig. 9 The relative expression of hSMN1 in wild-type mice by rAAV of the present invention (comprising various combinations of promoters and introns) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0029] 1. Definition

[0030] Unless otherwise indicated, all terms used herein have the same meanings as understood by one skilled in the art, and the practice of the present invention will employ conventional techniques of microbiology and recombinant DNA technology, which are within the knowledge of one skilled in the art.

[0031] As used herein, "SMN1", when referring to a gene, means the survival motor neuron gene 1, and when referring to a protein, means the protein encoded by the SMN1 gene. "hSMN1" means the human "SMN1" gene or protein.

[0032] Adeno-associated virus (AAV) is a member of the Parvoviridae family. It is a simple single-stranded DNA virus that requires a helper virus (such as adenovirus) for replication. The genome of wild-type AAV contains approximately 4.7 kilobases (kb) and includes the cap and rep genes between two inverted terminal repeat (ITR) sequences, which are approximately 145 nucleotides in length and have an interrupted palindromic sequence that can fold into a hairpin structure and function as a primer during the initiation of DNA replication. The cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of AAV. AAV can infect a variety of cells, and in the presence of the rep product, the viral DNA can be integrated into human chromosome 19.

[0033] As used herein, the term "inverted terminal repeat" or "ITR" refers to AAV viral cis elements, which are named because of their symmetry. These elements are very important for the efficient multiplication of the AAV genome. In the present invention, the term "ITR" refers to the ITRs of known natural AAV serotypes, chimeric ITRs formed by fusing ITR elements derived from different serotypes, and their functional variants.

[0034] The production of recombinant AAV particles may involve three plasmids, a transgene plasmid containing an expression construct for expressing an exogenous polynucleotide, a packaging plasmid encoding REP and / or CAP proteins, and a helper plasmid.

[0035] As used herein, the term "expression construct" refers to a single-stranded or double-stranded polynucleotide that is isolated from a naturally occurring gene or modified to contain a non-naturally occurring nucleic acid segment. The expression construct may contain control sequences required for expression of the coding sequence of the invention.

[0036] As used herein, the term "polynucleotide" generally refers to nucleic acid molecules (e.g., 100 bases and up to 30 kilobases in length) and sequences that are complementary (antisense) or identical (sense) to the sequences of messenger RNA (mRNA) or miRNA fragments or molecules. The term can also refer to transcribed or non-transcribed DNA or RNA molecules.

[0037] As used herein, the term "exogenous polynucleotide" refers to a nucleotide sequence that is not derived from the host in which it is located. It can be identical to the host's DNA or heterologous. An example is a sequence of interest inserted into a vector. This type of exogenous DNA sequence can be derived from a variety of sources, including DNA, cDNA, synthetic DNA, and RNA. Exogenous polynucleotides also encompass DNA sequences encoding antisense oligonucleotides.

[0038] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0039] "Control sequences" include all elements necessary or useful for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, an enhancer, a signal peptide sequence, and a transcription terminator. At a minimum, a control sequence includes a promoter and a signal to terminate transcription and translation.

[0040] For example, the control sequence can be a suitable promoter sequence, a nucleotide sequence that is recognized by the host cell to express a polynucleotide encoding the polypeptide of the present invention. The promoter sequence contains a transcription control sequence that mediates the expression of the polypeptide. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, for example, the lac operon of Escherichia coli. Promoters also include mutant, modified and hybrid promoters that can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0041] In some cases, introns may be included in the construct to improve expression of the coding sequence.A "modified" intron comprises a modification, such as a substitution, insertion or deletion of one or more nucleotides, within the internal region of the original intron.

[0042] As used herein, the term "operably linked" herein refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence so that the expression of the polypeptide coding sequence is directed by the control sequence.

[0043] The polynucleotide encoding the SMN1 protein can be manipulated in various ways to improve the expression of the polypeptide. Before inserting it into a vector, it is desirable or necessary to manipulate the polynucleotide, such as codon optimization, depending on the expression vector or host.

[0044] As used herein, the term "recombinant" refers to a nucleic acid, vector, polypeptide or protein that is produced using DNA recombination (cloning) methods and can be distinguished from a native or wild-type nucleic acid, vector, polypeptide or protein.

[0045] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids, including full-length proteins and fragments thereof.

[0046] As used herein, the term "host cell" refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as rAAV vector receptors. The term includes the progeny of the original cell that has been transduced. Therefore, as used herein, "host cell" generally refers to a cell that has been transduced with an exogenous DNA sequence. It should be understood that due to natural, accidental or artificial mutations, the progeny of a single parent cell may not necessarily be completely identical in morphology, or complementary to the original parent in genome or total DNA.

[0047] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and generally do not produce toxicity or allergic or similar adverse reactions such as gastric upset, dizziness, and the like when administered to humans.

[0048] The term "subject" as used herein includes, but is not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domesticated mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs, etc. The term does not denote a particular age or sex. Thus, it is intended to cover adult and newborn subjects as well as fetuses, whether male or female.

[0049] 2. Polynucleotides or constructs for expressing hSMN1

[0050] The purpose of gene therapy is to correct the defective genes that cause the development of the disease and introduce exogenous genes into the subject's cells of interest to express the products of exogenous genes that can be used to treat certain diseases such as SMA. Common methods for this purpose include delivering functional genes such as SMN1 to the cell nucleus. This gene can then be inserted into the genome of the cell of interest, or it can remain free. The delivery of functional genes to the subject's target cells can be carried out by many methods, including the use of viral vectors. Among the many available viral vectors (e.g., retroviruses, lentiviruses, adenoviruses, etc.), AAV is becoming increasingly popular in gene therapy as a versatile vector.

[0051] Vectors derived from AAV are particularly attractive for delivering genetic material because: (i) they are able to infect (transduce) a variety of non-dividing and dividing cell types, including muscle fibers and neurons; (ii) they have no viral structural genes, thereby eliminating natural host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses have never been associated with any pathology in humans; (iv) compared to wild-type AAVs that can integrate into the host cell genome, replication-defective AAV vectors are generally maintained as episomes, thus limiting the risk of insertional mutagenesis or activation of oncogenes; and (v) compared to other vector systems, AAV vectors do not trigger significant immune responses (see ii), thus allowing long-term expression of therapeutic transgenes (as long as their gene products are not rejected). AAV vectors can also be produced in high titers, and intra-arterial, intravenous or intraperitoneal injections have been reported to allow gene transfer to important muscle areas in rodents by a single injection.

[0052] Therefore, the present invention aims to provide a polynucleotide of interest, an expression construct, or a vector comprising a polynucleotide of interest or an expression construct for expressing SMN1 in a subject. In some embodiments, SMN1 can be from any animal species, including but not limited to humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domesticated mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs, etc.

[0053] In some embodiments, the SMN1 is human SMN1. In some embodiments, hSMN1 comprises the amino acid sequence of SEQ ID NO: 21 or an allelic variant thereof. In some embodiments, the SMN1 is a functional SMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21.

[0054] In some embodiments, the polynucleotide encoding SMN1 is codon optimized for improved expression.

[0055] In some embodiments, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. The inventors unexpectedly found that polynucleotides having nucleotide sequences of SEQ ID NOs: 4, 5, 6, 7, 8, 11, 16 and 19 achieved higher expression (1.5-4 times) of hSMN1 in cells than SEQ ID NO: 1, while in in vivo tests, SEQ ID NOs: 3, 6, 8 and 11 achieved higher expression in the spinal cord, but lower expression in the heart and liver, compared to SEQ ID NO: 1.

[0056] In some embodiments, the polynucleotide comprises SEQ ID NO: 2, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21.

[0057] In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0058] In some embodiments, the polynucleotide comprises SEQ ID NO:3 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0059] In some embodiments, the polynucleotide comprises SEQ ID NO: 4 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0060] In some embodiments, the polynucleotide comprises SEQ ID NO:5 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0061] In some embodiments, the polynucleotide comprises SEQ ID NO:6 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0062] In some embodiments, the polynucleotide comprises SEQ ID NO:7 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0063] In some embodiments, the polynucleotide comprises SEQ ID NO: 8, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0064] In some embodiments, the polynucleotide comprises SEQ ID NO:9 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0065] In some embodiments, the polynucleotide comprises SEQ ID NO: 10, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0066] In some embodiments, the polynucleotide comprises SEQ ID NO: 11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0067] In some embodiments, the polynucleotide comprises SEQ ID NO: 12, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0068] In some embodiments, the polynucleotide comprises SEQ ID NO: 13 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0069] In some embodiments, the polynucleotide comprises SEQ ID NO: 14, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0070] In some embodiments, the polynucleotide comprises SEQ ID NO: 15, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0071] In some embodiments, the polynucleotide comprises SEQ ID NO: 16, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0072] In some embodiments, the polynucleotide comprises SEQ ID NO: 17, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0073] In some embodiments, the polynucleotide comprises SEQ ID NO: 18, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0074] In some embodiments, the polynucleotide comprises SEQ ID NO: 19, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0075] In some embodiments, the polynucleotide comprises SEQ ID NO:20, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20, and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0076] The present invention also provides an expression construct for expressing SMN1, comprising a polynucleotide of interest encoding SMN1 operably linked to a promoter. In some embodiments, the SMN1 is human SMN1. In some embodiments, the SMN1 comprises the amino acid sequence of SEQ ID NO: 21 or an allelic variant thereof.

[0077] In some embodiments, the expression construct achieves higher expression of SMN1 polypeptide and / or mRNA in the spinal cord and / or lower expression of SMN1 polypeptide and / or mRNA in non-spinal cord tissues (e.g., heart and / or liver) compared to a control construct, such as an expression construct comprising the nucleotide sequence of SEQ ID NO: 1.

[0078] In some embodiments, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. The inventors unexpectedly discovered that polynucleotides having nucleotide sequences of SEQ ID NOs: 4, 5, 6, 7, 8, 11, 16 and 19 achieve higher expression (1.5-4 times) of hSMN1 in cells than SEQ ID NO: 1, while in in vivo tests, SEQ ID NOs: 3, 6, 8 and 11 achieve higher expression in the spinal cord, but lower expression in the heart and liver, compared to SEQ ID NO: 1.

[0079] In some embodiments, the polynucleotide comprises SEQ ID NO:2, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0080] In some embodiments, the polynucleotide comprises SEQ ID NO:3 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0081] In some embodiments, the polynucleotide comprises SEQ ID NO: 4 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0082] In some embodiments, the polynucleotide comprises SEQ ID NO:5 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0083] In some embodiments, the polynucleotide comprises SEQ ID NO:6 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0084] In some embodiments, the polynucleotide comprises SEQ ID NO:7 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0085] In some embodiments, the polynucleotide comprises SEQ ID NO: 8, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0086] In some embodiments, the polynucleotide comprises SEQ ID NO:9 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0087] In some embodiments, the polynucleotide comprises SEQ ID NO: 10, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0088] In some embodiments, the polynucleotide comprises SEQ ID NO: 11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0089] In some embodiments, the polynucleotide comprises SEQ ID NO: 12, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0090] In some embodiments, the polynucleotide comprises SEQ ID NO: 13 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0091] In some embodiments, the polynucleotide comprises SEQ ID NO: 14, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0092] In some embodiments, the polynucleotide comprises SEQ ID NO: 15, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0093] In some embodiments, the polynucleotide comprises SEQ ID NO: 16, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0094] In some embodiments, the polynucleotide comprises SEQ ID NO: 17, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0095] In some embodiments, the polynucleotide comprises SEQ ID NO: 18, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0096] In some embodiments, the polynucleotide comprises SEQ ID NO: 19, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, and encodes a polypeptide of SEQ ID NO: 21, or an allelic variant thereof.

[0097] In some embodiments, the polynucleotide comprises SEQ ID NO:20, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20, and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20, and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0098] Constructs are generally transferred to mammalian cells (such as human cells) for expression. Such constructs often include promoters-enhancers for high-level expression, such as SV40 promoter-enhancers, human cytomegalovirus (CMV) promoters, and long terminal repeats of Rous sarcoma virus (RSV). These promoters-enhancers are active in many cell types. Tissue and cell type promoters and enhancer regions can also be used for expression. Exemplary promoter / enhancer regions include, but are not limited to, promoter / enhancer regions from genes such as elastase I, insulin, immunoglobulin, mouse mammary cancer virus, albumin, alpha-fetoprotein, α1 antitrypsin, β-globin, myelin basic protein, myosin light chain 2, and gonadotropin-releasing hormone gene.

[0099] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter comprises SEQ ID NO:23 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23. In some embodiments, the promoter comprises SEQ ID NO:28 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28. In some embodiments, the promoter comprises SEQ ID NO:31 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31.

[0100] In some embodiments, the expression construct further comprises an enhancer. In some embodiments, the enhancer is upstream of the promoter. In some embodiments, the enhancer is a cytomegalovirus (CMV) early enhancer. In some embodiments, the enhancer comprises SEQ ID NO: 22 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 22.

[0101] It is also desirable to include introns (e.g., introns derived from eukaryotic organisms or artificial introns) in the construct to increase expression in eukaryotic cells. In some embodiments, the expression construct further comprises an intron. In some embodiments, the intron is upstream of the nucleotide sequence encoding SMN1. In some embodiments, the intron is downstream of the promoter.

[0102] In some embodiments, the intron is the PCI intron shown in SEQ ID NO: 24. In some embodiments, the intron is the chimeric intron (chicken β-actin (CBA intron and exon) + rabbit globulin intron) shown in SEQ ID NO: 29.

[0103] In some embodiments, the intron comprises SEQ ID NO:24 or 29, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24 or 29.

[0104] The expression construct further comprises a polyadenylation signal sequence for transcript processing. In some embodiments, the construct comprises a polyadenylation signal sequence downstream of the nucleotide sequence encoding hSMN1. In some embodiments, the polyadenylation signal sequence comprises SEQ ID NO: 25 or 30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25 or 30.

[0105] In some embodiments, the expression construct comprises, in 5' to 3' order, an enhancer, a promoter, an intron, a polynucleotide of interest encoding hSMN1, and a polyadenylation signal sequence.

[0106] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0107] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0108] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0109] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0110] SEQ ID NO:2 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2, and

[0111] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0112] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0113] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0114] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0115] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0116] SEQ ID NO:3 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, and

[0117] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0118] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0119] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0120] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0121] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0122] SEQ ID NO:4 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4, and

[0123] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0124] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0125] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0126] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0127] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0128] SEQ ID NO:5 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, and

[0129] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0130] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0131] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0132] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0133] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0134] SEQ ID NO:6 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6, and

[0135] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0136] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0137] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0138] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0139] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0140] SEQ ID NO:7 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, and

[0141] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0142] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0143] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0144] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0145] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0146] SEQ ID NO:8 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8, and

[0147] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0148] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0149] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0150] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0151] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0152] SEQ ID NO:9 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9, and

[0153] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0154] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0155] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0156] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0157] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0158] SEQ ID NO: 10 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10, and

[0159] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0160] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0161] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0162] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0163] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0164] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0165] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0166] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0167] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0168] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0169] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0170] SEQ ID NO: 12 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12, and

[0171] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0172] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0173] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0174] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0175] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0176] SEQ ID NO: 13 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, and

[0177] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0178] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0179] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0180] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0181] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0182] SEQ ID NO: 14 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14, and

[0183] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0184] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0185] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0186] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0187] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0188] SEQ ID NO: 15 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15, and

[0189] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0190] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0191] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0192] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0193] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0194] SEQ ID NO:16 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:16, and

[0195] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0196] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0197] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0198] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0199] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0200] SEQ ID NO: 17 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17, and

[0201] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0202] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0203] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0204] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0205] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0206] SEQ ID NO:18 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18, and

[0207] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0208] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0209] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0210] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0211] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0212] SEQ ID NO: 19 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 19, and

[0213] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0214] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0215] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0216] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0217] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0218] SEQ ID NO:20 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and

[0219] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0220] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0221] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0222] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0223] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0224] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0225] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0226] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0227] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0228] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0229] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0230] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0231] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0232] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0233] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0234] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0235] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0236] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0237] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0238] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0239] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0240] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0241] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0242] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0243] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0244] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0245] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0246] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0247] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0248] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0249] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0250] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0251] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0252] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0253] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0254] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0255] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0256] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0257] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0258] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0259] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0260] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0261] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0262] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0263] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0264] SEQ ID NO:2, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0265] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0266] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0267] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0268] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0269] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0270] SEQ ID NO:3, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0271] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0272] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0273] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0274] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0275] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0276] SEQ ID NO:4, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0277] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0278] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0279] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0280] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0281] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0282] SEQ ID NO:5, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0283] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0284] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0285] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0286] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0287] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0288] SEQ ID NO:6, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0289] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0290] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0291] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0292] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0293] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0294] SEQ ID NO:7, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0295] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0296] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0297] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0298] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0299] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0300] SEQ ID NO:8, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0301] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0302] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0303] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0304] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0305] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0306] SEQ ID NO:9, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0307] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0308] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0309] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0310] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0311] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0312] SEQ ID NO:10, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:10, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0313] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0314] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0315] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0316] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0317] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0318] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0319] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0320] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0321] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0322] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0323] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0324] SEQ ID NO:12, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0325] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0326] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0327] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0328] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0329] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0330] SEQ ID NO:13, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:13, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0331] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0332] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0333] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0334] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0335] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0336] SEQ ID NO:14, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:14, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0337] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0338] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0339] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0340] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0341] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0342] SEQ ID NO:15, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:15, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0343] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0344] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0345] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0346] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0347] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0348] SEQ ID NO:16, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:16, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0349] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0350] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0351] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0352] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0353] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0354] SEQ ID NO:17, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:17, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0355] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0356] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0357] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0358] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0359] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0360] SEQ ID NO:18, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0361] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0362] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0363] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0364] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0365] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0366] SEQ ID NO:19, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:19, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0367] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0368] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0369] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0370] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0371] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0372] SEQ ID NO:20, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0373] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0374] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0375] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0376] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0377] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0378] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0379] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0380] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0381] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0382] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0383] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0384] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0385] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0386] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0387] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0388] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0389] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0390] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0391] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0392] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0393] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0394] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0395] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0396] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0397] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0398] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0399] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0400] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0401] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0402] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0403] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0404] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0405] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0406] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0407] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0408] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0409] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0410] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0411] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0412] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0413] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0414] 3. Recombinant AAV, Pharmaceutical Compositions and Host Cells

[0415] The present invention further provides a recombinant AAV (rAAV) comprising a genome comprising an expression construct of the present invention. In some embodiments, the expression construct is flanked by the 5' and 3' inverted terminal repeats (ITRs) of AAV.

[0416] In some embodiments, the rAAV achieves higher expression of SMN1 polypeptide and / or mRNA in the spinal cord and / or lower expression of SMN1 polypeptide and / or mRNA in non-spinal cord tissues (e.g., heart and / or liver) compared to a control rAAV, such as a rAAV comprising SEQ ID NO: 1.

[0417] In some embodiments, the rAAV of the present invention can be selected from human serotype 1 AAV (hAAV1), hAAV2, hAAV3, hAAV4, hAAV5, hAAV6, hAAV7, hAAV8, hAAV9, hAAV10, and hAAV11. In some embodiments, the rAAV is hAAV9.

[0418] In some embodiments, the genome of the rAAV comprises an expression construct flanked by the 5' and 3' ITRs of AAV.

[0419] In some embodiments, the expression construct comprises a nucleotide sequence encoding SMN1 operably linked to a promoter. In some embodiments, the SMN1 is human SMN1. In some embodiments, the SMN1 comprises the amino acid sequence of SEQ ID NO: 21 or an allelic variant thereof.

[0420] In some embodiments, the construct comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. The inventors unexpectedly discovered that polynucleotides having the nucleotide sequences of SEQ ID NOs: 4, 5, 6, 7, 8, 11, 16 and 19 achieve higher expression of hSMN1 in cells than SEQ ID NO: 1 (1.5-4 times), while in in vivo testing, SEQ ID NOs: 3, 6, 8 and 11 achieve higher expression in the spinal cord, but lower expression in the heart and liver, compared to SEQ ID NO: 1.

[0421] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO:2 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0422] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO:3 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0423] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO:4 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0424] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO:5 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0425] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO:6 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0426] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO:7 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0427] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO:8 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0428] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO:9 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of a polypeptide of SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0429] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 10 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0430] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 11 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0431] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 12 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0432] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 13 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0433] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 14 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0434] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 15 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0435] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 16 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0436] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 17 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0437] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 18 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0438] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 19 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 19. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 19 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 19 and encodes a polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0439] In some embodiments, the nucleotide sequence is as set forth in SEQ ID NO: 20 or is a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 and encodes a functional hSMN1 polypeptide, such as an hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20 and encodes a polypeptide of SEQ ID NO:21 or an allelic variant thereof.

[0440] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter comprises SEQ ID NO:23 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23. In some embodiments, the promoter comprises SEQ ID NO:28 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28. In some embodiments, the promoter comprises SEQ ID NO:31 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31.

[0441] In some embodiments, the expression construct further comprises an enhancer. In some embodiments, the enhancer is upstream of the promoter. In some embodiments, the enhancer is a cytomegalovirus (CMV) early enhancer. In some embodiments, the enhancer comprises SEQ ID NO: 22 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 22.

[0442] It is also desirable to include introns (e.g., introns derived from eukaryotic organisms or artificial introns) in the construct to increase expression in eukaryotic cells. In some embodiments, the expression construct further comprises an intron. In some embodiments, the intron is upstream of the nucleotides encoding SMN1. In some embodiments, the intron is downstream of the promoter.

[0443] In some embodiments, the intron is the PCI intron shown in SEQ ID NO: 24. In some embodiments, the intron is the chimeric intron (chicken β-actin (CBA intron and exon) + rabbit globulin intron) shown in SEQ ID NO: 29.

[0444] In some embodiments, the intron comprises SEQ ID NO: 24 or 29, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24 or 29.

[0445] The expression construct further comprises a polyadenylation signal sequence for transcript processing. In some embodiments, the construct comprises a polyadenylation signal sequence downstream of the nucleotide sequence encoding hSMN1. In some embodiments, the polyadenylation signal sequence comprises SEQ ID NO: 25 or 30, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25 or 30.

[0446] In some embodiments, the expression construct comprises, in 5' to 3' order, an enhancer, a promoter, an intron, a polynucleotide of interest encoding hSMN1, and a polyadenylation signal sequence.

[0447] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0448] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0449] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0450] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0451] SEQ ID NO:2 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2, and

[0452] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0453] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0454] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0455] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0456] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0457] SEQ ID NO:3 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, and

[0458] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0459] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0460] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0461] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0462] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0463] SEQ ID NO:4 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4, and

[0464] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0465] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0466] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0467] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0468] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0469] SEQ ID NO:5 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, and

[0470] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0471] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0472] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0473] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0474] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0475] SEQ ID NO:6 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6, and

[0476] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0477] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0478] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0479] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0480] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0481] SEQ ID NO:7 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, and

[0482] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0483] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0484] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0485] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0486] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0487] SEQ ID NO:8 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8, and

[0488] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0489] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0490] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0491] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0492] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0493] SEQ ID NO:9 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9, and

[0494] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0495] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0496] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0497] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0498] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0499] SEQ ID NO: 10 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10, and

[0500] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0501] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0502] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0503] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0504] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0505] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0506] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0507] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0508] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0509] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0510] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0511] SEQ ID NO: 12 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12, and

[0512] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0513] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0514] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0515] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0516] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0517] SEQ ID NO: 13 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, and

[0518] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0519] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0520] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0521] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0522] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0523] SEQ ID NO: 14 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14, and

[0524] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0525] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0526] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0527] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0528] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0529] SEQ ID NO: 15 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15, and

[0530] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0531] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0532] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0533] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0534] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0535] SEQ ID NO:16 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:16, and

[0536] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0537] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0538] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0539] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0540] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0541] SEQ ID NO: 17 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17, and

[0542] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0543] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0544] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0545] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0546] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0547] SEQ ID NO:18 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18, and

[0548] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0549] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0550] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0551] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0552] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0553] SEQ ID NO: 19 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 19, and

[0554] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0555] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0556] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0557] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0558] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0559] SEQ ID NO:20 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and

[0560] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0561] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0562] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0563] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0564] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0565] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0566] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0567] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0568] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0569] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0570] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0571] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0572] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0573] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0574] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0575] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0576] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0577] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0578] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0579] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0580] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0581] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0582] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0583] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0584] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0585] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0586] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0587] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0588] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0589] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0590] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0591] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0592] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0593] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0594] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0595] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0596] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0597] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0598] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0599] SEQ ID NO: 11 or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and

[0600] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0601] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0602] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0603] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0604] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0605] SEQ ID NO:2, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0606] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0607] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0608] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0609] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0610] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0611] SEQ ID NO:3, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0612] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0613] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0614] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0615] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0616] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0617] SEQ ID NO:4, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0618] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0619] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0620] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0621] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0622] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0623] SEQ ID NO:5, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0624] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0625] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0626] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0627] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0628] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0629] SEQ ID NO:6, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0630] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0631] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0632] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0633] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0634] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0635] SEQ ID NO:7, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0636] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0637] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0638] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0639] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0640] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0641] SEQ ID NO:8, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0642] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0643] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0644] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0645] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0646] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0647] SEQ ID NO:9, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0648] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0649] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0650] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0651] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0652] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0653] SEQ ID NO:10, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:10, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0654] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0655] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0656] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0657] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0658] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0659] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0660] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0661] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0662] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0663] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0664] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0665] SEQ ID NO:12, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0666] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0667] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0668] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0669] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0670] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0671] SEQ ID NO:13, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:13, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0672] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0673] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0674] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0675] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0676] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0677] SEQ ID NO:14, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:14, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0678] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0679] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0680] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0681] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0682] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0683] SEQ ID NO:15, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:15, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0684] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0685] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0686] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0687] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0688] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0689] SEQ ID NO:16, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:16, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0690] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0691] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0692] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0693] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0694] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0695] SEQ ID NO:17, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:17, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0696] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0697] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0698] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0699] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0700] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0701] SEQ ID NO:18, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0702] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0703] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0704] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0705] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0706] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0707] SEQ ID NO:19, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:19, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0708] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0709] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0710] SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22,

[0711] SEQ ID NO:23 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23,

[0712] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0713] SEQ ID NO:20, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0714] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0715] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0716] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0717] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0718] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0719] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0720] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0721] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0722] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0723] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0724] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0725] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0726] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0727] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0728] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0729] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0730] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0731] SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28,

[0732] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0733] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0734] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0735] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0736] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0737] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0738] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0739] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0740] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0741] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0742] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0743] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0744] SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0745] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0746] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0747] SEQ ID NO:24 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24,

[0748] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0749] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0750] In some embodiments, the expression construct comprises, in 5' to 3' order,

[0751] SEQ ID NO:31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31,

[0752] SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29,

[0753] SEQ ID NO:11, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11, wherein the nucleotide sequence encodes the hSMN1 of SEQ ID NO:21 or an allelic variant thereof, or a functional hSMN1 polypeptide, such as an SMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21, and

[0754] SEQ ID NO:30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30.

[0755] The present invention also provides a method for preparing rAAV. In some embodiments, rAAV is prepared by a system containing: a transgenic plasmid comprising the rAAV genome, a packaging plasmid encoding REP and / or CAP proteins, and an auxiliary plasmid, for example, a host cell such as a mammalian cell, which contains a transgenic plasmid comprising the rAAV genome, a packaging plasmid encoding REP and / or CAP proteins, and an auxiliary plasmid. Therefore, the present invention also provides a vector, such as a plasmid, comprising the genome of the rAAV of the present invention.

[0756] In some embodiments, rAAV can be packaged as described in Crosson SM et al. (Crosson SM, Dib P, Smith JK, Zolotukhin S. Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation. Mol Ther Methods Clin Dev. 2018; 10: 1-7).

[0757] The present invention also provides a vector comprising the genome of rAAV, wherein the genome of rAAV comprises the expression construct of the present invention, which is flanked by 5' and 3' ITRs. In some embodiments, the vector is a transgenic plasmid for packaging rAAV.

[0758] In some embodiments, the genome of the rAAV is a self-complementary vector genome (scAAV).

[0759] In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, 5'ITR, SEQ ID NO:31, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:25, and 3'ITR. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, 5'ITR, SEQ ID NO:31, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:25, and 3'ITR. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, 5'ITR, SEQ ID NO:31, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:30, and 3'ITR. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, 5'ITR, SEQ ID NO:31, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:30, and 3'ITR.

[0760] In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, 5'ITR, SEQ ID NO:28, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:25, and 3'ITR. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, 5'ITR, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:25, and 3'ITR. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, 5'ITR, SEQ ID NO:28, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:30, and 3'ITR. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, 5'ITR, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:30, and 3'ITR.

[0761] In some embodiments, the 5' ITR comprises SEQ ID NO: 26. In some embodiments, the 3' ITR comprises SEQ ID NO: 27.

[0762] In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:25, and SEQ ID NO:27. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:25, and SEQ ID NO:27. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:30, and SEQ ID NO:27. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:30, and SEQ ID NO:27.

[0763] In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 24, SEQ ID NO: 11, SEQ ID NO: 25, and SEQ ID NO: 27. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 11, SEQ ID NO: 25, and SEQ ID NO: 27. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 24, SEQ ID NO: 11, SEQ ID NO: 30, and SEQ ID NO: 27. In some embodiments, the genome of the rAAV comprises, in 5' to 3' order, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:30, and SEQ ID NO:27.

[0764] The present invention also provides a pharmaceutical composition comprising rAAV. The pharmaceutical composition containing the AAV of the present invention can be formulated in any conventional manner by mixing a selected amount of rAAV with one or more pharmaceutically acceptable carriers or excipients.

[0765] The choice of carrier or excipient is within the skill of the administration professional and depends on many parameters. These include, for example, the mode of administration (i.e., systemic, oral, topical, topical, or any other mode) and the disease to be treated. Pharmaceutical carriers or vehicles suitable for administration of rAAV provided herein include any such carriers known to those skilled in the art to be suitable for a particular mode of administration.

[0766] In some embodiments, the pharmaceutical composition is formulated for parenteral administration, e.g., intravenous, intramuscular, or subcutaneous injection. In some embodiments, the pharmaceutical composition is formulated for topical administration.

[0767] The inventors have found that the pharmaceutical composition of the present invention can achieve the desired safety, for example, when administered by intravenous injection. For example, safety can be evaluated by the expression level of hSMN1 in tissues other than the spinal cord, such as the liver and heart.

[0768] The present invention further provides a host cell comprising a polynucleotide, an expression construct and / or a vector of the present invention. In some embodiments, the host cell is a mammalian cell, such as a human cell. In some embodiments, the host cell is a HEK293T cell.

[0769] 4. Treatment of diseases

[0770] The present invention provides a method for treating a disease associated with SMN1 deficiency, comprising administering to a subject in need thereof a rAAV or a pharmaceutical composition of the present invention. In some embodiments, the disease is SMA, particularly type I SMA. In some embodiments, the rAAV or pharmaceutical composition is administered by intravenous injection.

[0771] The present invention further provides a use of a polynucleotide, expression construct, rAAV, pharmaceutical composition, vector or host cell of the present invention in the preparation of a medicament for treating a disease associated with SMN1 deficiency in a subject in need thereof. In some embodiments, the disease is SMA, particularly type I SMA. In some embodiments, the medicament is administered by intravenous injection.

[0772] The rAAV and / or pharmaceutical composition of the present invention is provided for use in treating a disease associated with SMN1 deficiency in a subject in need thereof. In some embodiments, the disease is SMA, particularly type I SMA. In some embodiments, the rAAV and / or pharmaceutical composition is administered by intravenous injection.

[0773] In some embodiments, the treatment increases hSMN1 activity in the subject. In some embodiments, the treatment increases hSMN1 activity in the spinal cord, thereby alleviating atrophy. In some embodiments, the subject is a human.

[0774] In some embodiments, the treatment cures, ameliorates, alleviates, blocks, or partially blocks the symptoms of SMA.

[0775] Implementation

[0776] Embodiment 1. A polynucleotide comprising a nucleotide sequence encoding an SMN1, preferably a human SMN1 (hSMN1) polypeptide, wherein the nucleotide sequence is codon-optimized for improved expression.

[0777] Option 2. The polynucleotide of Option 1, wherein the nucleotide sequence is as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or a variant thereof.

[0778] Option 3. The polynucleotide of Option 2, wherein the variant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0779] Option 4. The polynucleotide of any one of Option 1-3, wherein the SMN1 polypeptide is a hSMN1 polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more activity of the polypeptide of SEQ ID NO:21.

[0780] Item 5. The polynucleotide of any one of items 1 to 4, wherein the SMN1 polypeptide comprises SEQ ID NO: 21 or an allelic variant thereof.

[0781] Option 6. The polynucleotide of any one of Option 1-5, wherein the nucleotide sequence is as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 11, 16 or 19.

[0782] Option 7. The polynucleotide of any one of Option 1-6, wherein the nucleotide sequence is shown as SEQ ID NO: 3, 6, 8 or 11.

[0783] Option 8. The polynucleotide of any one of Option 1 to Option 7, wherein the nucleotide sequence is as shown in SEQ ID NO:11.

[0784] Scheme 9. An expression construct comprising the polynucleotide of any one of Schemes 1-8 operably linked to a promoter.

[0785] Option 10. The expression construct of Option 9, wherein the promoter comprises the nucleotide sequence of SEQ ID NO:23, 28 or 31 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23, 28 or 31.

[0786] Option 11. The expression construct of Option 9 or 10, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 31.

[0787] Scheme 12. The expression construct of any one of Schemes 9 to 11, wherein the construct further comprises an enhancer, preferably upstream of the promoter.

[0788] Option 13. The expression construct of Option 12, wherein the enhancer comprises the nucleotide sequence of SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22.

[0789] Scheme 14. The expression construct of any one of Schemes 9 to 13, wherein the construct further comprises an intron, preferably downstream of the promoter and upstream of the nucleotide sequence encoding the SMN1 polypeptide.

[0790] Option 15. The expression construct of Option 14, wherein the intron comprises the nucleotide sequence of SEQ ID NO:24 or 29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24 or 29.

[0791] Scheme 16. The expression construct of any one of Schemes 9 to 15, wherein the construct further comprises a polyadenylation signal sequence downstream of the nucleotide sequence encoding the SMN1 polypeptide.

[0792] Option 17. The expression construct of Option 16, wherein the polyadenylation signal sequence comprises the nucleotide sequence of SEQ ID NO:25 or 30 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25 or 30.

[0793] Embodiment 18. The expression construct of any one of embodiments 9 to 17, wherein the construct comprises, in 5' to 3' order, an enhancer, a promoter, an intron, a polynucleotide of interest encoding hSMN1, and a polyadenylation signal sequence.

[0794] Scheme 19. The expression construct of any one of Schemes 9 to 18, wherein the construct comprises, in 5' to 3' order,

[0795] i) SEQ ID NO:31, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:25;

[0796] ii) SEQ ID NO:31, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:25;

[0797] iii) SEQ ID NO:31, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:30;

[0798] iv) SEQ ID NO:31, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:30;

[0799] v) SEQ ID NO:28, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:25;

[0800] vi) SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:25;

[0801] vii) SEQ ID NO:28, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:30; or

[0802] viii) SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:30.

[0803] Scheme 20. A recombinant adeno-associated virus (rAAV) comprising a genome comprising the expression construct of any one of Schemes 9-19, flanked by 5' and 3' ITRs.

[0804] Scheme 21. The rAAV of Scheme 20, wherein the rAAV is selected from human serotype 1 AAV (hAAV1), hAAV2, hAAV3, hAAV4, hAAV5, hAAV6, hAAV7, hAAV8, hAAV9, hAAV10 and hAAV11, preferably, the rAAV is hAAV9.

[0805] Item 22. The rAAV of Item 20 or 21, wherein the genome of the rAAV, from the 5' end of the 5' ITR to the 3' end of the 3' ITR, has a length of about 4,700 nucleotides, such as 4,600-4,800, 4,620-4,780, 4,640-4,760, 4,650-4,750, 4,660-4,740, 4,670-4,730, 4,680-4,720 or 4,690-4,710 nucleotides.

[0806] Option 23. The rAAV of any one of Option 20-22, wherein the genome of the rAAV comprises a stuffer fragment, preferably downstream of the polyadenylation signal sequence.

[0807] Item 24. The rAAV of any one of Items 20-23, wherein the 5'ITR comprises SEQ ID NO:26.

[0808] Item 25. The rAAV of any one of Items 20-24, wherein the 3'ITR comprises SEQ ID NO: 27.

[0809] Item 26. The rAAV of any one of items 20-24, wherein the genome of the rAAV comprises, in 5' to 3' order,

[0810] i) SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27;

[0811] ii) SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27;

[0812] iii) SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:30, SEQ ID NO:27;

[0813] v) SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27;

[0814] vi) SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:27;

[0815] vii) SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:24, SEQ ID NO:11, SEQ ID NO:30, SEQ ID NO:27; or

[0816] viii) SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:30, SEQ ID NO:27.

[0817] Option 27. A vector comprising the genome of the rAAV of any one of Option 20-26.

[0818] Scheme 28. The vector of Scheme 27, which is a packaging plasmid used to package the rAAV.

[0819] Scheme 29. A host cell comprising the polynucleotide of any one of Schemes 1-8, the expression construct of any one of Schemes 9-19, the rAAV of any one of Schemes 20-26, or the vector of Scheme 27 or 28.

[0820] Scheme 30. The vector of Scheme 29, wherein the host cell is a mammalian cell, such as a human cell.

[0821] Scheme 31. The vector of Scheme 29 or 30, wherein the host cell is a HEK293T cell.

[0822] Embodiment 32. A pharmaceutical composition comprising the rAAV of any one of Embodiments 20-26.

[0823] Embodiment 33. The pharmaceutical composition of embodiment 32, further comprising a pharmaceutically acceptable carrier or excipient.

[0824] Embodiment 34. The pharmaceutical composition of embodiment 32 or 33, wherein the pharmaceutical composition is formulated for parenteral administration, such as intravenous, intramuscular or subcutaneous injection.

[0825] Embodiment 35. The pharmaceutical composition of any one of embodiments 32-34, wherein the pharmaceutical composition is formulated for topical administration.

[0826] Item 36. The rAAV of any one of Items 20-26 or the pharmaceutical composition of any one of Items 32-35, for treating a disease associated with SMN1 in a subject in need thereof.

[0827] Embodiment 37. A method for treating a disease associated with SMN1, comprising administering the rAAV of any one of Embodiments 20-26 or the pharmaceutical composition of any one of Embodiments 32-35 to a subject in need thereof.

[0828] Scheme 38. Use of the polynucleotide of any one of Schemes 1-8, the expression construct of any one of Schemes 9-19, the rAAV of any one of Schemes 20-26, the vector of Scheme 27 or 28, the host cell of any one of Schemes 29-31, or the pharmaceutical composition of any one of Schemes 32-35 in the preparation of a medicament for treating a disease associated with SMN1 in a subject in need thereof.

[0829] Item 39. The rAAV or pharmaceutical composition for use according to Item 36, the method of Item 37, or the use of Item 38, wherein the disease is SMA, particularly type I SMA.

[0830] 40. The rAAV or pharmaceutical composition for use according to 36, the method of 37, or the use of 38, wherein the subject is a human.

[0831] 41. The polynucleotide of any one of claims 1 to 9, the expression construct of any one of claims 10 to 19, or the rAAV of any one of claims 20 to 26, which achieves higher expression of SMN1 polypeptide and / or mRNA in the spinal cord, and / or lower expression of SMN1 polypeptide and / or mRNA in non-spinal cord tissues (e.g., heart and / or liver) compared to a polynucleotide, expression construct, or rAAV comprising the nucleotide sequence of SEQ ID NO: 1. Example

[0832] The following examples are provided for illustration only and are not intended to limit the present application in any way.

[0833] Example 1. Expression of hSMN1 in 293T cells

[0834] 1.1. Construction of transgenic plasmids

[0835] By Gibson assembly, the pGCB108 plasmid containing the cytomegalovirus early (CMVen) enhancer (SEQ ID NO: 22) and the chicken β-actin core (CB) promoter (SEQ ID NO: 23) was assembled with the PCI intron (SEQ ID NO: 24) and the coding sequence (SEQ ID NO: 2-20 or 36), and 20 constructs containing rAAV genomes were prepared, wherein the genome contained the codon-optimized coding sequence (CoSMN) of hSMN1 or the coding sequence of GFP. The Zolgensma construct (see WO 2020 / 113034) was introduced into the pGCB108 plasmid by Gibson assembly. The structure of the construct is shown in Figure 1 The transgenic plasmids are respectively referred to as plasmid 2-20 (comprising SEQ ID NOs: 2-20, respectively), plasmid Zol (comprising the Zolgensma construct), and plasmid GFP (comprising the GFP coding sequence).

[0836] As an example, Figure 2 A shows a transgenic plasmid map comprising the optimized coding sequence of hSMN1 of SEQ ID NO:2, and its sequence is shown as SEQ ID NO:37.

[0837] 1.2. Transfection of 293T cells

[0838] 293T cells (ATCC CRL-3216 TM) were cultured at 37°C and 5% CO2 according to the manufacturer's protocol. One day before transfection, 293T cells were plated at 5×10 5 Cells were seeded into 12-well tissue culture plates at a density of 10 cells / well. HD transfection reagent), and transfection was performed with the transgenic plasmids constructed in Example 1.1. Cell pellets were collected 72 hours after transfection. When transfection was performed, one-tenth of the amount of fLuc2 plasmid (see Figure 2 D) Served as a control for transfection efficiency.

[0839] Western blotting

[0840] The infected cells were treated with M-PER TM Mammalian protein extraction reagent (Thermo Fisher, catalog number 78501) was used for lysis. Equal amounts of lysate (10 μg) were loaded onto NuPAGE 4–12% Bis-Tris gels for PAGE (1 μl reducing agent + 9 μl 4Xli-cor protein loading solution + 30 μl supernatant, 200 V, 22 min). 2GelTransfer Device (Thermo Fisher Scientific) and 2Transfer Stack (nitrocellulose, regular size) was used to transfer the protein to the nitrocellulose membrane. After incubation with blocking buffer (8% milk in 1XTBST) for 1 hour and rinsing 3 times (1 min each time), the membrane was incubated with primary antibodies (SMN1 monoclonal antibody (2F1), Thermo MA5-15857, and Thermo α-tubulin polyclonal antibody, PA1-38814) diluted 1:2000 overnight at 4°C, and then rinsed 3 times (1 min each time). Then incubate with donkey anti-mouse HRP (Thermo Fisher, catalog number A16011) (1:5000 dilution) at RT for 2 hours. The immune reaction was observed using the Biorad ChemiDoc imaging system. Western blot images of hSMN1 are shown in Figure 2. Figure 3 Scanned images were quantified using ImageJ software.

[0841] Luciferase assay

[0842] Promega The luciferase assay system was used to detect fLuc2 activity. 10 μl of the transfected cell lysate (10 μg) was diluted in 90 μl of OptiMEM medium and 100 μl of Luciferase substrate was added to a black 96-well plate (CorningTM 3991, Corning TM 96-well, flat-bottom polystyrene NBS microplate without lid). The plate was shaken at RT for at least 5 minutes to mix, and then luminescence was detected in a SpectraMax luminometer (Molecular Devices SpectraMax i3x Multi-Mode Detection Platform, Manufacturer: Molecular Devices, Model: SpectraMax I3x). The luminescence of each transfected cell sample was as follows: Figure 4 shown.

[0843] The Western blot data were normalized according to luciferase activity. Figure 5 Eight of the codon-optimized sequences (CoSMN4, CoSMN5, CoSMN6, CoSMN7, CoSMN8, CoSMN11, CoSMN16, and CoSMN19) showed 1.5-4 times higher expression of hSMN1 than Zolgensma, while six of the codon-optimized sequences (CoSMN2, CoSMN 9, CoSMN 12, CoSMN 13, CoSMN 17, and CoSMN 20) showed relatively lower expression of hSMN1.

[0844] Example 2. Preparation of rAAV

[0845] rAAV vectors were prepared similarly to those described by Crosson SM et al. 2018. Briefly, 293VPC cells (Thermo, catalog number A35347) cultured at a density of 3E6 cells / ml in serum-free virus production medium OPM-293CD05 (Shanghai OPM Bioscience Co., Ltd., catalog number 81075-001) were triple-transfected with polyethyleneimine using the following helper plasmids, a packaging plasmid encoding rep / cap, and a transgene plasmid:

[0846] - Helper plasmid ("pHelper", containing Ad E2A, E4 and VARNA helper genes, as described by Crosson Sm et al., and its map is shown in Figure 2 B);

[0847] -Packaging plasmid, the map of which is as follows Figure 2 C, wherein "AAV9 Cap" is a nucleotide sequence encoding the Cap polypeptide of AAV9 (SEQ ID NO: 34), and "AAV2 Rep" is a nucleotide sequence encoding the Rep polypeptide of AAV2 (SEQ ID NO: 35);

[0848] - Transgenic plasmids, such as plasmids 3, 4, 5, 6, 7, 8, 10, 11, 14, 15, 16, 18 and 19 constructed in Example 1.1, and plasmid Zol.

[0849] After incubation at 37°C for 72 h, cells were harvested and viral particles were purified by the iodixanol gradient method (see Crosson SM et al.).

[0850] The resulting rAAVs were named SMN3, SMN4, SMN5, SMN6, SMN7, SMN8, SMN10, SMN11, SMN14, SMN15, SMN16, SMN18, SMN19, and Zolgensma, and the titers were tested by ddPCR, all of which were 10 13 Viral genome (vg) / mL level. Specifically, ddPCR was performed using Bio-Rad's QXDx AutoDG ddPCR System and the QXDx Universal Kit for AutoDG ddPCR System according to the manufacturer's instructions. The primers and probes for ddPCR were as follows:

[0851] Forward primer: CCCACTTGGCAGTACATCAA (SEQ ID NO: 38)

[0852] Reverse primer: GCCAAGTAGGAAAGTCCCATAA (SEQ ID NO: 39)

[0853] Probe: CATAATGCCAGGCGGGCCATTTAC (SEQ ID NO: 40).

[0854] Example 3. rAAV-mediated hSMN1 expression

[0855] This example was conducted to verify the expression of hSMN1 contained in the rAAV prepared in Example 2, and the results showed that the expression of hSMN1 was increased in the rAAV containing the codon-optimized hSMN1 coding sequence of the present invention.

[0856] HEK293T cells were infected with the rAAV prepared in Example 2 at an MOI of 1E6 and incubated at 37°C for 48 hours.

[0857] The infected cells were stained with M-PER TM Lysis was performed with Mammalian Protein Extraction Reagent (Thermo Fisher, catalog number 78501). Equal amounts of lysate (10 μg) were loaded onto SDS-PAGE gels and run at 200 V for 22 min. 2Gel Transfer Device (Thermo Fisher Scientific) and 2TransferStack (PVDF, regular size) was used to transfer the proteins to the PVDF membrane. After incubation with blocking buffer (8% milk in 1XTBST) for 1 hour and rinsing 3 times (1 min each time), the membrane was incubated with the primary antibody (Invitrogen MAS-15857 (2B1) SMN1 antibody (1:2,000 dilution)) in the same buffer at 4°C overnight, and then rinsed 3 times (1 min each time). 800CW donkey anti-mouse IgG (Licor; P / N: 926-32212), (1:10,000 dilution) was incubated for 2 h at RT. β-actin served as a housekeeping gene control for quantification of hSMN1 relative expression. DLx Imaging System for Visualizing Immune Responses. The software collects, organizes and analyzes scanned images.

[0858] like Figure 6 As shown, codon optimization was indeed effective, with relative expression of rAAV SMN4, 7, 11, 16, and 19 being 3- to 5-fold higher than that of Zolgensma in in vitro infection studies.

[0859] Example 4. rAAV-mediated hSMN1 expression in vivo in WT mice

[0860] This example was conducted to evaluate the in vivo expression level of hSMN1 mediated by the rAAV prepared in Example 2.

[0861] SMN3, SMN5, SMN6, SMN8, SMN10, SMN11, SMN14, SMN15, SMN16, SMN18, SMN19 and Zolgensma were injected into FVB mice (https: / / www.jax.org / strain / 005025) at a single intravenous (IV) dose of 5e10 vg / mouse (in PBS, n=5) on postnatal day (PND) 1-2. Heart, liver and spinal cord tissues were collected on day 28 after injection for evaluation of hSMN1 protein levels.

[0862] Using T-PER TM The tissue was lysed with tissue protein extraction reagent (Thermo, catalog number 78510), and 100 μl of proteinase inhibitor (Pierce TMProtease inhibitor mini tablets, EDTA-free, catalog number A32955) were added to T-PER reagent and then operated according to the instructions of catalog number 78510. As described in Example 3, 50 μg of tissue lysate was loaded for SDS-PAGE and subsequent Western blot analysis.

[0863] like Figure 7 As shown, rAAV SMN3 and SMN11 showed the lowest expression in the heart ( Figure 7 A); rAAV containing codon-optimized constructs expressed hSMN1 in the liver similarly to Zolgensma ( Figure 7 B); rAAV SMN11 showed the highest expression in the spinal cord ( Figure 7 C).

[0864] Example 5. rAAV-mediated hSMN1 expression in vivo in the SMA model

[0865] This example was conducted to investigate the expression level of hSMN1 mediated by rAAV in the SMNΔ7 mouse model, a widely used SMA model.

[0866] Newborn SMNΔ7 mice (https: / / www.jax.org / strain / 005025) were injected with a single IV dose of 5e10 vg / mouse (in PBS) of rAAV SMN6, SMN8, and SMN11 and Zolgensma (n=7) on PND1-2.

[0867] 14 days after injection, heart, liver and spinal cord tissues were collected from injected mice and hSMN1 protein levels were assessed by Western blotting as described in Example 4.

[0868] like Figure 8 As shown, rAAV SMN6, SMN8, and SMN11 achieved higher levels of hSMN1 expression in the spinal cord and significantly lower levels of hSMN1 expression in the liver and heart compared to Zolgensma, and rAAV SMN11 achieved the highest hSMN1 expression in the spinal cord. It is well known that overexpression of SMN1 in the liver and heart may cause adverse effects, so SMN11 was selected as the best codon-optimized hSMN1 for further development.

[0869] Example 6. Optimization of other elements in the rAAV genome

[0870] This example was conducted to obtain an improved rAAV based on rAAV SMN11 for the treatment of SMA.

[0871] 6.1. Construction of modified transgenic plasmids

[0872] The modified transgenic plasmid was constructed by Gibson assembly by replacing elements such as promoter, intron and polyA signal in plasmid 11. The elements in plasmid 11 and the modified transgenic plasmid are shown in Table 1.

[0873] Table 1. Modified transgenic plasmids

[0874]

[0875] 6.2. Preparation of rAAV

[0876] rAAV vectors were prepared according to a method similar to that described in Example 2, with plasmids 11-1, 11-2, 11-3 and 11-4 as transgenic plasmids, and the resulting rAAVs were named SMN11-1, SMN11-2, SMN11-3 and SMN11-4, respectively.

[0877] 6.3. rAAV-mediated hSMN1 expression in vivo

[0878] On PND1-2, newborn FVB mice were injected IV with rAAV SMN11-1, SMN11-2, SMN11-3 and SMN11-4 and Zolgensma (single dose). The dosing schedule is shown in Table 2 (n=6).

[0879] Table 2. Dosage regimen

[0880]

[0881] On day 28 after injection, tissues (heart, liver, and spinal cord) were collected and analyzed for the expression level of hSMN1. Specifically, the mRNA level of hSMN1 was detected. The RT-qPCR method used has been developed and validated with samples from major peripheral and central nervous system tissues. Total RNA was extracted using the QIAGEN RNeasy Mini kit with slight modifications (Qiazol chloroform was used for extraction). The total RNA concentration was determined by Nanodrop. For RT-qPCR analysis, Fast-RT-PCR was performed using 1 μg of total RNA and QuantiTect Reverse Transcription Kit 400 (Catalog No. 205314, including genomic DNA removal step). When performing qPCR, cDNA was mixed with qPCR reaction mixture (TaqMan Gene Expression Master Mix, 4369016), primers and probes (see Table 3), which correspond to the codon-optimized hSMN1 transgenic sequence (CoSMN11) and the housekeeping gene GAPDH. Briefly, qPCR was performed in triplicate in 10 μl reactions in 384-well plates using 120 ng of total cDNA, TaqMan Gene Expression Master Mix (Thermos Fisher Scientific), 900 nM of each primer, and 450 nM of probe. TM The reaction was performed using a real-time PCR system. The results were presented as the relative mRNA expression level of hSMN1 normalized to the housekeeping gene GAPDH.

[0882] Table 3. Primers and probes for qPCR

[0883]

[0884] like Fig. 9 As shown, the results indeed showed that these combinations had a significant effect on the expression of hSMN1 in different tissues. In particular, rAAV containing promoter 2 achieved higher hSMN1 expression in the spinal cord, and rAAV containing promoter 2 and intron 2 achieved the highest expression, which was about 2-3 times that of Zolgensma; rAAV containing promoter 1 expressed higher levels than Zolgensma, but lower than rAAV containing promoter 2 ( Fig. 9 C). In the liver and heart, the rAAV of the present invention showed lower but significant expression levels than Zolgensma ( Fig. 9 A and 9B).

[0885] Since overexpression of hSMN1 in the heart and liver may lead to adverse effects, lower but functional expression in the heart and liver would be safer while still effectively rescuing the SMA phenotype.

[0886] In addition, the expression level of hSMN1 mRNA is dose-dependent, and at the same dose, the expression level is higher than that of Zolgensma, which suggests that lower doses can be used to transduce motor neurons in the spinal cord, reduce their degeneration and death, and thereby delay the onset of the disease or even cure the disease.

[0887] Based on the above results, the combination of CoSMN11 (SEQ ID NO: 11), Promoter 2 (SEQ ID NO: 31) and Intron 2 (SEQ ID NO: 29) was proposed as a lead transgene.

[0888] sequence

[0889] SEQ ID NO:1WT hSMN1

[0890] ATGGCGATGAGCAGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAGGAGGATTCCGTGCTGTTCCGGCGCGGCACAGGCCAGAGCGATGATTCTGACATTTGGGATGATACAGCACTGATAAAAGCATATGATAAAGCTGTGGCTTCATTTAAGCATGCTCTAAAGAATGGTGACATTTGTGAAACTTCGGGTAAACCAAAAACCACACCTAAAAGAAAACCTGCTAAGAAGAATAAAAGCCAAAAGAAGAATACTGCAGCTTCCTTACAACAGTGGAAAGTTGGGGACAAATGTTCTGCCATTTGGTCAGAAGACGGTTGCATTTACCCAGCTACCATTGCTTCAATTGATTTTAAGAGAGAAACCTGTGTTGTGGTTTACACTGGATATGGAAATAGAGAGGAGCAAAATCTGTCCGATCTACTTTCCCCAATCTGTGAAGTAGCTAATAATATAGAACAGAATGCTCAAGAGAATGAAAATGAAAGCCAAGTTTCAACAGATGAAAGTGAGAACTCCAGGTCTCCTGGAAATAAATCAGATAACATCAAGCCCAAATCTGCTCCATGGAACTCTTTTCTCCCTCCACCACCCCCCATGCCAGGGCCAAGACTGGGACCAGGAAAGCCAGGTCTAAAATTCAATGGCCCACCACCGCCACCGCCACCACCACCACCCCACTTACTATCATGCTGGCTGCCTCCATTTCCTTCTGGACCACCAATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGAAGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATGGGTTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATTAA

[0891] SEQ ID NO:2CoSMN2

[0892] ATGGCGATGAGCAGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAGGAGGATTCCGTGCTGTTCCGGCGCGGCACAGGCCAGAGCGATGATTCTGACATTTGGGATGATACAGCACTGATAAAAGCATATGATAAAGCTGTGGCTTCATTTAAGCATGCTCTAAAGAATGGTGACATTTGTGAAACTTCGGGTAAACCAAAAACCACACCTAAAAGAAAACCTGCTAAGAAGAATAAAAGCCAAAAGAAGAATACTGCAGCTTCCTTACAACAGTGGAAAGTTGGGGACAAATGTTCTGCCATTTGGTCAGAAGACGGTTGCATTTACCCAGCTACCATTGCTTCAATTGATTTTAAGAGAGAAACCTGTGTTGTGGTTTACACTGGATATGGAAATAGAGAGGAGCAAAATCTGTCCGATCTACTTTCCCCAATCTGTGAAGTAGCTAATAATATAGAACAAAATGCTCAAGAGAATGAAAATGAAAGCCAAGTTTCAACAGATGAAAGTGAGAACTCCAGGTCTCCTGGAAATAAATCAGATAACATCAAGCCCAAATCTGCTCCATGGAACTCTTTTCTCCCTCCACCACCCCCCATGCCAGGGCCAAGACTGGGACCAGGAAAGCCAGGTCTAAAATTCAATGGCCCACCACCGCCACCGCCACCACCACCACCCCACTTACTATCATGCTGGCTGCCTCCATTTCCTTCTGGACCACCAATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGAAGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATGGGTTTCAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATTAA

[0893] SEQ ID NO:3CoSMN3

[0894] ATGGCAATGTCTTCCGGTGGATCTGGGGGCGGAGTCCCAGAACAGGAAGACTCCGTATTGTTCAGAAGAGGGACAGGGCAATCTGACGACTCCGATATTTGGGATGACACGGCCCTCATTAAGGCATATGACAAGGCAGTGGCCAGTTTCAAACATGCTCTGAAAAATGGCGACATTTGCGAAACATCCGGGAAGCCGAAGACAACACCAAAACGTAAGCCCGCAAAGAAAAATAAGTCTCAGAAGAAGAACACTGCTGCCAGTCTTCAGCAGTGGAAAGTGGGAGACAAATGTTCCGCAATCTGGTCTGAGGACGGGTGTATTTACCCCGCCACGATCGCTTCTATCGACTTTAAGAGAGAAACTTGTGTGGTGGTGTATACCGGCTACGGGAACCGCGAAGAGCAAAACCTGTCCGACCTGCTGTCCCCCATCTGCGAGGTGGCCAACAATATCGAGCAGAACGCTCAGGAGAACGAAAACGAGAGTCAAGTGTCTACAGATGAAAGCGAGAATTCTCGGTCTCCAGGAAATAAATCTGACAATATCAAACCGAAAAGCGCCCCCTGGAATTCCTTTTTGCCCCCTCCACCTCCAATGCCTGGGCCTAGACTGGGTCCTGGGAAGCCCGGCCTCAAGTTCAATGGACCGCCTCCCCCACCCCCCCCTCCCCCGCCCCATCTCCTCAGTTGTTGGCTCCCCCCCTTCCCATCCGGGCCCCCAATCATTCCTCCGCCGCCCCCAATTTGTCCAGACAGCCTGGACGACGCTGACGCTTTAGGGTCCATGCTCATCTCTTGGTATATGAGTGGCTATCATACCGGTTATTACATGGGTTTCAGGCAGAACCAGAAGGAGGGCCGGTGTTCGCATAGCCTAAATTAG

[0895] SEQ ID NO:4CoSMN4

[0896] ATGGCCATGAGCAGCGGCGGATCTGGCGGCGGCGTGCCTGAGCAGGAGGATAGCGTGCTGTTCCGGAGAGGCACCGGACAGTCCGATGATTCTGACATCTGGGACGACACCGCCCTGATCAAGGCCTACGACAAGGCCGTGGCTTCCTTCAAGCACGCTCTGAAAAATGGCGACATCTGTGAAACCAGCGGCAAGCCCAAGACAACCCCTAAGAGAAAGCCTGCCAAGAAAAACAAGTCTCAAAAGAAGAACACCGCCGCTTCTCTGCAGCAGTGGAAGGTGGGCGATAAGTGCAGCGCTATCTGGTCCGAGGACGGCTGTATCTACCCTGCCACCATCGCCAGCATCGACTTCAAGCGGGAAACATGCGTGGTGGTCTACACAGGCTACGGCAACCGGGAAGAACAAAACCTGAGCGATCTGCTCAGCCCTATCTGCGAGGTGGCCAACAACATCGAGCAGAATGCCCAGGAAAACGAGAATGAAAGCCAGGTTTCCACCGACGAGTCTGAGAACAGCCGCAGCCCCGGAAACAAGAGCGACAACATCAAGCCTAAGTCCGCCCCTTGGAACAGCTTTCTGCCTCCTCCGCCCCCCATGCCTGGCCCTAGACTGGGCCCCGGCAAACCTGGACTGAAATTCAACGGCCCCCCCCCACCCCCCCCTCCTCCTCCACCTCACCTGCTGAGCTGCTGGCTGCCTCCCTTCCCTTCTGGGCCTCCAATTATCCCTCCACCTCCTCCTATCTGCCCCGACAGCCTGGACGACGCCGACGCCCTGGGCAGCATGCTGATCAGCTGGTACATGAGCGGCTACCACACAGGCTACTACATGGGCTTTAGACAGAACCAGAAAGAGGGCAGATGCAGCCATTCTCTGAATTGA

[0897] SEQ ID NO:5CoSMN5

[0898] ATGGCCATGTCCTCCGGTGGGTCAGGTGGGGGGGTCCCTGAACAGGAAGACAGTGTCTTGTTTCGACGCGGGACGGGCCAAAGTGACGATTCTGATATTTGGGACGATACCGCACTCATTAAGGCTTACGATAAGGCTGTGGCGAGCTTCAAGCACGCTTTGAAGAACGGAGATATTTGCGAGACGTCTGGCAAGCCGAAAACAACCCCGAAGCGCAAGCCCGCTAAGAAGAATAAGTCCCAGAAAAAAAACACAGCTGCCAGTCTTCAGCAGTGGAAGGTGGGAGATAAATGCTCTGCCATATGGTCTGAGGATGGGTGTATTTACCCGGCAACAATCGCCAGCATCGACTTCAAACGAGAAACGTGCGTGGTCGTTTATACCGGGTATGGGAACAGGGAGGAACAGAACCTCAGCGATCTTCTTTCTCCCATTTGTGAGGTGGCCAACAACATCGAACAAAATGCGCAGGAAAATGAGAATGAGAGTCAAGTGTCTACCGATGAAAGTGAAAATTCTCGCAGCCCGGGAAACAAGTCTGACAATATCAAGCCCAAGTCTGCACCATGGAACAGTTTCTTGCCGCCTCCGCCACCGATGCCGGGGCCTAGATTGGGGCCGGGCAAACCGGGACTTAAGTTCAACGGACCCCCTCCACCGCCCCCTCCTCCGCCACCGCACTTGTTGTCATGTTGGCTCCCTCCATTTCCCAGTGGTCCTCCTATCATACCACCTCCTCCGCCGATTTGTCCCGATAGCCTTGATGACGCTGATGCTTTGGGATCCATGCTTATCTCTTGGTATATGTCAGGATACCATACCGGGTATTACATGGGATTCAGACAGAACCAGAAAGAGGGGCGGTGCAGCCACTCCCTCAACTGA

[0899] SEQ ID NO:6CoSMN6

[0900] ATGGCCATGTCTAGTGGCGGATCTGGTGGCGGAGTGCCCGAGCAAGAAGATAGCGTCCTGTTCAGAAGAGGCACCGGCCAGAGCGACGACAGCGACATCTGGGATGATACAGCCCTGATCAAGGCCTACGACAAGGCCGTGGCCAGCTTTAAGCACGCCCTGAAGAACGGCGATATCTGCGAGACAAGCGGCAAGCCCAAGACCACACCTAAGAGAAAGCCCGCCAAGAAGAACAAGAGCCAGAAGAAGAATACCGCCGCCAGCCTGCAGCAGTGGAAAGTGGGCGATAAGTGCAGCGCCATTTGGAGCGAGGACGGCTGTATCTACCCTGCCACAATCGCCAGCATCGACTTCAAGCGGGAAACCTGCGTGGTGGTGTACACAGGCTACGGCAACAGAGAGGAACAGAACCTGAGCGACCTGCTGAGCCCAATTTGCGAGGTGGCCAACAACATCGAGCAGAACGCCCAAGAGAACGAGAACGAGTCCCAGGTGTCCACCGACGAGAGCGAGAATAGCAGAAGCCCCGGCAACAAGAGCGACAACATCAAGCCTAAGAGCGCCCCTTGGAACAGCTTCCTGCCTCCTCCTCCACCAATGCCTGGACCTAGACTCGGACCTGGAAAGCCCGGCCTGAAGTTCAATGGACCTCCACCACCGCCACCACCTCCGCCTCCACATCTTCTGTCTTGTTGGCTGCCTCCATTTCCTAGCGGCCCTCCAATCATCCCGCCACCTCCACCTATCTGCCCCGACAGTCTGGATGATGCTGATGCCCTGGGCTCCATGCTGATCTCTTGGTACATGAGCGGCTACCACACCGGCTACTACATGGGCTTCAGACAGAACCAGAAAGAGGGCCGTTGCAGCCACAGCCTGAACTGA

[0901] SEQ ID NO:7 CoSMN7

[0902] ATGGCTATGAGCAGCGGAGGAAGCGGAGGCGGAGTGCCCGAACAAGAGGATTCCGTGCTGTTTAGAAGGGGAACCGGACAGTCCGATGATAGCGACATCTGGGACGACACCGCTCTGATCAAGGCCTACGACAAAGCCGTGGCCTCCTTCAAGCATGCTCTGAAGAACGGCGACATCTGCGAAACCAGCGGCAAGCCCAAAACAACCCCCAAGAGGAAGCCCGCCAAGAAGAACAAGTCCCAGAAGAAGAACACCGCCGCTTCTCTGCAGCAGTGGAAGGTGGGCGACAAGTGTAGCGCCATCTGGTCCGAGGATGGCTGCATCTATCCCGCCACCATTGCCTCCATCGACTTCAAGAGGGAGACATGCGTGGTGGTGTACACCGGCTACGGCAATAGAGAGGAACAGAATCTGTCCGATCTGCTGAGCCCTATCTGCGAGGTGGCCAACAACATTGAGCAGAACGCCCAAGAGAACGAGAACGAAAGCCAAGTGTCCACCGACGAGAGCGAAAACAGCAGAAGCCCCGGCAACAAGAGCGACAACATCAAGCCCAAGTCCGCCCCTTGGAACTCCTTTCTGCCCCCTCCTCCCCCTATGCCCGGCCCTAGACTGGGACCCGGCAAGCCCGGACTGAAGTTCAACGGACCCCCCCCTCCCCCCCCCCCCCCTCCTCCTCATCTGCTGAGCTGTTGGCTCCCTCCTTTCCCCAGCGGCCCTCCTATCATCCCTCCTCCTCCTCCCATCTGCCCCGATTCTCTGGATGATGCCGATGCTCTGGGCAGCATGCTGATCAGCTGGTACATGAGCGGCTATCACACCGGCTACTACATGGGCTTCAGACAGAACCAGAAGGAGGGCAGATGCTCCCACTCTCTGAACTGA

[0903] SEQ ID NO:8 CoSMN8

[0904] ATGGCTATGTCCTCTGGAGGGTCCGGCGGAGGCGTGCCGGAGCAGGAAGACTCCGTTCTGTTCCGCCGTGGCACAGGGCAGTCAGACGATTCCGACATCTGGGATGACACTGCCCTCATCAAGGCCTACGATAAGGCTGTCGCTAGCTTTAAGCATGCCCTGAAGAACGGAGACATCTGTGAGACATCCGGTAAGCCCAAGACCACGCCCAAGCGGAAGCCCGCCAAAAAGAACAAGAGTCAGAAAAAGAACACCGCTGCATCTCTTCAACAGTGGAAAGTGGGCGACAAGTGCTCCGCTATCTGGAGCGAGGACGGGTGCATCTATCCAGCTACCATCGCATCTATCGACTTTAAGCGCGAGACCTGCGTAGTGGTCTATACTGGTTACGGCAATAGGGAGGAACAGAACTTGTCTGATCTTCTGTCCCCGATCTGCGAAGTGGCCAATAACATTGAACAAAACGCCCAGGAGAACGAGAACGAGAGCCAGGTCTCCACGGACGAGAGCGAGAACAGTCGCTCCCCTGGCAACAAGTCCGACAACATCAAACCGAAGTCTGCGCCCTGGAACTCATTCCTCCCACCTCCGCCCCCAATGCCTGGTCCCCGCCTGGGCCCCGGCAAGCCGGGGCTCAAGTTTAATGGACCTCCCCCGCCCCCACCCCCACCCCCTCCCCACCTGCTCTCTTGCTGGTTGCCCCCATTCCCCTCCGGGCCACCTATCATTCCCCCACCGCCTCCAATCTGCCCAGACTCTCTGGACGATGCCGATGCCCTGGGCTCTATGCTGATCTCCTGGTATATGTCCGGCTATCATACCGGCTATTACATGGGATTCCGCCAGAACCAAAAGGAGGGCCGCTGTAGCCACTCCCTGAACTGA

[0905] SEQ ID NO:9 CoSMN9

[0906] ATGGCGATGAGCAGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAGGAGGATTCCGTGCTGTTCCGGCGCGGCACAGGCCAGAGCGATGATTCTGACATTTGGGATGATACAGCACTGATAAAAGCATATGATAAAGCTGTGGCTTCATTTAAGCATGCTCTAAAGAATGGTGACATTTGTGAAACTTCGGGTAAACCAAAAACCACACCTAAAAGAAAACCTGCTAAGAAGAATAAAAGCCAAAAGAAGAATACTGCAGCTTCCTTACAACAGTGGAAAGTTGGGGACAAATGTTCTGCCATTTGGTCAGAAGACGGTTGCATTTACCCAGCTACCATTGCTTCAATTGATTTTAAGAGAGAAACCTGTGTTGTGGTTTACACTGGATATGGAAATAGAGAGGAGCAAAATCTGTCCGATCTACTTTCCCCAATCTGTGAAGTAGCTAATAATATAGAACAGAATGCTCAAGAGAATGAAAATGAAAGCCAAGTTTCAACAGATGAAAGTGAGAACTCCAGGTCTCCTGGAAATAAATCAGATAACATCAAGCCCAAATCTGCTCCATGGAACTCTTTTCTCCCTCCACCACCCCCCATGCCAGGGCCAAGACTGGGACCAGGAAAGCCAGGTCTAAAATTCAATGGCCCACCACCGCCACCGCCACCACCACCACCCCACTTACTATCATGCTGGCTGCCTCCATTTCCTTCTGGACCACCAATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGAAGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATGGGTTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATTAA

[0907] SEQ ID NO:10 CoSMN10

[0908] ATGGCCATGTCGTCCGGAGGCTCCGGTGGTGGGGTGCCTGAGCAAGAGGATTCCGTCCTCTTCAGGCGCGGGACCGGGCAAAGTGATGACTCGGATATCTGGGACGATACTGCCTTAATCAAAGCCTACGATAAAGCCGTTGCTAGCTTTAAACACGCTCTGAAGAATGGCGACATCTGCGAGACAAGTGGGAAGCCCAAGACCACCCCAAAGCGGAAGCCTGCAAAAAAGAATAAGTCACAGAAAAAAAACACAGCCGCGAGCCTGCAGCAGTGGAAGGTCGGCGATAAGTGTTCTGCCATTTGGTCTGAGGACGGTTGTATCTACCCTGCGACCATCGCCTCAATTGACTTTAAACGGGAGACCTGCGTGGTAGTCTACACAGGCTACGGCAACCGCGAGGAACAGAACCTGTCAGATTTGCTTAGCCCGATCTGTGAGGTGGCCAATAACATTGAACAGAATGCACAGGAAAATGAAAATGAATCCCAGGTGAGCACCGACGAGTCTGAAAATTCTCGGTCCCCCGGTAATAAATCGGACAATATAAAACCAAAAAGCGCCCCGTGGAACTCTTTCCTCCCACCTCCTCCCCCCATGCCAGGGCCACGCCTTGGCCCAGGTAAGCCGGGCCTCAAGTTCAACGGCCCCCCACCCCCCCCCCCTCCCCCTCCCCCGCACTTACTGAGCTGTTGGCTACCCCCATTTCCATCAGGCCCACCCATCATTCCGCCACCCCCTCCCATCTGTCCTGATTCACTAGATGACGCGGATGCCCTCGGCTCTATGCTAATCTCGTGGTATATGAGCGGCTATCACACAGGTTACTACATGGGCTTCCGGCAAAATCAGAAAGAGGGCCGCTGCTCGCACTCTCTGAACTAA

[0909] SEQ ID NO:11 CoSMN11

[0910] ATGGCCATGAGCAGCGGCGGCAGCGGCGGCGGCGTGCCTGAGCAGGAGGACAGCGTGCTGTTCAGGAGGGGCACCGGCCAGAGCGACGACAGCGACATCTGGGACGACACCGCCCTGATCAAGGCCTACGACAAGGCCGTGGCCAGCTTCAAGCACGCCCTGAAGAACGGCGACATCTGCGAGACCAGCGGCAAGCCTAAGACCACCCCTAAGAGGAAGCCTGCCAAGAAGAACAAGAGCCAGAAGAAGAACACCGCCGCCAGCCTGCAGCAGTGGAAGGTGGGCGACAAGTGCAGCGCCATCTGGAGCGAGGACGGCTGCATCTACCCTGCCACCATCGCCAGCATCGACTTCAAGAGGGAGACCTGCGTGGTGGTGTACACCGGCTACGGCAACAGGGAGGAGCAGAACCTGAGCGACCTGCTGAGCCCTATCTGCGAGGTGGCCAACAACATCGAGCAGAACGCCCAGGAGAACGAGAACGAGAGCCAGGTGAGCACCGACGAGAGCGAGAACAGCAGGAGCCCTGGCAACAAGAGCGACAACATCAAGCCTAAGAGCGCCCCTTGGAACAGCTTCCTGCCTCCTCCTCCTCCTATGCCTGGCCCTAGGCTGGGCCCTGGCAAGCCTGGCCTGAAGTTCAACGGCCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCACCTGCTGAGCTGCTGGCTGCCTCCTTTCCCTAGCGGCCCTCCTATCATCCCTCCTCCTCCTCCTATCTGCCCTGACAGCCTGGACGACGCCGACGCCCTGGGCAGCATGCTGATCAGCTGGTACATGAGCGGCTACCACACCGGCTACTACATGGGCTTCAGGCAGAACCAGAAGGAGGGCAGGTGCAGCCACAGCCTGAACTGA

[0911] SEQ ID NO:12 CoSMN12

[0912] ATGGCTATGTCCAGCGGTGGATCTGGGGGCGGCGTTCCCGAGCAGGAGGACTCCGTGCTGTTCCGCAGGGGTACTGGCCAGAGTGATGACTCTGACATATGGGATGACACCGCCCTCATTAAGGCTTACGACAAAGCAGTGGCCAGTTTCAAGCACGCTCTGAAAAACGGTGACATTTGTGAGACTTCTGGCAAACCTAAAACCACTCCTAAAAGAAAACCCGCTAAGAAGAACAAATCACAAAAGAAGAATACAGCCGCCTCCTTACAGCAGTGGAAGGTGGGCGACAAGTGCTCAGCTATCTGGTCCGAGGACGGGTGCATTTACCCTGCCACCATCGCTTCAATCGATTTCAAAAGGGAGACCTGCGTGGTTGTGTATACTGGGTACGGAAATCGAGAAGAACAGAATCTGTCCGATCTGCTCTCCCCCATATGCGAGGTCGCAAACAACATAGAACAGAATGCACAGGAGAACGAAAATGAGTCACAGGTTTCAACTGATGAAAGTGAGAATAGTAGGTCCCCTGGTAATAAAAGCGACAACATAAAGCCCAAAAGCGCACCATGGAATAGCTTCCTGCCACCCCCCCCACCAATGCCAGGGCCAAGGCTGGGGCCAGGCAAGCCGGGTCTAAAGTTTAATGGTCCGCCCCCACCGCCCCCCCCACCCCCACCCCACTTGCTGTCATGTTGGCTTCCGCCATTCCCTTCCGGTCCACCTATCATTCCTCCACCCCCACCAATTTGCCCAGACAGCTTAGACGACGCAGACGCCTTGGGCTCGATGCTGATCAGCTGGTATATGTCGGGATACCATACAGGCTACTATATGGGTTTCCGTCAGAACCAAAAGGAAGGCCGGTGTTCCCATTCCCTGAATTGA

[0913] SEQ ID NO:13 CoSMN13

[0914] ATGGCCATGTCTTCAGGAGGATCAGGTGGGGGCGTCCCTGAACAGGAGGACTCTGTGCTCTTCCGGCGAGGTACGGGCCAAAGCGATGACAGCGACATTTGGGATGACACCGCGCTCATCAAGGCCTATGATAAAGCTGTTGCATCCTTCAAGCACGCTCTTAAAAATGGTGACATTTGTGAAACTTCCGGGAAACCCAAAACCACACCCAAGAGGAAGCCCGCCAAGAAGAATAAATCTCAGAAGAAAAACACTGCTGCCTCCCTGCAGCAGTGGAAGGTGGGGGACAAGTGCAGCGCTATATGGTCTGAAGATGGCTGCATCTATCCAGCAACCATTGCGTCCATTGACTTCAAAAGAGAGACCTGTGTGGTGGTATACACAGGATATGGCAACCGGGAGGAGCAAAACCTGAGCGACCTGCTATCACCCATTTGTGAGGTCGCCAACAACATCGAGCAGAATGCCCAGGAAAACGAGAATGAAAGCCAAGTTTCCACAGATGAAAGTGAGAACTCCAGGAGTCCAGGGAACAAGTCAGACAATATAAAACCAAAAAGTGCACCTTGGAATAGCTTTCTCCCTCCACCACCACCCATGCCTGGGCCCCGTTTGGGTCCTGGCAAGCCTGGCCTGAAGTTTAATGGACCTCCTCCCCCACCACCACCACCGCCCCCTCATTTATTGAGCTGCTGGCTGCCCCCCTTTCCTAGTGGACCGCCCATCATCCCCCCGCCACCTCCAATCTGCCCTGATTCTCTGGATGATGCTGATGCATTAGGCAGCATGCTGATATCCTGGTACATGAGTGGCTACCACACTGGTTACTATATGGGGTTCCGCCAGAACCAGAAAGAAGGCAGATGTTCGCATTCACTTAATTGA

[0915] SEQ ID NO:14 CoSMN14

[0916] ATGGCCATGAGCTCAGGGGGTTCTGGAGGCGGTGTACCAGAACAAGAAGACTCTGTTCTCTTCAGGAGAGGAACAGGGCAATCTGATGACAGCGACATCTGGGACGACACTGCTTTAATAAAGGCGTATGACAAGGCTGTCGCTTCCTTCAAGCATGCACTTAAAAATGGAGACATTTGTGAGACTAGTGGGAAACCTAAGACAACTCCAAAGAGGAAGCCTGCCAAGAAGAACAAAAGCCAGAAAAAAAACACAGCAGCATCCTTGCAGCAGTGGAAAGTGGGTGATAAATGTTCAGCAATATGGAGCGAAGATGGCTGCATCTACCCAGCCACCATTGCCAGCATTGATTTTAAAAGAGAGACCTGTGTGGTCGTTTACACCGGCTATGGCAACCGGGAGGAGCAAAACCTGAGTGACCTGTTGTCTCCGATCTGTGAAGTGGCCAACAACATTGAGCAGAATGCTCAGGAGAATGAAAATGAGTCCCAGGTGTCCACCGATGAATCTGAGAACTCCCGTTCACCCGGAAATAAATCGGACAACATCAAGCCCAAGAGTGCGCCCTGGAATTCCTTCCTTCCTCCGCCGCCTCCCATGCCTGGGCCTCGATTAGGCCCTGGGAAGCCAGGGCTCAAGTTCAATGGCCCACCCCCACCACCACCTCCACCGCCCCCCCACCTACTGTCGTGCTGGCTGCCTCCTTTTCCTTCTGGTCCCCCCATTATCCCTCCTCCCCCACCCATCTGCCCAGACAGTCTGGATGATGCTGATGCCCTGGGAAGCATGCTGATCTCCTGGTACATGAGTGGATATCACACGGGTTACTACATGGGCTTTCGGCAGAACCAGAAGGAGGGCCGCTGCAGTCATAGCCTCAACTGA

[0917] SEQ ID NO:15 CoSMN15

[0918] ATGGCCATGAGCTCCGGGGGAAGTGGAGGGGGAGTTCCTGAGCAAGAAGACAGCGTCCTCTTTAGGAGGGGTACAGGCCAATCCGATGATTCAGATATTTGGGACGACACTGCTCTGATTAAAGCATACGACAAAGCGGTGGCAAGTTTCAAGCATGCATTGAAAAATGGTGATATATGCGAGACTTCAGGCAAGCCTAAAACGACACCCAAGAGAAAGCCAGCTAAGAAAAACAAGAGCCAAAAGAAGAACACAGCCGCCAGCCTCCAGCAGTGGAAGGTCGGTGATAAATGTTCAGCTATATGGTCAGAGGACGGGTGTATTTATCCAGCAACCATCGCCAGCATCGACTTCAAACGGGAGACCTGTGTAGTGGTTTACACCGGATACGGCAACCGGGAAGAGCAGAACCTGTCCGACCTTCTGTCTCCAATCTGTGAGGTGGCTAACAATATCGAGCAGAATGCCCAGGAAAATGAGAATGAATCCCAGGTGTCGACGGATGAGTCGGAAAACTCTCGAAGTCCCGGGAATAAGTCCGATAATATCAAACCAAAAAGTGCCCCCTGGAACTCTTTCCTCCCACCTCCTCCACCTATGCCCGGGCCCCGCCTGGGCCCCGGGAAGCCTGGATTGAAGTTTAACGGCCCCCCTCCGCCACCCCCGCCCCCACCGCCTCATCTGCTAAGTTGCTGGCTGCCTCCGTTTCCGTCTGGCCCACCGATTATCCCCCCTCCGCCACCCATTTGCCCAGACAGCCTTGACGATGCGGACGCCTTAGGTTCCATGTTAATCAGCTGGTATATGTCCGGCTATCACACCGGCTACTACATGGGATTCAGACAGAATCAGAAGGAAGGCCGCTGCAGCCACTCTCTGAACTGA

[0919] SEQ ID NO:16 CoSMN16

[0920] ATGGCAATGAGTAGTGGTGGTAGTGGTGGTGGAGTGCCTGAGCAGGAAGATTCCGTGCTGTTCCGGAGAGGCACAGGCCAGTCTGACGACTCTGATATCTGGGATGACACCGCTCTGATCAAGGCCTACGACAAGGCCGTGGCCAGCTTTAAGCACGCCCTGAAGAATGGCGACATTTGTGAAACCTCTGGCAAACCCAAGACCACCCCTAAGCGGAAGCCTGCCAAGAAGAACAAGTCCCAGAAGAAAAACACCGCCGCTTCTCTGCAGCAATGGAAGGTGGGCGACAAGTGCTCTGCCATCTGGTCCGAGGACGGCTGCATCTACCCTGCTACCATCGCCTCTATCGACTTCAAGAGAGAGACATGTGTGGTCGTGTACACCGGCTACGGCAACCGGGAGGAACAGAACCTGTCCGACCTGCTCTCTCCCATCTGCGAGGTGGCTAACAACATCGAGCAAAACGCCCAGGAAAACGAGAACGAGTCCCAGGTGTCCACCGACGAATCCGAGAATTCCCGCTCTCCTGGCAATAAGTCCGACAACATCAAGCCTAAAAGCGCTCCTTGGAACTCCTTCCTGCCTCCACCTCCTCCAATGCCTGGTCCTAGACTGGGCCCCGGCAAGCCCGGCCTGAAGTTCAACGGCCCTCCTCCACCTCCCCCGCCCCCACCCCCTCACCTGCTGTCCTGCTGGCTGCCTCCTTTCCCTTCTGGCCCCCCCATCATCCCTCCACCTCCTCCTATCTGCCCTGATAGCCTGGACGATGCCGACGCTCTGGGATCTATGCTGATCTCCTGGTATATGTCCGGATACCACACCGGCTACTACATGGGCTTCAGACAGAACCAGAAAGAGGGCAGATGCAGCCATTCCCTGAACTGA

[0921] SEQ ID NO:17 CoSMN17

[0922] ATGGCAATGAGTTCCGGGGGGTCAGGTGGAGGTGTGCCAGAACAAGAAGATAGTGTGCTCTTCCGTCGGGGCACAGGCCAATCAGATGATAGTGACATCTGGGATGACACCGCACTTATCAAAGCATATGATAAAGCAGTAGCTTCTTTCAAGCATGCTCTAAAAAATGGAGACATATGTGAAACGTCAGGGAAACCAAAAACAACTCCTAAAAGGAAGCCTGCGAAGAAAAACAAGTCTCAGAAGAAGAACACTGCAGCCAGTTTGCAGCAGTGGAAAGTGGGAGACAAGTGCAGTGCTATTTGGTCTGAGGATGGCTGCATTTACCCTGCTACCATAGCATCCATTGATTTTAAGAGAGAGACTTGTGTCGTTGTCTACACAGGTTATGGCAACAGAGAAGAGCAGAACCTCTCAGACTTACTTAGCCCCATTTGTGAGGTTGCCAACAATATTGAACAGAATGCTCAAGAGAATGAAAATGAGAGTCAGGTATCTACAGATGAATCTGAGAACAGCAGGAGCCCTGGAAATAAATCAGACAATATAAAGCCCAAAAGCGCCCCCTGGAATTCTTTTTTGCCTCCTCCGCCCCCAATGCCAGGTCCACGACTGGGCCCAGGAAAACCTGGGCTAAAGTTTAATGGGCCTCCTCCACCCCCCCCTCCGCCACCGCCTCATCTGCTGTCCTGCTGGCTTCCACCATTTCCAAGTGGACCTCCCATCATTCCTCCCCCACCACCAATCTGTCCAGATTCGTTAGACGATGCAGATGCCCTGGGATCTATGCTGATATCCTGGTACATGTCTGGATACCACACAGGGTATTATATGGGTTTCAGACAAAACCAGAAAGAAGGACGCTGCTCACACAGCTTGAACTGA

[0923] SEQ ID NO:18CoSMN18

[0924] ATGGCCATGAGCTCCGGTGGTAGTGGCGGAGGCGTACCTGAACAAGAAGACTCTGTTTTGTTTCGGAGGGGCACCGGCCAAAGCGATGATTCAGATATATGGGACGACACCGCGTTAATCAAGGCCTACGATAAAGCTGTGGCATCTTTCAAACATGCATTGAAGAATGGAGACATCTGCGAGACCAGCGGAAAACCCAAGACAACGCCAAAGCGCAAACCTGCCAAGAAGAATAAGAGCCAGAAGAAAAATACTGCTGCCTCACTGCAGCAGTGGAAGGTGGGGGATAAATGTAGCGCCATATGGAGCGAGGATGGCTGCATCTATCCAGCTACAATTGCATCCATTGACTTCAAGAGGGAAACTTGCGTGGTGGTCTACACCGGATATGGTAACAGAGAGGAACAGAATCTCTCTGACCTTCTGTCCCCAATTTGCGAGGTCGCCAACAACATAGAGCAGAATGCCCAGGAGAACGAAAATGAATCACAAGTTTCGACAGATGAAAGTGAGAACAGCCGATCCCCGGGGAACAAATCTGACAACATCAAACCAAAGAGTGCGCCCTGGAATTCATTTCTGCCCCCACCCCCTCCCATGCCTGGACCACGTCTAGGTCCTGGGAAACCTGGCCTTAAGTTCAATGGCCCCCCTCCTCCACCCCCACCTCCACCGCCTCACCTGCTGTCTTGTTGGCTGCCGCCCTTCCCCAGTGGGCCGCCCATCATTCCTCCGCCCCCCCCAATCTGTCCAGACAGCCTGGATGACGCTGATGCACTAGGCTCCATGCTCATTTCCTGGTATATGTCTGGGTATCACACTGGATACTACATGGGGTTTCGGCAGAACCAGAAAGAGGGCAGATGTTCCCATTCTCTCAACTGA

[0925] SEQ ID NO:19CoSMN19

[0926] ATGGCCATGTCTTCGGGCGGCTCGGGTGGCGGTGTCCCCGAACAGGAGGACTCCGTGCTGTTCCGGCGCGGGACTGGACAGAGCGATGACAGTGACATATGGGATGACACCGCCCTGATCAAGGCGTACGACAAAGCCGTAGCTTCTTTCAAGCACGCCCTGAAGAATGGGGACATCTGCGAGACAAGCGGCAAGCCCAAAACCACTCCTAAGCGCAAGCCTGCCAAGAAAAACAAGTCCCAGAAGAAGAACACGGCAGCCAGCTTGCAACAGTGGAAGGTGGGTGACAAGTGTTCTGCTATCTGGTCTGAAGACGGCTGTATTTATCCAGCGACCATCGCATCCATTGACTTTAAGAGAGAGACCTGCGTGGTGGTGTACACCGGCTACGGCAACCGCGAGGAGCAGAACCTGTCCGACCTGCTGTCGCCCATCTGCGAGGTGGCCAACAACATTGAGCAGAATGCGCAGGAGAATGAGAACGAGTCCCAGGTGTCCACCGACGAGAGCGAGAACTCGCGTAGCCCAGGCAACAAGTCCGACAACATCAAACCCAAGTCAGCTCCGTGGAACTCCTTCCTGCCTCCTCCACCGCCAATGCCTGGACCCCGCCTGGGTCCGGGCAAGCCCGGTCTCAAATTTAACGGCCCTCCACCCCCCCCCCCGCCTCCCCCTCCCCACCTGCTTTCATGCTGGCTTCCGCCCTTCCCGTCTGGGCCGCCCATCATCCCGCCTCCGCCCCCCATCTGCCCAGATTCCTTGGATGACGCGGATGCTCTGGGATCTATGTTAATCAGTTGGTACATGAGCGGCTACCACACGGGCTATTACATGGGCTTCCGCCAGAACCAGAAGGAGGGCAGGTGTAGCCACTCCCTCAACTGA

[0927] SEQ ID NO:20CoSMN20

[0928] ATGGCCATGTCGTCAGGGGGCTCAGGAGGTGGAGTTCCTGAGCAGGAGGATAGTGTCCTGTTCCGAAGGGGCACCGGACAAAGTGATGACTCTGACATTTGGGATGACACCGCGTTGATAAAGGCATATGACAAAGCAGTGGCTAGCTTCAAACATGCTCTTAAAAATGGTGACATCTGTGAAACTTCTGGGAAGCCAAAAACGACACCCAAAAGGAAGCCGGCCAAGAAGAATAAATCTCAAAAGAAAAACACTGCAGCGTCCCTGCAGCAGTGGAAGGTTGGAGACAAGTGTTCAGCCATCTGGAGCGAAGATGGCTGCATCTATCCTGCCACCATCGCCTCCATTGACTTTAAACGGGAGACCTGTGTAGTGGTCTACACTGGCTATGGTAATAGAGAGGAGCAAAATCTTTCAGACCTCCTCTCTCCCATTTGTGAGGTGGCCAACAACATCGAGCAGAATGCCCAGGAAAATGAGAATGAAAGCCAGGTGAGTACAGATGAAAGTGAAAACTCCCGCTCTCCCGGCAACAAGAGTGACAACATTAAGCCCAAGTCAGCTCCCTGGAACTCCTTTTTACCACCACCTCCGCCCATGCCTGGGCCTCGGCTAGGCCCAGGTAAACCCGGTCTGAAGTTTAATGGGCCTCCACCTCCACCCCCGCCTCCCCCACCCCACCTGCTCAGCTGCTGGCTGCCCCCTTTCCCTTCGGGACCGCCCATAATTCCACCTCCACCACCGATCTGCCCCGACAGCTTAGATGATGCTGATGCACTGGGGAGCATGCTGATCTCCTGGTACATGAGCGGCTACCACACAGGATACTACATGGGCTTCAGACAGAACCAGAAAGAGGGCCGCTGCTCCCATTCTTTGAACTGA

[0929] SEQ ID NO: 21 hSMN1 amino acid sequence

[0930] MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN*

[0931] SEQ ID NO:22 CMV IE enhancer

[0932] CTAGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTAC

[0933] SEQ ID NO:23 Chicken β-actin core (CB) promoter

[0934] TGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGG

[0935] SEQ ID NO:24 PCI intron

[0936] GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG

[0937] SEQ ID NO:25 RGb polyA signal

[0938] GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCG

[0939] SEQ ID NO:26 5’ITR105

[0940] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTG

[0941] SEQ ID NO:27 3’ITR130

[0942] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0943] SEQ ID NO:28 hPGK promoter

[0944] GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG

[0945] SEQ ID NO:29 chimeric intron CBA + rabbit globin

[0946] GGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTT

[0947] SEQ ID NO:30 bGH polyA signal

[0948] CTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA

[0949] SEQ ID NO:31 hSyn promoter

[0950] CTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG

[0951] SEQ ID NO:32 AAV9 capsid amino acid sequence

[0952] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0953] SEQ ID NO:33 AAV2 Rep amino acid sequence

[0954] MPGFYEIVIKVPSDLDEHLPGISDSFVNWVAEKEWELPPDSDMDLNLIEQAPLTVAEKLQRDFLTEWRRVSKAPEALFFVQFEKGESYFHMHVLVETTGVKSMVLGRFLSQIREKLIQRIYRGIEPTLPNWFAVTKTRNGAGGGNKVVDECYIPNYLLPKTQPELQWAWTNMEQYLSACLNLTERKRLVAQHLTHVSQTQEQNKENQNPNSDAPVIRSKTSARYMELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRYQNKCSRHVGMNLMLFPCRQCERMNQNSNICFTHGQKDCLECFPVSESQPVSVVKKAYQKLCYIHHIMGKVPDACTACDLVNVDLDDCIFEQ

[0955] SEQ ID NO:34 AAV9 capsid amino acid sequence

[0956]

[0957] SEQ ID NO:35 AAV2 Rep coding sequence

[0958]

[0959] SEQ ID NO:36 GFP coding sequence

[0960] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATTTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0961] SEQ ID NO:37 Transgenic plasmid containing CoSMN2

[0962]

[0963] SEQ ID NO:38 Forward primer for ddPCR

[0964] CCCACTTGGCAGTACATCAA

[0965] SEQ ID NO:39 Reverse primer for ddPCR

[0966] GCCAAGTAGGAAAGTCCCATAA

[0967] SEQ ID NO:40 ddPCR probe

[0968] CATAATGCCAGGCGGGCCATTTAC

[0969] SEQ ID NO:41 Forward primer for qPCR of mouse GAPDH

[0970] CCACTCACGGCAAATTCAAC

[0971] SEQ ID NO:42 Reverse primer for qPCR of mouse GAPDH

[0972] ACCAGTAGACTCCACGACATA

[0973] SEQ ID NO:43 qPCR probe for mouse GAPDH

[0974] TTGTCATCAACGGGAAGCCCATCA

[0975] SEQ ID NO:44 Forward primer for qPCR of CoSMN11

[0976] CTATCATCCCTCCTCCTCCTC

[0977] SEQ ID NO:45 Reverse primer for qPCR of CoSMN11

[0978] GCCTGAAGCCCATGTAGTAG

[0979] SEQ ID NO:46 qPCR probe for CoSMN11

[0980] CTGGTACATGAGCGGCTACCACAC

Claims

1. A recombinant adeno-associated virus (rAAV) comprising a genome, the genome comprising an expression construct, the expression construct comprising a polynucleotide of interest, the polynucleotide of interest comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 and operably linked to a promoter.

2. The rAAV of claim 1, wherein the construct further comprises an intron.

3. The rAAV of claim 2, wherein the intron is between the promoter and the polynucleotide of interest.

4. The rAAV of claim 2 or 3, wherein the intron comprises a nucleotide sequence selected from SEQ ID NOs: 24 and 29.

5. The rAAV of any one of claims 1-4, wherein the construct further comprises an enhancer.

6. The rAAV of claim 5, wherein the enhancer is upstream of the promoter.

7. The rAAV of claim 5 or 6, wherein the enhancer comprises the nucleotide sequence of SEQ ID NO:

22.

8. The rAAV of any one of claims 1-7, wherein the promoter comprises a nucleotide sequence selected from SEQ ID NOs: 23, 28, and 31.

9. A pharmaceutical composition comprising the rAAV of any one of claims 1-8.

10. The rAAV of any one of claims 1 to 8 or the pharmaceutical composition of claim 9 for use in treating a disease associated with a defect in the SMN1 gene.

11. The rAAV or pharmaceutical composition of claim 10, wherein the disease is spinal muscular atrophy (SMA).

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