WO2024165839A1

WO2024165839A1 - Transgenes

Info

Publication number: WO2024165839A1
Application number: PCT/GB2024/050308
Authority: WO
Inventors: Rafael YÁÑEZ-MUÑOZ; Daniel HAYLER-POUNTNEY; Ellie CHILCOTT; Neda ALI MOHAMMADI NAFCHI
Original assignee: Royal Holloway and Bedford New College
Current assignee: Royal Holloway and Bedford New College
Priority date: 2023-02-06
Filing date: 2024-02-05
Publication date: 2024-08-15
Anticipated expiration: 2025-08-06

Abstract

The invention provides a nucleic acid molecule comprising a codon optimised nucleotide sequence encoding for a functional human survival motor neuron 1 protein, the nucleotide sequence having at least 96% identity with SEQ ID NO:1.

Description

TRANSGENES

Technical Field of the Invention

The present invention relates to novel transgenes for spinal muscular atrophy disease.

Background to the Invention

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, which results in muscle weakness and causes problems with movement. It is a serious disease which can get worse over time.

The disease is chiefly characterised by degeneration of motor neurons from the ventral horn of the spinal cord. Survival motor neuron (SMN) 1 gene is a SMA-determining gene, being absent in 95% of patients with SMA and mutated in the remaining 5%. SMN2 is a highly similar gene with only five nucleotide mismatches, which result in 90% truncated transcripts lacking exon 7 (SMNA7), producing only low levels of SMN protein. SMN2 copy number is a strict determinant of disease severity, whereby patients with only two copies of the gene present with the severe type I form of SMA while patients with a greater number of SMN2 copies have less severe symptoms. Full-length SMN is a ubiquitous and essential cellular protein that has roles in RNA metabolism, cytoskeletal maintenance, transcription, cell signalling and DNA repair. For many years, it was thought that motor neurons were the only affected cells, but recent evidence suggests a wide range of systemic pathologies are also caused by low levels of SMN protein. Therefore, an effective and successful therapy for SMA is likely to involve the consideration of SMA as a multi-system disorder.

In the past five years, three therapies for SMA patients have been approved by regulatory bodies: Spinraza, Zolgensma and Evrysdi, the first two of which are genetic therapies.

Survival motor neuron (SMN) 1 gene mutations cause SMA, and gene addition strategies to replace the faulty SMN1 copy are a therapeutic option. Spinraza is an antisense oligonucleotide (ASO) that increases the level of full-length SMN protein by binding and altering the splicing of SMN2 pre-mRNA, enhancing the inclusion of exon 7. Zolgensma is an adeno-associated viral vector of serotype 9 (AAV9) vector containing the cDNA of the human SMN1 gene under the control of the cytomegalovirus enhancer/chicken-P-actin-hybrid promoter. Evrysdi is a small molecule that modulates SMN2 RNA splicing by binding to two unique sites in SMN2 pre-mRNA: 5' splice site of intron 7 and an exonic splicing enhancer 2 in exon 7, therefore promoting inclusion of exon 7. Evrysdi is an oral medicine expected to be taken for the duration of the individual’s life, while Spinraza requires repeated delivery through intrathecal injections and Zolgensma is a one-off intravenous infusion.

Gene therapy is a technology that allows the modification of gene expression with one possible strategy being the introduction of transgenes for therapeutic purposes. In this context, the efficient delivery of therapeutic genes, or other gene therapy agents, is a critical requirement for the development of an effective treatment. Vectors derived from lentiviruses have proven to be efficient gene delivery vehicles as they integrate into the host’s chromosomes and show continued expression for a long time. They also have a relatively large cloning capacity, which is sufficient for most clinical purposes. Lentiviral vectors can transduce different types of cells, including quiescent cells, have low immunogenicity upon in vivo administration, lead to stable gene expression and can be pseudotyped with alternative envelopes to alter vector tropism.

Due to their unique advantages, lentiviral vectors are important gene delivery systems for research and clinical applications. Lentiviral vectors have been utilised to treat symptoms in several animal models, such as X-linked severe combined immunodeficiency (SCID-X1), P-thalassemia, Wiskott-Aldrich syndrome, metachromatic leukodystrophy, haemophilia, Fanconi anaemia and liver disease, as well as being used in clinical applications. Although the integrative nature of lentiviral vectors provides long-term transgene expression, integration events carry the risk of insertional mutagenesis. Intensive study of the genome and analysis of integration strategies of lentiviral vectors has led to the development of a number of strategies to minimise these risks. These include the use of viral vectors with a safer integration pattern, the utilisation of self-inactivating vectors and the design of integrationdeficient lentiviral vectors (IDLVs). IDLVs are non-integrative due to an engineered class I mutation in the viral integrase gene, most commonly involving an amino acid change at position D64 within the catalytic core domain. Whilst the SMA therapies developed thus far have their advantages, there exists a need for further safe and effective or more effective treatments for SMA.

It is an aim of embodiments of the present invention to overcome or mitigate at least one problem of the prior art, whether expressly described herein or not.

Summary of the Invention

According to a first aspect of the invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding for a functional human survival motor neuron 1 protein, the nucleotide sequence having at least 96% identity with SEQ ID NO:1.

The novel codon optimised sequence nucleic acid molecule is produced by a method that involves making silent changes to the nucleotide sequence of the human survival motor neuron 1 gene. This codon-optimised sequence provides for enhanced translational efficiency of the corresponding mRNA, without modifying the sequence of the protein encoded. The silent changes provide for removal of cryptic sequences that could be detrimental to transgene expression.

Thus, the novel nucleic acid molecule of the invention provides a codon-optimized human survival motor neuron 1 cDNA (Co-hSMNl), which results in increased expression of the SMN1 protein compared to the wild-type hSMNl cDNA.

The term “codon-optimized” refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of a host organism without altering the polypeptide encoded by the DNA. Such optimization may comprise replacing or altering at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

The nucleic acid molecule can be easily and effectively engineered into a range of vectors/delivery systems. The nucleic acid molecule can be produced or engineered using any suitable vector. Suitable vectors may be one or more selected from the group consisting of: standard integrating lentivectors, integration-deficient lentiviral vectors (IDLVs) and adeno-associated virus (AAV) vectors. The vector may be used in conjunction with a promoter. Suitable promoters include existing promoters and those engineered for use with the specific vector used.

The nucleic acid molecule allows for highly efficient, effective and safe production of survival motor neuron (SMN) protein in target cells.

The nucleic acid molecule also surprisingly helps reduce the dose of gene therapy vector necessary to achieve a therapeutic effect in spinal muscular atrophy, greatly minimising the risk of toxicity. cDNA refers to complementary DNA - i.e. DNA synthesised from a single-stranded RNA template.

In some embodiments, the nucleotide sequence has at least 96.5% identity with SEQ ID NO:1, or at least 97, 97.5, 98, 98.5, 99, or at least 99.5, 99.6, 99.7, 99.8, or at least 99.9% identity with SEQ ID NO:1. In some embodiments, the Co-hSMNl cDNA has 100% identity with SEQ ID NO:1.

In some embodiments, the nucleotide sequence encodes a SMN protein which provides at least about 50%, at least about 75%, at least about 80%, at least about 90%, or about the same, or greater than 100% of the biological activity level of the native survival of human motor neuron protein SMN1, or a natural variant or polymorph thereof which is not associated with disease.

In some embodiments, the encoded SMN 1 protein is an isoform D protein.

According to a second aspect of the invention, there is provided a vector for expressing human survival motor neuron 1 protein comprising the nucleic acid molecule of the first aspect of the invention.

The vector may be an integrating lentivector, integration-deficient lentiviral vector (IDLV) or adeno-associated viral (AAV) vector.

Statements of invention above for the first aspect of the invention may also be applied mutatis mutandis to the second aspect of the invention. Statements of invention below for the second aspect of the invention may also be applied mutatis mutandis to the other aspects of the invention.

In some embodiments, the vector is a single- stranded vector. In some embodiments, the vector comprises a complete virus particle, such as a wildtype (wt) virus particle.

In some embodiments, the vector is a recombinant vector.

In some embodiments, the vector comprises an external component and an internal DNA genome. The external component may be a capsid. The capsid may comprise the nucleic acid molecule of the invention, preferably flanked by two inverted terminal repeats (ITRs). The capsid may further comprise one or more of the group comprising: an enhancer, a promoter, an intron, a polyA signal, and combinations thereof.

In some embodiments, the vector is a lentivector or lentiviral vector. Such vectors are capable of being administered locally and are capable of integrating the sequence into the genome.

The vector may be an integrating lentivector. The integrating lentivector may be an integration-proficient lentivector.

The vector may be an integration-deficient lentiviral vector.

In some embodiments, the vector is an adeno-associated viral (AAV) vector. AAVs are able to penetrate the blood-brain barrier and can be used to deliver the sequence to the nucleus of a cell.

In some embodiments, the AAV vector comprises a vector derived from an adeno- associated virus serotype. In some embodiments, the vector comprises at least one AAV vector that is independently selected from the group comprising: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and combinations thereof. At least one AAV vector may have one or more of the AAV wild-type genes deleted in whole or part, which may be the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences allow for rescue, replication, and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences that in cis provide for replication and packaging (e.g. functional ITRs) of the virus. The ITRs may or may not be wild-type nucleotide sequences, and may be altered, such as by insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. In some embodiments, the vector is an AAV-9 vector, with AAV-2 derived ITRs. In some embodiments, the vector comprises at least one self-complementary adeno- associated viral vector (scAAV), which is a viral vector engineered from the naturally occurring adeno-associated virus (AAV) for use in gene therapy. scAAV is termed “self-complementary” because the coding region has been designed to form an intramolecular double-stranded DNA template.

In some embodiments, the vector is produced from at least one plasmid. At least one plasmid may preferably be independently selected from the group comprising: pRRLsc_CMV_co_hSMNl_mW (SEQ ID NO: 8), pRRLsc_hSYN_co_hSMNl_mW (SEQ ID NO: 9), pRRLsc_hPGKp_co_hSMNl_mW (SEQ ID NO: 10), pAAV_CAG_hSMNlopt_mWPRE_sensl (SEQ ID NO: 11), and combinations thereof.

In some embodiments, the integrating lentivector or IDLV is produced from at least one plasmid. At least one plasmid may preferably be independently selected from the group comprising: pRRLsc_CMV_co_hSMNl_mW (SEQ ID NO: 8), pRRLsc_hS YN_co_hSMNl_mW (SEQ ID NO: 9), pRRLsc_hPGKp_co_hSMNl_mW (SEQ ID NO: 10), and combinations thereof. In some embodiments, the integrating lentivector or IDLV is produced from a pRRLsc_CMV_co_hSMNl_mW (SEQ ID NO: 8) plasmid. Vectors produced from such plasmids were found to be particularly effective at transgenic expression in cell culture.

In some embodiments, the AAV vector is produced from at least one plasmid. At least one plasmid may preferably comprise pAAV_CAG_hSMN lopt_mWPRE_sens 1 (SEQ ID NO: 11). Vectors produced from such as plasmid demonstrated expression in cell culture and strong transgenic expression in vivo.

In some embodiments, the vector is associated with at least one transfer, packaging or helper plasmid. In embodiments in which the vector is a lentiviral vector the vector may be produced in association with at least one plasmid selected from the group consisting of: a transfer plasmid, a packaging plasmid, a rev plasmid and an envelope plasmid, or any combination thereof. Suitable transfer plasmids include one or more selected from the group consisting of pRRLsc_CMV_co_hSMNl_mW (SEQ ID NO: 8), pRRLsc_hS YN_co_hSMNl_mW (SEQ ID NO: 9), and pRRLsc_hPGKp_co_hSMNl_mW (SEQ ID NO: 10). Suitable packaging plasmids include pMDLg/pRRE (SEQ ID NO: 12) for integrating lentivectors or pMDLg/pRRintD64V (SEQ ID NO: 13) for integration-deficient lentivectors. Suitable rev plasmids include pRSV-rev (SEQ ID NO: 14). Suitable envelope plasmids include pMD2.VSVG (SEQ ID NO: 15). Such plasmids may be used in cell culture to make or engineer the lentiviral vector that will then be delivered in vivo.

In some embodiments, the vector further comprises at least one promoter. In some embodiments, at least one promoter is independently selected from the group comprising: cytomegalovirus (CMV); phosphoglycerate kinase (PGK), preferably human phosphoglycerate kinase (hPGK); CAG; synapsin (SYN), preferably human synapsin (hSYN); and combinations thereof. In some embodiments, at least one vector promoter is independently selected from the group comprising: CMV, hSYN, hPGK, and combinations thereof.

In some preferred embodiments, the vector is a lentivector or lentiviral vector and the vector comprises at least one vector promoter that is independently selected from the group comprising: CMV, hSYN, hPGK, and combinations thereof. In some preferred embodiments, the vector is a lentivector or lentiviral vector and the vector comprises a CMV vector promoter.

In some preferred embodiments, the vector is an integration-deficient lentiviral vector comprising a CMV vector promoter. Such a vector and promoter combination has been shown to lead to significant but safe expression of the optimised sequence.

In preferred embodiments, the vector is an integrating lentivector comprising a CMV vector promoter. Such a vector and promoter combination has been shown to result in very high production of functional SMN protein.

In some embodiments, the vector comprises at least one expression cassette. The nucleic acid molecule of the invention may be contained in at least one expression cassette. The term “expression cassette” refers to a nucleic acid molecule which comprises the sequence or nucleic acid molecule of interest and one or more regulatory sequences (e.g. selected from a promoter, enhancer, polyA etc.). The expression cassette may be packaged into a capsid of the vector. Typically, such an expression cassette for generating a viral vector contains the cDNA sequence flanked by packaging signals of the viral genome and other expression control sequences.

In some embodiments, at least one expression cassette further comprises at least one expression control sequence. At least one expression control sequence may direct expression of the Co-hSMNl cDNA nucleic acid molecule of the invention in a host cell.

In some embodiments, at least one expression control sequence comprises a promoter, preferably as described in statements of invention above.

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

In addition to a promoter, an expression cassette and/or a vector may contain one or more other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA for example WPRE; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, e.g., SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyAs. An example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include those that are appropriate for CNS indications. In one embodiment, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or may differ from one another. For example, an enhancer may include a CMV immediate early enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of the enhancer may be separated by one or more sequences. In still another embodiment, the expression cassette further contains an intron, e.g, the chicken beta-actin intron. Other suitable introns include those known in the art, e.g., such as are described in WO 2011/126808. Optionally, one or more sequences may be selected to stabilize mRNA. An example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence.

These control sequences are "operably linked" to the hSMN gene sequences. As used herein, the term "operably linked" refers to both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

According to a third aspect of the invention, there is provided a host cell comprising the nucleic acid molecule of the first aspect of the invention or the vector of the second aspect of the invention.

As used herein, the term "host" refers to organisms and/or cells which harbour a nucleic acid molecule or a vector of the invention, as well as organisms and/or cells that are suitable for use in expressing a recombinant gene or protein. It is not intended that the present invention be limited to any particular type of cell or organism. Indeed, it is contemplated that any suitable organism and/or cell will find use in the present invention as a host. A host cell may be in the form of a single cell, a population of similar or different cells, for example in the form of a culture (such as a liquid culture or a culture on a solid substrate), an organism or part thereof.

A host cell according to the invention may permit the expression of the nucleic acid molecule of the invention. The host cell may be, for example, a bacterial, a yeast, an insect or a mammalian cell. According to a fourth aspect of the invention, there is provided a non-human transgenic animal comprising cells comprising the nucleic acid molecule of the first aspect of the invention or the vector of the second aspect of the invention.

Statements of invention above for the first and second aspects of the invention may also be applied mutatis mutandis to the third and fourth aspects of the invention.

According to a fifth aspect of the invention, there is provided a pharmaceutical composition comprising the nucleic acid molecule of the first aspect of the invention.

In some embodiments, the pharmaceutical composition comprises a vector comprising the nucleic acid molecule. The vector is preferably the vector of the second aspect of the invention.

The vector may preferably comprises an integrating lentivector, integration-deficient lentiviral vector (IDLV) or adeno-associated viral (AAV) vector of the second aspect of the invention.

Statements of invention above for the first and second aspects of the invention may also be applied mutatis mutandis to the fifth aspect of the invention. Statements of invention below for the fifth aspect of the invention may also be applied mutatis mutandis to the other aspects of the invention.

The composition may comprise a pharmaceutically acceptable carrier, excipient and/or preservative.

In some embodiments, the nucleic acid molecule or vector is suspended in solution, preferably an aqueous solution.

The nucleic acid molecule or vector may be dissolved or suspended in an aqueous carrier, which may be independently selected from the group comprising: water, buffered water, saline, and combinations thereof.

The composition may further comprise at least one tonicifier to render the solution iso- osmotic or isotonic. At least one tonicifier may be independently selected from a salt and/or a sugar.

In some embodiments, wherein the vector is an AAV, the composition comprises between 1 x 10¹³ vg/mL and 1 x 10¹⁵ vg/mL of the vector, or between 1-10 x 10¹³ vg/mL, or between 1-8 x 10¹³ vg/mL of the nucleic acid molecule or vector, or between 1.5-6 x 10¹³ vg/mL, or between 1.5-5 x 10¹³ vg/mL, or between 1.5-4.5 x 10¹³ vg/mL of the nucleic acid molecule or vector.

In some embodiments, the composition has less than about 10% empty viral capsids, or less than about 8, 7, or less than about 5% empty viral capsids

The composition may have a pH of between 4-11, or between 5-10, 6-9, 6.5-8.5, 7-8, or of between 7.2 to 7.8.

According to a sixth aspect of the invention, there is provided a nucleic acid molecule of the first aspect of the invention or a vector of the second aspect of the invention for use in therapy.

Statements of invention for the other aspects of the invention may also be applied mutatis mutandis to the sixth aspect of the invention.

According to a seventh aspect of the invention, there is provided a nucleic acid molecule of the first aspect of the invention or a vector of the second aspect of the invention for use in the treatment of a neuromuscular disorder.

According to an eighth aspect of the invention, there is provided a nucleic acid molecule of the first aspect of the invention or a vector of the second aspect of the invention for use in a method of treating a neuromuscular disorder comprising administering a therapeutically effective amount of the nucleic acid molecule or the vector to a patient suffering the neuromuscular disorder.

Statements of invention for the other aspects of the invention may also be applied mutatis mutandis to the seventh and eighth aspects of the invention. Statements of invention below for the seventh and eighth aspects of the invention may also be applied mutatis mutandis to the other aspects of the invention.

The following statements apply to both the seventh and eighth aspects of the invention.

The neuromuscular disorder may be independently selected from the group comprising: spinal bulbar muscular atrophy, spinal cerebellar ataxia, traumatic spinal cord injury, spinal muscular atrophy, and combinations thereof. In particularly preferred embodiments, the neuromuscular disorder is spinal muscular atrophy (SMA).

In some embodiments, the SMA is Type II or Type III SMA.

According to a ninth aspect of the invention, there is provided a method for treating a neuromuscular disorder in a subject, said method comprising administering the nucleic acid molecule of the first aspect of the invention or the vector of the second aspect of the invention to a subject in need thereof.

Statements of invention for the other aspects of the invention may also be applied mutatis mutandis to the ninth aspect of the invention. Statements of invention below for the ninth aspect of the invention may also be applied mutatis mutandis to the other aspects of the invention.

In some embodiments, the method comprises administering the pharmaceutical composition of the fifth aspect of the invention to the subject.

In some embodiments, the step of administering the nucleic acid molecule or vector may comprise intravenously and/or intrathecally administering the vector to the subject. The nucleic acid molecule or vector may be injected into the spinal cord of the subject. The nucleic acid molecule or vector may be delivered to a plurality of sites in the spinal cord.

Intrathecal administration provides nucleic acid molecule or vector delivery past the blood-brain barrier directly to the cerebrospinal fluid.

In some embodiments, the nucleic acid molecule or vector may be administered by an administration route independently selected from the group comprising: oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, and by another parental route.

The subject is preferably a mammal, and more preferably a human. In some embodiments, the subject is an adult. In other embodiments, the subject is a child under the age of 18.

In some embodiments, the subject has a neuromuscular disease, preferably SMA.

In some embodiments, the subject has SMA type II or III. In some embodiments, the subject comprises bi-allelic SMN1 null mutations or inactivating deletions, optionally wherein the mutations comprise deletion of exon seven of SMN1. In some embodiments, the subject has three copies of SMN2. In some embodiments, the subject does not have a c.859G>C substitution in exon 7 on at least one copy of the SMN2 gene. In some embodiments, the subject in need thereof is determined by one or more genomic tests.

In some embodiments, the nucleic acid molecule or the vector may be delivered by a method that is independently selected from: transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection, and protoplast fusion.

In some embodiments, the nucleic acid molecule or the vector is administered with another therapeutic. In some embodiments, the method further comprises administering a second therapeutic agent to the patient concomitantly or consecutively with the administration of the nucleic acid molecule or vector. In some embodiments, the second therapeutic agent comprises a muscle enhancer or neuroprotector. In some embodiments, the second therapeutic agent comprises an antisense oligonucleotide or antisense oligonucleotides targeting SMN1 and/or SMN2. In some embodiments, the second therapeutic agent comprises nusinersen and/or stamulumab.

In some embodiments, the nucleic acid molecule or vector is administered together with a contrast medium, optionally wherein the contrast medium comprises iohexol. In some embodiments, the volume of contrast medium administered is about 1-2 mL. In some embodiments, the contrast medium is mixed with the nucleic acid molecule or vector prior to administration. The contrast medium may be mixed with the nucleic acid molecule or vector less than 24 hours prior to administration, or less than 12, 6, 5, 4, 3, 2, or less than 1 hour prior to administration, or less than 30 minutes prior to administration or immediately prior to administration. In some embodiments, the contrast medium and nucleic acid molecule or vector are administered sequentially. In some embodiments, the nucleic acid molecule or vector may be administered after the contrast medium. In some embodiments, the nucleic acid molecule or vector may be administered before the contrast medium. In some embodiments, the nucleic acid molecule or vector is administered at a dosage of from IxlO¹⁰ GC/g brain mass to about 3xl0¹⁴ GC/g brain mass. The nucleic acid molecule or vector may be administered at a dosage of about 5xl0¹³ GC. The nucleic acid molecule or vector may be administered at a dosage of around 1.85xl0¹⁴ GC.

In some embodiments, the nucleic acid molecule or vector is administered at a dosage of from IxlO¹³ vg to 5xl0¹⁴ vg. In some embodiments, the nucleic acid molecule or vector is administered at a dosage of from 5xl0¹³ vg to 3xl0¹⁴ vg. In some embodiments, the nucleic acid molecule or vector is administered at a dosage of up to 6xl0¹³ vg, or of about 6xl0¹³ vg. In some embodiments, the nucleic acid molecule or vector is administered at a dosage of up to 1.2xl0¹⁴ vg, or of about 1.2xl0¹⁴ vg. In some embodiments, the nucleic acid molecule or vector is administered at a dosage of up to 2.4xl0¹⁴ vg, or of about 2.4xl0¹⁴ vg.

In some embodiments, the nucleic acid molecule or vector is administered in a unit dose comprising from 1X10¹³-9.9X10¹⁴ vg. In some embodiments, the nucleic acid molecule or vector is administered in a unit dose comprising from IxlO¹³ to 5xl0¹⁴ vg. In some embodiments, the nucleic acid molecule or vector is administered in a unit dose comprising 5xl0¹³ to 3xl0¹⁴ vg. In some embodiments, the nucleic acid molecule or vector is administered in a unit dose comprising 6xl0¹³ vg. In some embodiments, the nucleic acid molecule or vector is administered in a unit dose comprising about 1.2xl0¹⁴ vg. In some embodiments, the nucleic acid molecule or vector is administered in a unit dose comprising about 2.4xl0¹⁴ vg.

In some embodiments, the method comprises administering the nucleic acid molecule or vector more than once. The method may comprise administering the nucleic acid molecule or vector twice, three times, four times, or more.

According to a tenth aspect of the invention there is provided a protein encoded by the nucleic acid molecule of the first aspect of the invention.

According to an eleventh aspect of the invention there is provided use of the nucleic acid molecule of the first aspect of the invention in the treatment of SMA, preferably SMA type II or SMA type III. Statements of invention for the other aspects of the invention may also be applied mutatis mutandis to the tenth and eleventh aspects of the invention.

Detailed Description of the Invention

In order that the invention may be more clearly understood embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1 Maps displaying features of the transfer plasmids encoding Co-hSMNl or control eGFP. The constructs used in transfer plasmids to produce (A-D) lentiviral or (E,F) adeno-associated viral (AAV) vectors are shown.

Figure 2 Characterisation of cortical and motor neurons in culture. (A) 6 day-old mouse cortical neuron cultures fixed and stained with neuron marker (NeuN). Nuclei stained with DAPI. (B) 72-hours post-seeding, rat motor neurons fixed and immunostained for a common motor neuronal marker (ChAT). Scale bars = 100 pm.

Figure 3 Lentiviral vector-mediated hSMNl and Co-hSMNl expression in mouse primary cortical neurons and rat primary motor neurons. (A) Western blot analysis of qPCR MOI 30 and 100 used to transduce mouse cortical neuronal cultures with IPLVs and IDLVs encoding CMN _hSMNl, CMV_Co-hSMNl, hS N JiSMNl or hS N_Co-hSMNl cassettes. (B) SMN expression (arbitrary units) versus vector promoter and MOI for mouse primary cortical neuronal cultures transduced with IPLVs and IDLVs encoding CMNJiSMNl, CMV_Co-hSMNl, hS N JiSMNl or hSYN _Co-hSMNl cassettes. (C) Immunofluorescence images showing examples of motor neurons transduced at MOI 60, 72h posttransduction. Scale bars = 20 pm. (D) Quantification of SMN immunofluorescence in cell bodies of transduced or control E14 rat primary motor neurons. Error bars represent standard deviation. N=3 biological replicates were collected in each case. Figure 4 Assessment of SMN protein levels in iPSC motor neurons. (A) Representative images of mature, SMA type I iPSC-derived motor neurons at both high and low seeding density. Scale bar = 100 pm (high density, top image) and 50 pm (low density, bottom image). (B) Immunofluorescence images of control and IDL _CM _Co-hSMNl- transduced SMA type I iPSC motor neurons. Scale bar = 20 pm (top image) and 50 pm (bottom image). (C) Quantification of western blots showing SMN expression (arbitrary units) versus vector. Error bars represent standard deviation. Significance represented by stars on transduced samples indicates a comparison to the control SMN levels in that particular line. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. N=3 biological replicates were collected for each line, as well as three independent lines for each genotype used.

Figure 5 Characterisation of iPSC-derived motor neurons. Representative images of motor neuron cells at different stages of the differentiation protocol. (A) OLIG2-positive motor neuron progenitors at day 16 of differentiation. (B-D) Mature motor neurons express (B) SMI-32 and pill-tubulin, (C) HB9 and (D) ChAT. All counterstained with DAPI.

Figure 6 Determining SMN transcript origin and SMN protein levels in iPSC- derived motor neurons. (A) RT PCR showing full length SMN (FL- SMN) products (504bp) and SMNA7 transcripts (450bp). (B) RT PCR showing amplification of two control gene products (GAPDH: 184bp and P-actin: 295bp). The same lane order is present in all gels. (C) RT PCR of the two bands at 504 and 450bp in (A) Cleavage products: FL- SMN2 (504bp) = 382 and 122bp, SMN2A7 (450bp) = 328 and 122bp. (D) Quantification of western blots showing SMN expression (arbitrary units) versus iPSC line.

Figure 7 SMN levels in primary SMA type I patient fibroblasts following IDEV transduction. (A) Representative immunofluorescent images of wildtype and SMA type I fibroblasts after IDL _CM _Co-hSMNl transduction at qPCR MOI 75 and 100, plus control. Scale bars = 50 pm in all images. (B) Western blots from cells harvested 72h posttransduction with IDLVs at MOI 75 and 100. (C) Quantification of western blots. Error bars represent standard deviation. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. N=3 biological replicates were collected in each case.

Figure 8 Restoration of gems in SMA type I fibroblasts transduced with lentiviral vectors encoding hSMNl or Co-hSMNl . Quantification of gems in control human fibroblasts, non-transduced and SMA type I cells transduced at MOI 100 is displayed. Error bars represent standard deviation. N=3 biological replicates were collected in each case.

Figure 9 The effect of IDLV _CM V _Co -hSMNl transduction on yH2AX foci in SMA type I fibroblasts. (A) Quantification of the number of foci per cell and (B) percentage of foci-positive cells. Error bars represent standard deviation. * P<0.05, ** P<0.01. N=3 biological replicates were collected in each case with each technical replicate quantifying at least n=25 cells.

Figure 10 ATM and pATM in wild-type and SMA type I fibroblasts and SMA type I iPSC-derived motor neurons. Quantification of western blots using protein lysates from wild-type, SMA type I fibroblasts and SMA type I fibroblasts treated with 200 pM hydrogen peroxide (H2O2) for 2 hours prior to lysis assessing (A) ATM and (B) pATM levels. (C) Transduction of SMA type I fibroblasts with either IDLV_CM V_<?G/-7^J or IDLV _CM V _Co-hSMNl (both MOI 75) for either 3 or 7 days before harvest and pATM western blot. (D,E) Quantification of ATM and pATM western blots from three independent lines of SMA type I iPSC- derived motor neurons transduced at maturity with IDLV_CMV_Co- hSMNl (MOI 75) and harvested 3 days post-transduction. Error bars represent standard deviation. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. N=3 biological replicates were collected in each case..

Figure 11 Analysis of SMN levels following in vivo neonatal administration of AAV9 vectors expressing Co-hSMNl. (A) Results for liver. (B) Results for spinal cords. SMN protein levels were normalised to those in wild- type samples in all cases. Error bars represent standard deviation. * P<0.05, ** P<0.01. Wild-type n=4, untreated Smn^2B/' n=3, Smn^2B/' + AAV9_CAG_cGT7^J n=5, Smn^2B/- + AAV9_CAG_Co-hSMNl n=5 biological replicates.

Figure 12 Levels and distribution of SMN in heart tissues from Smn^2B/~ mice following administration of AAV9 vectors expressing Co-hSMNl . (A) Representative western blots of SMN levels in heart tissues from untreated Smn^2B/~ mice, Smn^2B/~ mice following AAV9-mediated treatment with and without SMN1 plus a corresponding age-matched WT mouse (Pl 8). The bar graph represents average SMN levels expressed relative to the corresponding WT mouse. (B) Representative immunofluorescence for SMN staining within heart tissues from untreated Smn^2B/~ mice and Smn^2B/~ mice following treatment with AAV9_Co-hSMNl . ***p<0.001. Scale bars represent 75 pm.

Figure 13 Levels and distribution of desmin in heart tissues from Smn^2B/~ mice following administration of AAV9 vectors expressing Co-hSMNl. (A) representative western blots of desmin levels in heart tissues from WT mice (Pl 8), untreated Smn^2B/~ mice, and Smn^2B/~ mice following AAV9- _Co-hSMNl treatment. The bar graph represents average desmin levels expressed relative to WT mice. (B) Representative western blots of desmin levels in heart tissues from untreated Smn^2B/~ mice and Smn^2B/~ mice following AAV9 treatment without SMN1 (control, AAV9- _eG P). The bar graph represents average desmin levels expressed relative to untreated Smn^2B/~ mice. (C) Representative immunofluorescence for desmin staining within heart tissues from WT mice (Pl 8), untreated Smn^2B/~ mice and Smn^2B/~ mice following AAV9- _Co-hSMNl treatment. Corresponding bar graph reflects the area of cells stained for desmin corrected for number of cells present (DAPI stain) as determined by ImageJ analysis and expressed relative to corresponding WT mice. Dashed line represents the average desmin levels in WT mice and error bars represent the standard deviation from the mean. * p<0.05; **p<0.01; ***p<0.001. Scale bars represent 75 pm (panels a-c), and 25 pm (panels e-g).

In the description above, the term "identity" is used to refer to the similarity of two sequences. For the purpose of this invention, it is defined here that in order to determine the percent identity of two nucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first nucleic acid for optimal alignment with a second nucleic acid sequence). The nucleotide residues at nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e. overlapping positions) x 100).

Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used.

Materials and Methods

Optimisation ofhSMNl sequence

The wild-type sequence of the human SMN1 transcript was codon-optimised to generate a nucleic acid molecule of the invention comprising a codon-optimised nucleotide sequence encoding human survival motor neuron 1 protein having at least 96% identity with SEQ ID NO:1. In this particular embodiment, the nucleotide sequence had 100% identity with SEQ ID NO: 1 - however, in other embodiments, the nucleic acid molecule of the invention may have anywhere between 96 and 100% identity with SEQ ID NO:1. The nucleic acid molecule was cloned into lentiviral and AAV transfer plasmid using standard molecular biology procedures.

Fibroblast cell culture

Low passage, primary human fibroblasts from wild-type (GM04603) and SMA type I (GM00232) donors were obtained from Coriell Institute for Medical Research and used to assess overall lentiviral transduction efficiency, yH2AX and caspase 3 foci, and ATM and pATM levels. Similar wild-type and SMA type I fibroblast cell lines were also obtained from E. Tizzano and used to assess restoration of gems following transduction. All fibroblasts were cultured in 65% DMEM+Glutamax, 21% M199, 10% FBS, 10 ng/ml FGF2, 25 ng/ml EGF and 1 pg/ml gentamicin.

Isolation and culture of El 8 mouse cortical neurons

Preparation of primary cortical cultures from El 8 mouse embryos followed the protocol described in Lu-Nguyen et al. Human Gene Therapy. 2015;26:719-33.

Preparation of embryonic rat motor neuron primary cultures

The isolation and culture of primary rat motor neurons was achieved by following the protocol previously described in Peluffo et al. Gene Ther. 2013;20(6):645-57. iPSC culture and motor neuron differentiation

Six iPSC lines were used in this project; three wild-type (4603, derived in house from GM04603 fibroblasts; 19-9-7T, from WiCell and AD3-CL1, gifted by Majlinda Lako) and three SMA type I (SMA-19, gifted by Majlinda Lako; CS13iSMALnxx and CS32iSMALnxx, obtained from Cedars-Sinai). Undifferentiated iPSCs were seeded at a density of 20,000 cells/cm² onto Matrigel-coated cultureware in mTeSR™l or mTeSR™ Plus media for general growth. iPSCs were grown until 90% confluent in 6 well plates then clump passaged with 0.5mM EDTA to Matrigel-coated 10cm dishes until 60-70% confluent. A protocol adapted from Maury et al. Nature Biotechnol. 2015;33(l):89-96 was used to differentiate iPSCs into motor neurons. Basal medium (IX DMEM/F12, IX Neurobasal, IX B27, IX N2, IX antibiotic-antimycotic, IX /i-mercaptoethanol and 0.5 pM ascorbic acid) was used throughout the 28-day protocol. Basal medium was supplemented at specific stages with additional compounds: 3 pM Chir99021 (days 0- 3), 1 pM Compound C (days 0-3), 1 pM retinoic acid (day 3+), 500 nM SAG (day 3+), 0.5 pg/ml laminin (day 16+), 10 ng/ml each of IGF1, CNTF, BDNF, GDNF (all day 16+) and 10 pM DAPT (days 16-23). Single cell passaging on days 9, 13 (1:3 split ratio) and 16 (at appropriate density for final assay) was performed using Accutase and cells were re-seeded onto Matrigel-coated cultureware in the presence of 10 pM ROCK inhibitor for 24 hours.

Viral vector production

A 3^rd generation, transient transfection system was used to generate self-inactivating HIV- 1-based lentiviral vectors by calcium phosphate co-transfection of HEK293T/17 cells with pMDLg/pRRE (SEQ ID NO: 12) or pMDLg/pRRE_intD64V (SEQ ID NO: 13) (for integrating and non-integrating vectors, respectively), pRSV_REV (SEQ ID NO: 14), pMD2_VSV-G (SEQ ID NO: 15) and a transfer plasmid containing the promoter of interest and either hSMNl , Co-hSMNl of the invention or eGFP at a 1 : 1 : 1 :2 ratio, respectively. Other suitable plasmids may be used instead of the above-mentioned plasmids, and such plasmids may be obtained from Addgene, Massachusetts, USA, for example). Supernatants were harvested at 48- and 72-hours post-transfection and lentiviral vectors were concentrated by ultracentrifugation. Vectors were titrated by qPCR and where possible, by flow cytometry.

AAV _CAG_Co-hSMNl vectors of the invention and AAV _CAG_eGFP control vectors were produced and were titrated by qPCR against the inverted terminal repeats (ITRs).

Viral transduction in cell culture

For transduction of cell lines and primary fibroblasts, cells were seeded in appropriate media 24 hours prior to transduction. Lentiviral vectors were diluted in fresh media at the desired qPCR MOI then added to cells in the minimum volume needed to cover cells. 1 hour after transduction, media was topped up to an appropriate volume. All cells were incubated for 72-hours before analysis. Fibroblasts were transduced in the presence of 2 pg/ml polybrene. iPSC-derived motor neurons were transduced at day 28 of differentiation to ensure maturity of cells. Transduction of primary motor neurons was carried out 2 hours post-seeding, while for primary cortical neurons it was three weeks post-seeding. Lentiviral vectors were diluted in conditioned media at the desired qPCR MOI. Analyses were performed three days post-transduction.

Viral transduction in vivo

Single-stranded AAV9 vectors (AAN9_C AG _Co-hSMNl (of the invention) & AAV9_CAG_eGFP (control)) were administered intravenously through the facial vein to post-natal day (P) 0 Smn^2B/~ SMA mice at a dose of 8E10 vg/pup. Liver and spinal cord were harvested at Pl 8 from untreated Smn^2B/~ mice (n=6), Smn^2B/~ mice treated with AAV9_CAG_eGFP (n=5) or AAV9_CAG_Co-hSMNl (n=5) and age-matched wild-type controls (n=4). At P18 there are overt symptoms in untreated Smn^2B/~ mice.

Single-stranded AAV9 vectors (AAV9_C AG _Co-hSMNl (of the invention) & AAV9_CAG_eGFP (control)) were administered intravenously through the facial vein to post-natal day (P) 0 Smn^2B/~ SMA mice at a dose of 5E10 vg/pup - 7E10 vg/pup. Following CO2 anesthesia and exsanguination, heart tissues were harvested at P18 from untreated Smn^2B/' mice (n=6), Smn^2B/' mice treated with AAV9_CAG_eGFP (n=5) or AA 9 _C AG _Co-hS VI N I (n=5) and age-matched wild-type controls (n=4).

Experimental procedures were authorized and approved by the Keele University Animal Welfare Ethical Review Body (AWERB) and UK Home Office (Project Licence P99AB3B95) in accordance with the Animals (Scientific Procedures) Act 1986.

RT-PCR

An RT-PCR was performed using cDNA extracted from SMA iPSC motor neurons to identify the origins of SMN transcripts. The primers used to amplify a region between exons 6-8 of the SMN genes, plus [3-actin and GAPDH as housekeeping genes were as follows:

Exon6_F CTCCCATATGTCCAGATTCTCTTG (SEQ ID NO:2)

Exon8_R CTACAACACCCTTCTCACAG (SEQ ID NOG) p-actin_F TCACCCACACTGTGCCCATCTACGA (SEQ ID NO:4) p-actin_R CAGCGGAACCGCTCATTGCCAATGG (SEQ ID N0:5) 189_mGapdhex4_Fw AAAGGGTCATCATCTCCGCC (SEQ ID N0:6) 190_mGapdhex4-5_Rv ACTGTGGTCATGAGCCCTTC (SEQ ID N0:7)

SMN RT-PCR amplicons were digested with Dde to reveal FL-SMN1 (504bp), FL- SMN2 (382+122bp) and SMN2A7 (328+122bp) transcripts.

Immunofluorescence

Fibroblasts were fixed with 4% PFA before being concurrently permeabilised and blocked in 5% normal goat serum in PBS with 0.25% Triton X-100. Primary and secondary antibodies were incubated with samples overnight at 4°C or 1 hour at room temperature, respectively. iPSC motor neurons were seeded at a density of 25,000 cells on day 16 of differentiation onto 13 mm coverslips coated with 15 pg/ml poly-omithine and Matrigel. 4% PFA and 5% normal goat serum in PBS with 0.25% Triton X-100 were used to fix, permeabilise and block coverslips before antibody incubation at room temperature for both primary (2 hours) and secondary (1 hour). All cells were counterstained with 1 pg/ml DAPI, mounted using Fluoromount™ Aqueous mounting medium then imaged using a Zeiss Axio Observer DI fluorescent microscope (Germany).

Primary antibodies: anti-gemin2 (Abeam, ab6084, 2.5 pg/ml), anti-SMN (BD Biosciences, 610646, 0.6 pg/ml), anti-OEIG2 (Santa Cruz, sc-515947, 2 pg/ml), anti- SMI-32 (Biolegend, 801701, 10 pg/ml), anti-/>III-tubulin (Sigma, T2200, 10 p/ml), anti-choline acetyltransferase (Abeam, abl81023, 5.4 pg/ml), anti-HB9 (DSHB, 81.5cl0, 1:50). Secondary antibodies: goat anti-mouse IgG Alexa Fluor 488 (Invitrogen, A-11001, 2 pg/ml), goat anti-mouse IgG Alexa Fluor 555 (Invitrogen, A- 21424, 2 pg/ml), goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen, A- 11034, 2 pg/ml).

Heart tissues were flash frozen in liquid nitrogen and stored at -80°C then sectioned (7 pm) on a rotary cryostat, collected onto polylysine-coated slides and stored at -20°C. Prior to staining, slides were brought to room temperature. All subsequent steps were carried out at room temperature and PBS used for each wash step (3 x 5 min) whilst blocking buffer (1% FBS, 1% HS, 0.1% BSA in PBS) was used for antibody dilution. Sections were washed, then blocked for 30 min prior to being incubated either for 2 h at room temperature or overnight at 4°C with primary antibody (mouse anti-SMN (MANSMA12 2E6; 1:4), rabbit monoclonal anti-desmin (Abeam; ab227651; 1:100)). Following washing, 5 pg/ml of secondary antibody (Molecular Probes; goat anti-rabbit IgG ALEXA Flour 488; Al 1034) was applied for 1 h. Sections were washed prior to being stained with 4’,6-diamidino-2-phenylindole (DAPI; D9542; 0.4 pg/mL) for 10 min, then washed, mounted and imaged with a Leica TCS SP5 spectral confocal microscope (Leica Microsystems, Milton Keynes, UK). Measurement ofSMN intensity by immunofluorescence

Analyses of all samples were performed blind to vector type, gene of interest and MOI. Fluorescence pixel intensities (background corrected) were measured in a region of interest around the motor neuron cell body and are expressed as arbitrary units (a.u.) 2 per pm .

Western blotting

Cultured cells were lysed in RIPA buffer supplemented with Halt Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 3 and the concentration of resulting protein lysates was determined using the Bio-Rad DC protein assay according to manufacturer’s instructions. SMN western blots used 4-15% Tris-Glycine gels and PageRuler™ Plus Prestained Protein Ladder, whilst ATM and phosphorylated ATM western blots used NuPAGE™ 3-8% Tris-Acetate gels and HiMark™ Pre-stained protein standard. Western blots containing samples from iPSC motor neurons were subjected to total protein staining immediately after transfer using REVERT Total Protein Stain and Wash, as per manufacturer’s instructions. Nitrocellulose membranes were blocked in an appropriate buffer (Intercept® 1:1 PBS, 5% milk/PBS or 5% BSA/PBS) for 1 hour at room temperature. Primary and secondary antibodies were diluted in blocking buffer 0.1% Tween-20, with incubations overnight at 4°C or 1 hour at room temperature, respectively. Western blots were imaged using the Odyssey CLx (LLCOR Biosciences, US) in 700nm and 800nm channels. Quantification of protein signals was achieved using Image Studio Lite.

Primary antibodies: anti-SMN (BD Biosciences, 610646, 0.05 pg/ml), anti-ATM (Abeam, ab32420, 0.12 pg/ml), anti-ATM phospho (Abeam, ab81292, 0.28 pg/ml), anti-alpha tubulin (Abeam, ab4074, 0.33 pg/ml). Secondary antibodies: IRDye 800CW goat anti-mouse IgG (LiCor, 926-32210, 0.5 pg/ml), goat anti-rabbit IgG Alexa Fluor 680 (Invitrogen, A-21076, 0.4 pg/ml).

Western blots were carried out on liver and spinal cord tissues from Sinn²¹¹' mice and heart tissues from Smn^2B/' mice, which were extracted as previously described (Soltic D et al. Brain Sciences. 2018;8(212)) using 2X modified RIPA buffer (2% NP-40, 0.5% deoxycholic acid, 2 mM EDTA, 300 mM NaCl and 100 mM Tris-HCl (pH 7.4)). Firstly, the tissues were diced and added to the extraction buffer and homogenized with pellet pestles, then, after 5 minutes on ice, the tissues were sonicated at 5 microns for 10 s. This process was repeated a further 2 times. The tissue extracts were centrifugated at 13,000 RPM (MSE, Heathfield, UK; MSB010.CX2.5 Micro Centaur) for 5 minutes at 4°C and their protein concentrations calculated using a BCA protein assay (PierceTM, 23227). Following adjustment of protein levels, the tissue extracts were heated for 3 minutes at 95°C in 2X SDS sample buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.125 M Tris-HCl (pH 6.8) and bromophenol blue) then loaded onto 4-12% Bis-Tris polyacrylamide gels for SDS-PAGE. The gel was excised along the horizontal axis at a molecular weight greater than that expected for SMN (38 kDa) and the proteins in the lower half of the gel were transferred onto a nitrocellulose membrane overnight via western blot then blocked with 4% powdered milk in PBS. The membranes were probed for SMN with the mouse anti-SMN antibody (MANSMA122E6), at either 1:50 or 1:100 (and rabbit anti-desmin (Abeam; ab227651; 1:1000) for desmin analysis) for 2 hours and subsequently incubated with HRP-labelled rabbit anti-mouse Ig (DAKO, P0260), or goat anti-rabit Ig (DAKO, P0488), at 0.25 ng/ml for Ih. Both incubations were at room temperature and antibodies prepared in diluent (1% FBS, 1% horse serum (HS), 0.1% bovine serum albumin (BSA) in PBS with 0.05% Triton X-100).

Following incubation with West Pico, SMN-positive bands were imaged with the Gel Image Documentation system (Bio-Rad). Total protein was assessed in the upper half of the gel via Coomassie blue staining, and this data was used as the internal loading control for each sample. ImageJ Fiji software (vl.51) was used to analyse both antibody reactive and Coomassie- stained gel bands.

Statistical analyses Data are presented as mean ± standard deviation. For all experiments where replicate data are presented, at least n = 3 biological replicates were used, unless otherwise stated in specific sections. A range of statistical tests were used, with the most appropriate test for each dataset being determined individually. Data were tested for a normal distribution wherever possible, and appropriate parametric and non-parametric tests were used accordingly.

Results

Lentiviral andAAV9 vectors used for over-expression o/hSMNl

To test whether production of SMN could be improved by codon-optimisation of hSMN 7, a wild-type hSMNl cDNA was used and an optimised form of the invention was engineered using a customised commercial procedure. Both cDNAs were cloned into several lentiviral plasmid backbones under the control of CMV, hSYN and hPGK promoters and in all cases, followed by a mutated form of the WPRE sequence (to prevent putative expression of woodchuck hepatitis virus X protein; Fig. 1A-C). These transfer plasmids were used to produce integrating and integration-deficient lentiviral vectors. Finally, an embodiment of a nucleic acid molecule of the invention comprising a codon optimised nucleotide sequence encoding human survival motor neuron 1 protein which had 100% identity with SEQ ID NO:1 was also cloned into an AAV plasmid backbone under the control of the CAG promoter, followed by a mutated WPRE element (Fig. IE). In other embodiments, nucleic acid molecules of the invention may have nucleotide sequences having at least 96% identity with SEQ ID NO:1. The plasmid into which the nucleic acid molecule of the invention was cloned, as well as a control AAV_CAG_eGFP plasmid (Fig. IF), were used to produce singlestranded AAV9 vectors for in vivo use. Each plasmid encoded the Co-hSMNl transgene of the invention or eGFP control transgene, flanked upstream by a promoter (CMV, hSYN, hPGK or chicken beta-actin CMV hybrid (CAG)) and downstream by woodchuck hepatitis post-transcriptional regulatory element (WPRE; mutated in constructs A-C and E), a post-transcriptional element that improves transgene expression (except in the case of AAV_CAG_eGFP (Fig. IF)).

Over-expression of a codon-optimised nucleotide sequence of the invention encoding hSMN 1 protein in primary neuronal cultures Mouse cortical neuron cultures and rat motor neuron cultures were characterised as shown in Fig. 2, demonstrating the expected morphology and the presence of relevant markers. Integration-proficient (IPLV) and integration-deficient (IDLV) lentiviral vectors driven by the CMV or hSYN promoters, encoding either wild-type hSMNl or a codon-optimised nucleotide sequence of the invention encoding hSMNl protein and having 100% identity with SEQ ID NO:1, were used to transduce the cultures (Fig. 3). 3-week old mouse primary cortical cultures and isolated motor neuron cultures from E15 rat embryos were transduced with IPLVs and IDLVs encoding CMV_hSMNl, CMV_Co-hSMNl, hSYN_hSMNl or hSYN_Co-hSMNl cassettes, with cells collected at 72h post-transduction. Dose-dependent increases in mean SMN fluorescence intensity were seen by western blot in cortical neurons and immunofluorescence in motor neurons (Fig. 3B,D). IPEV delivery led to higher expression levels than with IDEVs, but SMN protein levels from the latter were also considerably elevated. In terms of the promoter, CMV resulted in higher SMN levels regardless of vector integration proficiency. The codon-optimised nucleotide sequence of the invention led to significant increases in SMN production in all cases, highlighting the improvements that this technology can afford for transgenic gene expression.

Characterisation of IDLVs of the invention in human iPSC-derived motor neurons

Three different wild-type and three SMA type I iPSC clones were differentiated into motor neurons with high efficiency, exhibiting a characteristic neural network and individual cellular morphology (Fig. 4A) with >90% OEIG2 positive MN progenitors at day 16 and 77.3% SMI-32-, 61.4% HB9- and 90.1% ChAT -positive motor neurons at maturity (Fig. 5). An RT PCR was performed using primers to amplify a region between exons 6-8 of the SMN genes in iPSC-derived MNs. -RT = minus reverse transcriptase control reaction. A lack of full-length SMN1 transcripts (Fig. 6) and an 18-fold reduction in SMN protein (Fig. 6) were evident in SMA type I motor neurons compared to wild-type cells (P<0.0001).

Transduction of SMA type I iPSC-derived motor neurons with IDLV _Co-hSMNl (a vector of the invention) driven by CMV, hSYN or PGK promoters led to an increase in SMN protein levels, detected by both immunofluorescence (Fig. 4B) and western blot (Fig. 4C). Quantitation of western blot data showed that SMN protein was increased in all transduced samples compared to untransduced counterparts (Fig. 4C). IDLVs expressing survival motor neuron protein 1 and comprising a nucleic acid molecule of the invention (comprising a nucleotide sequence having 100% identity with SEQ ID NO:1) under the transcriptional control of either CMV or hPGK promoters were able to significantly increase SMN protein production in all iPSC motor neuron lines (Fig. 4C). IDLV_hSYN_Co-/i.S'A7A^// of the invention led to a significant increase in CS13iSMAI-nxx. Maximal SMN protein levels were observed with IDLVs expressing survival motor neuron protein 1 and comprising a nucleic acid molecule of the invention (comprising a nucleotide sequence having at least 96% identity with SEQ ID NO:1) under the transcriptional control of CMV (line SMA-19: 79.8-fold, P<0.0001; CS13iSMAI-nxx: 14.5-fold, P<0.0001; CS32iSMAI-nxx: 42.8-fold, P<0.0001). When levels were compared to those in wild-type iPSC motor neurons, supraphysiological SMN protein was evident in SMA-19 and CS32iSMAI-nxx lines, but not in CS13iSMAI-nxx.

Transduction and rescue of human SMA type I fibroblasts by lentiviral vectors of the invention

Cultured human wild-type or type I SMA fibroblasts were transduced with IDLVs encoding wild-type hSMNl or expressing survival motor neuron protein 1 and comprising a nucleic acid molecule of the invention (comprising a nucleotide sequence having 100% identity with SEQ ID NO:1) under CMV, hSYN or hPGK promoters. A clear increase in cytoplasmic SMN was seen by immunofluorescence in both wild-type and SMA type I fibroblasts following IDLV transduction (Fig. 7A) and a statistically significant increase was confirmed by western blot (Fig. 7B,C). Analysis of total SMN levels in transduced fibroblasts (Fig. 7C) corroborated the pattern of expression seen in SMA type I iPSC-motor neurons (Fig. 4D), where CMV-driven vectors were able to increase SMN expression to the highest extent, followed by hPGK and then hSYN- driven vectors.

SMA type I fibroblasts were transduced with IPLVs and IDLVs to determine the effectiveness of each vector to restore SMN-expressing nuclear gems, which are largely absent in SMA type I samples. Cultured human SMA type I fibroblasts were transduced with IPLVs or IDLVs encoding CMV_hSMNl, CMV_Co-hSMNl, hS N JiSMNl or hSYN _Co-hSMNl cassettes at qPCR MOI 30, 60 or 100. The number of gems present in 100 nuclei was quantified 72h post-transduction. All vectors were able to restore the presence of gems in transduced cells (Fig. 8 A) in an MOI-dependent manner (Fig. 8B). At the highest MOI tested (MOI 100), no visible changes in cell morphology were seen, suggesting absence of vector-mediated toxicity. IPLV transduction led to a 1.6-fold greater number of gems than in IDLV-transduced cells (P=0.0015), regardless of promoter or transgene (Fig. 8B). Moreover, hSMNl encoded by a codon optimised nucleotide sequence of the invention having at least 96% identity with SEQ ID NO:1 led to the restoration of a significantly higher number of gems than wild-type hSMNl (1.7-fold, P=0.0005). With regards to choosing the optimal promoter, CMV-driven vectors were able to increase gem number by 1.8-fold compared to hSYN-driven vectors (P= 0.0003). In some cases, a higher number of gems was seen in transduced SMA type I fibroblasts than in healthy cells.

Analysis of downstream DNA damage markers following in vitro IDLV transduction

The molecular links between SMN and DNA damage- and apoptosis-related proteins are not completely clear but learning how SMN interacts with these pathways may be important in understanding why SMA motor neurons degenerate and how this could be modulated by treatment with a YWA-cncoding vector. It is also important to understand the consequences of SMN restoration to wild-type or supraphysiological levels, and what effect this might have on cells that have always been severely deficient in SMN.

The effect of IDLV _CMV _Co -hSMNl transduction on yH2AX foci in SMA type I fibroblasts was assessed. SMA type I fibroblasts were immunostained for yH2AX 72h post-transduction with IDLV _CMV _Co-hSMNl at MOI 75. yH2AX foci are hallmarks of DNA damage and immunofluorescent detection of these in untreated wild-type and SMA type I fibroblasts revealed distinct foci in nuclei of both genotypes, but these were seen more frequently in SMA type I cells. Both the number of foci per cell and the percentage of cells exhibiting any number of foci were significantly higher in SMA type I samples (Fig. 9A,B; P=0.0057 and P=0.0069, respectively). Upon transduction of SMA type I fibroblasts with IDLV _CMV _Co-hSMNl (the IDLV vector of the invention shown to be most potent in previous experiments), signs of DNA damage were increased further as the number of yH2AX foci, and yH2AX foci-positive cells increased significantly, compared to mock-treated SMA type I cells (Fig. 9A,B; P=0.0134 and P=0.0068, respectively).

ATM, specifically its phosphorylated form, acts as a chief mobiliser of cellular DNA damage and apoptotic pathways that may be active in SMA cells. Levels of total ATM were found to be equal in both wild-type and SMA type I fibroblasts according to quantitated western blots (Fig. 10A; P=0.6662), with the phosphorylated form only showing a trend for increased signal in the mutant cells (Fig. 10B; P>0.05). Phosphorylated ATM could be significantly increased by treatment of the cells with 200 pM hydrogen peroxide for 2 hours (Fig. 10B; wild-type vs SMA+H2O2 P<0.01, SMA vs SMA+H2O2 P<0.05). Following transduction of SMA type I fibroblasts with either IDLV_CMV_eGFP (control) or IDLV _CMV _Co-hSMNl (of the invention), phosphorylated ATM was assessed. At 3 days post-transduction, pATM was significantly increased in IDLV_CMV_eGFP treated cells, but not in IDLV_CMV_Co- hSMNl (Fig. 10C; P=0.0160 and P=0.4983, respectively). pATM remained relatively high in IDLV_CMV_eGFP treated cells at 7 days post-transduction (Fig. 10C; P=0.0002), whereas in IDLV_CMV_Co-/i.S'A7A/ -transduced cells dropped below that of mock samples (Fig. 10C; P=0.0256). Of note, no increase in levels of cleaved caspase 3, a marker of DNA damage and apoptosis, were observed in ID N _Co-hSMNl- transduced SMA type I fibroblasts. ATM and pATM levels were also measured in SMA type I iPSC-derived motor neurons, mock-transduced or treated with IDLV_CMV_Co- hSMNl . No effect of transduction on total ATM was observed, but a significant increase in pATM was seen in two out of three SMA type I iPSC-MN lines at 3 days posttransduction (Fig. 10D,E; SMA-19 P<0.0001, CS13iSMAI-nxx P=0.0003, CS32iSMAI-nxx P=0.0160).

In vivo expression from AAV _CAG_Co-hSMNl (vector of the invention) in the Smn^2B/' mouse model of SMA

To test the expression of hSMNl encoded by a nucleotide sequence of the invention which had at least 96% identity with SEQ ID NO: 1, in vivo, we chose the Smn^2B/~ mouse model of SMA, where over-expression of the transgene would be easily detected above low background levels of the protein. An AAV9 vector driven by the CAG promoter and including a mutated WPRE element was produced, and an AAV9_CAG_eGFP vector used as a control. These vectors were delivered to neonatal mice and SMN expression assessed in liver and spinal cord samples harvested at the symptomatic timepoint of Pl 8.

Smn^2B/' neonatal (PO) mice were administered AAV9_CAG_eGFP or A A V9_CAG_Co-/i.S'A7A/ and their livers and spinal cords harvested at the symptomatic time-point of Pl 8 for protein analysis Livers of untreated and AAV9_CAG_eGFP- treated Smn^2B/~ mice showed significantly less SMN than wild-type controls (Fig. 11 A; P=0.0377 and P=0.0118, respectively), whereas those treated with a AAV9_CAG_Co- hSMNl vector of the invention exhibited 1.7-fold of wild-type levels (Fig. 11 A; SMN vs wild-type P=0.0725, SMN vs Smn^2B/' P=0.0005). Data from spinal cord samples showed similarly low levels of SMN in Smn^2B/~ mice, and more variability in AAN9_CAG_Co-hSMNl treated mice, but a 2.6-fold increase above wild-type SMN levels was still seen (Fig. 11B; SMN vs wild-type P=0.5260, SMN vs Smn^2B/' P=0.0162).

Smn^2B/~ neonatal (PO) mice were administered with AVV9_CAG_eGFP or AV 9 _CAG_Co-hSMNl and their heart tissue harvested at the symptomatic timepoint of P18 for protein analysis. Delivery of AAN9_CAG_Co-hSMNl resulted in enhanced levels of SMN (169-fold; p=0.0002) compared to untreated Smn^2B/~ mice with increased levels of SMN beyond that expected in WT mice (~13.50-fold) (Figure 12A). There was no statistically significant difference in SMN expression levels between untreated Smn^2B/' mice and vehicle controls (i.e., AAV9_CAG_eGFP (2.1-fold; p=0.14) (Figure 12A)). Immunohistochemistry analysis of heart tissues from Smn^2B/~ mice revealed increased SMN immunoreactivity following SMN replacement, particularly in those treated with AAN9_CAG_Co-hSMNl (Figure 12B).

Analysis of desmin levels in heart tissues from Smn^2B/' mice following in vitro AAV- mediated SMN1 delivery

Studies have shown widespread protein dysregulation in Smn^2B/~ mouse hearts compared to age-matched WT mice. In previous studies, the intermediate filament protein, lamin A/C has been shown to have increased expression in Smn^2B/~ mice heart tissue, whereas desmin, another filament protein was found to be decreased. The effect of AAV-mediated SMN1 delivery on desmin levels in heart tissues from Smn^2B/~ mice was assessed. Western blot and immunochemistry analysis revealed Smn^2B/~ mice administered with AAV9_CAG_Co-/i.S'A7A restored the decreased expression of desmin in Smn^2B/~ mouse hearts towards WT levels. From western blots, desmin expression levels following delivery of AAV9_CAG_Co-/i.S'A7A were 1.74-fold (p=0.0095) compared to untreated Smn^2B/~ mice and 1.06-fold (p=0.44) compared to WT mice (Figure 13A). There was statistically no significant difference in desmin expression levels between Smn^2B/~ mice and the control AAV9_CAG_eG P (0.99-fold (p=0.88)) (Figure 13B). Desmin immunoreactivity in heart sections from the WT mice was typically present at the Z-disc (Figure 13C). Desmin immunoreactivity in heart sections from Smn^2B/~ mice was clearly reduced, although some striations could still be seen, the staining appearing to be variable in intensity and disorganised. Quantification of desmin immunoreactivity across each heart section from the Smn^2B/~ mice found desmin expression to be significantly decreased compared to age-matched (Pl 8) WT mice (0.61 ± 0.12-fold (p<0.001)), but treatment with AAV9_CAG_Co-hSMN confirmed that desmin levels in Smn^2B/~ mice were increased compared to the untreated Smn^2B/' mice (1.75 ± 0.19-fold (p<0.0001)) (Figure 13C).

Discussion

Gene therapy allows the modification of gene expression for therapeutic purposes, whereby gene addition involves the introduction of a functional transgene into the appropriate cells of the host. Therefore, the efficient delivery of therapeutic genes and appropriate gene expression systems are critical requirements for the development of an effective treatment. Benefits of an optimised system include significant reduction of vector dose needed to maintain transgene expression and lead to sufficient levels of protein production. Therefore, this study aimed to optimise a novel expression cassette for SMA, assessing integrative ability, promoters and transgene sequences for their effect on vector expression.

The in vitro SMN restoration data provides results in line with those reported for existing lentiviral and adenoviral transduction as well as plasmid lipofection and gene targeting. Limited use of lentiviral vectors for in vivo treatment of SMA has previously been reported. The results obtained evidence that a lentiviral expression system can efficiently restore SMN protein levels, especially when expressing the nucleic acid molecule of the invention, comprising a nucleotide sequence encoding human survival motor neuron 1 protein and having at least 96% identity with SEQ ID NO:1. Four seminal papers first demonstrated that viral vector-mediated expression of SMN1 in vivo on the day of birth provides amelioration of SMA phenotype, all of which used AAV vectors. Whilst these provided invaluable data and later led to the approval of Zolgensma as a licensed SMA therapy, no curative treatment has yet been made available for SMA. The invention has allowed for development of a novel expression cassette, implemented in lentiviral vectors for cell culture testing and localised delivery in vivo, and in AAV vectors for widespread in vivo distribution.

Both IPLV and IDLV configurations encoding SMN1 variants are efficient at transducing various in vitro models. Generally, IPLVs resulted in higher expression levels compared to their IDLV counterparts, although significant expression could still be obtained with the latter. The expression levels mediated by the IDLVs may actually be more adequate, as it has come to light that supraphysiological levels of SMN may be toxic, and IDLVs are a safer option without the potential risk of insertional mutagenesis from IPLVs. Transgenic expression levels of SMN1 can also be controlled through the choice of promoter. The in vitro experiments revealed that the ubiquitous CMV promoter directed the most robust transgene expression from lentiviral vectors. The strong and constitutive nature of this promoter lends itself to the systemic nature of SMA, as CMV can mediate gene expression in a remarkably broad range of cells. Intermediate transgenic expression levels were achieved with the ubiquitous hPGK promoter, while the neuron- specific hSYN promoter appeared the weakest of the three, despite the use of relevant neuronal systems as well as human fibroblasts.

Codon-optimisation of the nucleotide sequence encoding human survival motor neuron 1 protein to provide the nucleotide sequence of the invention had a significant positive impact on the efficiency of the transgenic expression in all the cell culture systems evaluated. Implementation of the optimised nucleotide sequence of the invention in an AAV9 vector for in vivo delivery in Smn^2B/~ mice demonstrated robust expression in liver and spinal cord, at somewhat variable levels that on average were not significantly different from wild-type. Cell culture experiments have shown dose-dependent expression from lentiviral vectors, which presumably could be replicated in vivo to titrate expression levels to an optimum. This is important, given the potential toxicity of SMN over-production. Several groups have found proteins associated with DNA damage and apoptosis to be dysregulated in SMA systems, including cleaved caspase 3, pATM , DNA-PKcs, senataxin, CHK2, pBRCAl, p53 and yH2AX. Signals indicative of genomic instability caused by DNA double strand breaks are transduced by ATM and downstream proteins including H2AX, leading to DNA repair by proteins such as BRCA1; or if damage is too severe, apoptosis. Evidence of SMN restoration being able to revert some molecular signatures of the DNA damage response has been reported in the literature. In contrast, it has been shown here that lentiviral transduction caused an increase in pATM levels, in the percentage of SMA fibroblasts that exhibited yH2AX foci as well as in the number of foci per cell, indicative of activation of the DNA damage response pathway. However, it was observed that the nucleotide sequence of the invention encoding human survival motor neuron 1 protein and having at least 96% identity with SEQ ID NO: 1 had a protective effect in fibroblasts compared to eGFP-expressing vector regarding the induction of pATM. A possible explanation for increase in yH2AX foci and pATM following IDLV transduction could be shortterm initiation of host anti-viral responses which then activate the DNA damage response pathway. Lentiviral vector transduction is likely to trigger host anti-viral responses causing an increase in Toll-like receptor- and type I interferon- signaling. Endocytosis of vectors, presence of the RNA genome acting as a pathogen-associated molecular pattern, or presence of plasmid contamination in laboratory-grade vector preparations could all alert the cell to presence of the viral vector. Interferon-y treatment has been shown to activate ATM, a process that involves autophosphorylation thus leading to increased pATM, like that seen here in SMA type I cells. Interferon-y also increases yH2AX, potentially explaining why lentiviral vector transduction increased levels of this protein further above elevated SMA levels.

Although SMA is typically considered a motor neuron disease, research has shown the effects of reduced levels of SMN in peripheral tissues and organs, particularly the impairment of cardiac function in mouse models of SMA. The function of SMN protein in cardiac muscle remains largely unknown, however, previous studies show widespread protein dysregulation in Smn^2B/~ mice compared to aged-matched WT mice. One known intermediate filament protein dysregulated is desmin, which levels are decreased.

The implementation of the optimised nucleotide sequence of the invention in an AAV9 vector for in vivo delivery in Smn^2B/~ mice demonstrated enhanced expression of the human SMN1 gene in Smn^2B/~ mice hearts. The treatment of Smn^2B/~ mice with AAV9_CAG_Co-/i.S'A7A also showed restoration of desmin levels in mice hearts to that of WT mice. In previous studies, desmin appeared to be associated with sarcomere damage, thus it is reasonable that the improved levels of desmin in AAV9_CAG_Co- hSMN treated Smn^2B/~ mice result in improved sarcomere structure. SMN has been found to be localised to the I- and M-bands of sarcomeres in normal human skeleton fibres and the Z-disc in mice which suggests that sarcomeric SMN may directly or indirectly interact with the cytoskeleton and thus be involved in sarcomere structure of its maintenance.

Although not discussed, it may also be possible for other reported dysregulated proteins with a known role in heart function, such as the intermediate filament protein lamin A/C, to also be restored using the AAV-mediated SMN1 therapy according to this invention.

The results indicate the development of a new strategy focused on delivery of a codon- optimised transgene, Co-hSMNl . Lentiviral-mediated expression of Co-hSMNl is able to rescue SMN expression in multiple in vitro cell systems and AAV9 delivery leads to strong expression in the Smn^2B/~ mouse model of SMA.

The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention as defined in the appended claims. Sequence Listings

SEQ ID NO:1 (Co-hSMNl )

ATGGCCATGAGCAGCGGCGGCTCTGGCGGCGGAGTGCCCGAGCAGG

AAGATAGCGTCCTGTTCAGACGGGGCACCGGCCAGAGCGACGACAG

CGACATCTGGGACGACACCGCCCTGATCAAGGCCTACGACAAGGCC

GTGGCCAGCTTCAAGCACGCCCTGAAGAACGGCGACATCTGCGAGA

CAAGCGGCAAGCCCAAGACCACCCCCAAGCGGAAGCCCGCCAAGA

AGAACAAGAGCCAGAAGAAGAACACCGCCGCCAGCCTCCAGCAGT

GGAAAGTGGGCGACAAGTGCAGCGCCATTTGGAGCGAGGACGGCT

GCATCTACCCCGCCACAATCGCCAGCATCGACTTCAAGCGGGAAAC

CTGCGTGGTGGTGTACACCGGCTACGGCAACAGAGAGGAACAGAAC

CTGAGCGACCTGCTGAGCCCCATCTGCGAGGTGGCCAACAACATCG

AGCAGAACGCCCAGGAAAACGAGAACGAGAGCCAGGTGTCCACCG

ACGAGAGCGAGAACAGCAGAAGCCCCGGCAACAAGAGCGACAACA

TCAAGCCTAAGAGCGCCCCCTGGAACAGCTTCCTGCCCCCTCCCCCA

CCAATGCCTGGCCCTAGACTGGGCCCTGGCAAGCCCGGCCTGAAGT

TCAACGGCCCTCCCCCCCCACCTCCACCACCCCCTCCACATCTGCTG

AGCTGCTGGCTGCCCCCATTCCCCAGCGGCCCTCCCATCATTCCCCC

TCCACCCCCCATCTGCCCCGACAGCCTGGATGATGCCGACGCCCTGG

GCAGCATGCTGATCAGCTGGTACATGAGCGGCTACCACACAGGCTA

CTACATGGGCTTCCGGCAGAACCAGAAAGAGGGCCGCTGCTCCCAC

AGCCTGAACTGAC

SEQ ID N0:2 (Exon6_E primer)

CTCCCATATGTCCAGATTCTCTTG

SEQ ID NO: 3 (Exon8_R primer)

CTACAACACCCTTCTCACAG

SEQ ID N0:4 (/3-actin_F primer)

TCACCCACACTGTGCCCATCTACGA

SEQ ID N0:5 ([)-actin_R primer)

CAGCGGAACCGCTCATTGCCAATGG SEQ ID N0:6 (189_mGapdhex4_Fw primer)

AAAGGGTCATCATCTCCGCC

SEQ ID N0:7 (190_mGapdhex4-5 primer)

ACTGTGGTCATGAGCCCTTC

SEQ ID N0:8 (pRRLsc_CMV_co_hSMNl_mW)

GGTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTAT

TCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAAT

GCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTC

CTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCG

TTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACC

CCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGAC

TTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCT

GCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAA

TTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCG

CCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTC

CCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCC

GGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTC

GGATCTCCCTTTGGGCCGCCTCCCCGCGAATTCGAGCTCGGTACCTT

TAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTA

AAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGA

CAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGAT

CTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAG

CCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCT

GTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAG

TGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAG

TATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACT

TGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACA

AATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTG

TCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCG

CCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACT

AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGC

TATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC

GTCGAGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGC

TCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGT

TACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGC

GTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCG

CAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCGCATTA

AGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTG

CCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCG CCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT

TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACT

T

_GG TA T_TT TT TA CG GG CG CT CG TA TT TG GG AT CT GC TA TC GG GT AA GG TT CG CG AG CC GC TA TT CC TG T_TC AC ACT T_AGA GT TA GG GA AC C_TG C

TTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTT

TGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATG

AGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAAC

GTTTACAATTTCCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC

CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT

GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG

AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG

GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT

AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA

CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAG

AACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCG

GTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCA

TACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA

AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTG

CCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAAC

GATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGG

GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAG

CCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGC

AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTT

CCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG

ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATA

AATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACT

GGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACG

GGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAG

ATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTA

CTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAG

GATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT

AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT

CAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTT

GCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGAT

CAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG

CGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCAC

CACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAAT

CCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG

GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGG

CTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACC

TACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCA

CGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCA

GGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACG

CCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAG CGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA

ACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCT

TTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAAC

CGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAAC

GACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCC

AATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCA

GCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAA

CGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAA

CAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCG

CAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTA

ATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGAT

GAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGC

CGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGC

AACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCA

TTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGG

GTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAAC

TAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTT

CAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATC

CCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCC

GAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACG

CAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCG

GCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAG

GAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATT

AGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAA

AAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAAC

GATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAG

ACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAA

GAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCA

TCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGAT

AGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGC

CGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAG

AAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGA

GTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAA

AGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAG

CAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGC

CAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTG

AGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGG

GCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCT

AAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTC

ATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATC

TCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGA

GAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAAT

CGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAG ATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCT

GTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTT

TAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAG

GGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGG

ACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGA

CAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGG

TTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG

AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAAT

TACAAAAACAAATTACAAAATTCAAAATTTTATCGATAAGCTTGGG

AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG

CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT

AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTAT

TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC

AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTA

CATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC

AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCC

AAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAA

ATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGAC

GCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGA

GCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCT

GTTTTGACCTCCATAGAAGACACCGACTCTAGAGGATCCACCGGAC

CGGTGAATTCGCCACCATGGCCATGAGCAGCGGCGGCTCTGGCGGC

GGAGTGCCCGAGCAGGAAGATAGCGTCCTGTTCAGACGGGGCACCG

GCCAGAGCGACGACAGCGACATCTGGGACGACACCGCCCTGATCAA

GGCCTACGACAAGGCCGTGGCCAGCTTCAAGCACGCCCTGAAGAAC

GGCGACATCTGCGAGACAAGCGGCAAGCCCAAGACCACCCCCAAG

CGGAAGCCCGCCAAGAAGAACAAGAGCCAGAAGAAGAACACCGCC

GCCAGCCTCCAGCAGTGGAAAGTGGGCGACAAGTGCAGCGCCATTT

GGAGCGAGGACGGCTGCATCTACCCCGCCACAATCGCCAGCATCGA

CTTCAAGCGGGAAACCTGCGTGGTGGTGTACACCGGCTACGGCAAC

AGAGAGGAACAGAACCTGAGCGACCTGCTGAGCCCCATCTGCGAGG

TGGCCAACAACATCGAGCAGAACGCCCAGGAAAACGAGAACGAGA

GCCAGGTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCA

ACAAGAGCGACAACATCAAGCCTAAGAGCGCCCCCTGGAACAGCTT

CCTGCCCCCTCCCCCACCAATGCCTGGCCCTAGACTGGGCCCTGGCA

AGCCCGGCCTGAAGTTCAACGGCCCTCCCCCCCCACCTCCACCACCC

CCTCCACATCTGCTGAGCTGCTGGCTGCCCCCATTCCCCAGCGGCCC

TCCCATCATTCCCCCTCCACCCCCCATCTGCCCCGACAGCCTGGATG

ATGCCGACGCCCTGGGCAGCATGCTGATCAGCTGGTACATGAGCGG

CTACCACACAGGCTACTACATGGGCTTCCGGCAGAACCAGAAAGAG

GGCCGCTGCTCCCACAGCCTGAACTGACCTGCA SEQ ID N0:9 (pRRLsc_hSYN_co_hSMN 1 _mW)

GGTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTAT

TCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAAT

GCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTC

CTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCG

TTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACC

CCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGAC

TTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCT

GCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAA

TTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCG

CCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTC

CCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCC

GGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTC

GGATCTCCCTTTGGGCCGCCTCCCCGCGGTACCTTTAAGACCAATGA

CTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGG

GGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTT

TTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGA

GCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCT

TGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCT

GGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTC

TAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGC

AAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCT

TATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATA

AAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCA

ATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGC

CCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTT

ATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTA

GTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCGTCGAGACGTACC

CAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCG

TTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAAT

CGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAG

AGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG

CGAATGGCGCGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGT

GTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAG

CGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG

GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGA

TTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGA

TGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT

TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACT

GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGG

GATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAAC

AAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCC

CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTA TTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACC

CTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATT

CAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTT

CCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTG

AAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAA

CAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAA

TGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGT

ATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTC

AGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTAC

GGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATG

AGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGAC

CGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAAC

TCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAAC

GACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGC

GCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAA

TTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGC

GCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCC

GGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATG

GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGC

AACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCA

CTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACT

TTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGA

AGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTT

TCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTT

CTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAA

AAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC

CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACC

AAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA

ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCA

GTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTC

AAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG

GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAA

CTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG

AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA

CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCT

TTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT

GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC

GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG

TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCC

TTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCA

GCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAAC

CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGAC

AGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATG TGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTT CCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACAC

AGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCT

CACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTAATGTAGTCTTA

TGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAAC

ATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGA

AGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGT

CTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATA

TTGTATTTAAGTGCCTAGCTCGATACAATAAACGGGTCTCTCTGGTT

AGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCA

CTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTG

TGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCT

TTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGAC

CTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGC

TTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGA

GTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATG

GGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATG

GGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATT

AAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTT

AATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGG

GACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATC

ATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAG

AGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGC

AAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCA

GACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTAT

ATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCAC

CAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGG

AATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACT

ATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTAT

TGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGA

GGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAG

CTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAAC

AGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT

GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGA

TTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAA

TTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAG

CAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCA

AGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAA

ATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTT

TTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCA

TTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGC

CCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGAT

CCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAA

AGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTA

GACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAA ATTACAAAATTCAAAATTTTATCCTAGACTCGAGCTGCAGAGGGCC

CTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGT

GGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACC

CAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAG

GGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCA

CCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCC

GCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCA

AACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCC

CAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGC

GCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTC

TGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGTCG

AATTCGCCACCATGGCCATGAGCAGCGGCGGCTCTGGCGGCGGAGT

GCCCGAGCAGGAAGATAGCGTCCTGTTCAGACGGGGCACCGGCCAG

AGCGACGACAGCGACATCTGGGACGACACCGCCCTGATCAAGGCCT

ACGACAAGGCCGTGGCCAGCTTCAAGCACGCCCTGAAGAACGGCGA

CATCTGCGAGACAAGCGGCAAGCCCAAGACCACCCCCAAGCGGAA

GCCCGCCAAGAAGAACAAGAGCCAGAAGAAGAACACCGCCGCCAG

CCTCCAGCAGTGGAAAGTGGGCGACAAGTGCAGCGCCATTTGGAGC

GAGGACGGCTGCATCTACCCCGCCACAATCGCCAGCATCGACTTCA

AGCGGGAAACCTGCGTGGTGGTGTACACCGGCTACGGCAACAGAGA

GGAACAGAACCTGAGCGACCTGCTGAGCCCCATCTGCGAGGTGGCC

AACAACATCGAGCAGAACGCCCAGGAAAACGAGAACGAGAGCCAG

GTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCAACAAG

AGCGACAACATCAAGCCTAAGAGCGCCCCCTGGAACAGCTTCCTGC

CCCCTCCCCCACCAATGCCTGGCCCTAGACTGGGCCCTGGCAAGCCC

GGCCTGAAGTTCAACGGCCCTCCCCCCCCACCTCCACCACCCCCTCC

ACATCTGCTGAGCTGCTGGCTGCCCCCATTCCCCAGCGGCCCTCCCA

TCATTCCCCCTCCACCCCCCATCTGCCCCGACAGCCTGGATGATGCC

GACGCCCTGGGCAGCATGCTGATCAGCTGGTACATGAGCGGCTACC

ACACAGGCTACTACATGGGCTTCCGGCAGAACCAGAAAGAGGGCCG

CTGCTCCCACAGCCTGAACTGACCTGCA

SEQ ID NO: 10 (pRRLsc_hPGKp_co_hSMNl_mW)

GGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG

CTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTT

CCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGG

GGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCC

CAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCC

TGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAA

TAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGG

TCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGT

TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAA

AATATTAACGTTTACAATTTCCCAGGTGGCACTTTTCGGGGAAATGT GCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA

A _CTA TA TA TG TG TA GA C_GG GA CG ATA T_TT TG TA GG CT CA TT TT CC CA TA GC TA TT TT TT TC GC CG TT CG AT CC CG CC AC GC AT AT AA CT GT CC TC GGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGT TACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCG CCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTAT

GTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGG TCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAG

TCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATG CAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTC TGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAA CATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTG

AATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAG CAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTAC

TCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAA GTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTAT TGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATT GCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCT ACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGA

TCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGAC CAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAA AATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATC

TGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTT GCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCA GCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTT AGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTC TGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT

CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGC GGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCG

AACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTA AGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGG GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTG

ACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTAT GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGC TGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTG GATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAG CCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA

GCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATT AATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAG GCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAG

CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCC

AAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGC

AAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGG

TAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCG

TGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTA

GGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAAT

TGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAAT

AAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTG

GCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTG

AGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACT

AGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT

GGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCT

CTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGA

GGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGC

TAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGG

AGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGA

AAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAG

CTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAG

GCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGG

ATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATT

GTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAG

ACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGC

AAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACA

ATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACC

ATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAG

AGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTG

GGAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACG

GTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACA

ATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCAC

AGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAA

AGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTG

GAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGT

AATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGT

GGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAAT

TGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATT

GGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACA

AATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTT

GGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAG

TTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACC

CCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGA

GAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGAC

GGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAG

TGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAAC TAAAGAATTACAAAAACAAATTACAAAATTCAAAATTTTATCGATC

ACGAGACTAGCCTCGAGAAGCTTGATATCGAATTCCCACGGGGTTG

GGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCG

GCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGG

GTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCG

CCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTA

AGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAA

CGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCA

GGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGC

GGCTGCTCAGCGGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCG

GTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGC

CCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGC

AGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGGGGG

ATCCACCGGACCGGTGAATTCGCCACCATGGCCATGAGCAGCGGCG

GCTCTGGCGGCGGAGTGCCCGAGCAGGAAGATAGCGTCCTGTTCAG

ACGGGGCACCGGCCAGAGCGACGACAGCGACATCTGGGACGACAC

CGCCCTGATCAAGGCCTACGACAAGGCCGTGGCCAGCTTCAAGCAC

GCCCTGAAGAACGGCGACATCTGCGAGACAAGCGGCAAGCCCAAG

ACCACCCCCAAGCGGAAGCCCGCCAAGAAGAACAAGAGCCAGAAG

AAGAACACCGCCGCCAGCCTCCAGCAGTGGAAAGTGGGCGACAAG

TGCAGCGCCATTTGGAGCGAGGACGGCTGCATCTACCCCGCCACAA

TCGCCAGCATCGACTTCAAGCGGGAAACCTGCGTGGTGGTGTACAC

CGGCTACGGCAACAGAGAGGAACAGAACCTGAGCGACCTGCTGAG

CCCCATCTGCGAGGTGGCCAACAACATCGAGCAGAACGCCCAGGAA

AACGAGAACGAGAGCCAGGTGTCCACCGACGAGAGCGAGAACAGC

AGAAGCCCCGGCAACAAGAGCGACAACATCAAGCCTAAGAGCGCC

CCCTGGAACAGCTTCCTGCCCCCTCCCCCACCAATGCCTGGCCCTAG

ACTGGGCCCTGGCAAGCCCGGCCTGAAGTTCAACGGCCCTCCCCCC

CCACCTCCACCACCCCCTCCACATCTGCTGAGCTGCTGGCTGCCCCC

ATTCCCCAGCGGCCCTCCCATCATTCCCCCTCCACCCCCCATCTGCC

CCGACAGCCTGGATGATGCCGACGCCCTGGGCAGCATGCTGATCAG

CTGGTACATGAGCGGCTACCACACAGGCTACTACATGGGCTTCCGG

CAGAACCAGAAAGAGGGCCGCTGCTCCCACAGCCTGAACTGACCTG

CAGGTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGG

TATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTT

AATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTC

CTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGC

CCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCA

ACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGG

GACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCG

CCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGAC

AATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCT

CGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACG

TCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTG CCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAG

TCGGATCTCCCTTTGGGCCGCCTCCCCGCGAATTCGAGCTCGGTACC

TTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTT

TAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAA

GACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAG

ATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTA

AGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGT

CTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTC

AGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTC

AGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAA

CTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCA

CAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGT

TTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCC

CGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGA

CTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGA

GCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTT

GCGTCGAGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGC

GCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGC

GTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTG

GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTG

CGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGC

SEQ ID NO: 11 (pAAV_CAG_hSMNlopt_mWPRE_sensl )

TGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCC

CGGGCGTCGGGCGACCTTTGGTCGGGCGGCCTCAGTGAGCGAGCGA

GCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGT

AGTTAATGATTAACCCGCCATGCTACTTATCTACGACATTGATTATT

GACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCC

CATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT

GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGT

ATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG

GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATG

GCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA

CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAG

GTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCA

CCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATG

GGGGCGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG

GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCC

AATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGC

GGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAG

TCTGGCGCGCTCGCTTCGCCCCGTGCCCCCTGCCGCCGCCGCCTCGC

GCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAG CGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGG CTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGT GCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCC CGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTC CGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGG TGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTG TGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGC AACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCT TCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGC CGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGC CGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCC GGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCT TTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAA TCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAG CGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGG CGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTC TCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGG ACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCT AGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTC CTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGA ATTGATTAATTCGAGCGAACGCGTCGAGTCGCTCGGTACGATTTAA ATTGAATTCGCCACCATGGCCATGAGCAGCGGCGGCTCTGGCGGCG GAGTGCCCGAGCAGGAAGATAGCGTCCTGTTCAGACGGGGCACCGG CCAGAGCGACGACAGCGACATCTGGGACGACACCGCCCTGATCAAG GCCTACGACAAGGCCGTGGCCAGCTTCAAGCACGCCCTGAAGAACG GCGACATCTGCGAGACAAGCGGCAAGCCCAAGACCACCCCCAAGC GGAAGCCCGCCAAGAAGAACAAGAGCCAGAAGAAGAACACCGCCG CCAGCCTCCAGCAGTGGAAAGTGGGCGACAAGTGCAGCGCCATTTG GAGCGAGGACGGCTGCATCTACCCCGCCACAATCGCCAGCATCGAC

TTCAAGCGGGAAACCTGCGTGGTGGTGTACACCGGCTACGGCAACA GAGAGGAACAGAACCTGAGCGACCTGCTGAGCCCCATCTGCGAGGT GGCCAACAACATCGAGCAGAACGCCCAGGAAAACGAGAACGAGAG CCAGGTGTCCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCAA CAAGAGCGACAACATCAAGCCTAAGAGCGCCCCCTGGAACAGCTTC CTGCCCCCTCCCCCACCAATGCCTGGCCCTAGACTGGGCCCTGGCAA GCCCGGCCTGAAGTTCAACGGCCCTCCCCCCCCACCTCCACCACCCC CTCCACATCTGCTGAGCTGCTGGCTGCCCCCATTCCCCAGCGGCCCT CCCATCATTCCCCCTCCACCCCCCATCTGCCCCGACAGCCTGGATGA TGCCGACGCCCTGGGCAGCATGCTGATCAGCTGGTACATGAGCGGC TACCACACAGGCTACTACATGGGCTTCCGGCAGAACCAGAAAGAGG GCCGCTGCTCCCACAGCCTGAACTGACCTGCAGGTAATCAACCTCTG GATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGC TATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTG

GTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTG

GCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGC

ATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTC

CCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTG

GACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCG

GGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTG

GATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATC

CAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTT

CCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGC

CGCCTCCCCGCGAATTCGAATGGCCATGGGACGTCGACCTGAGGTA

ATTATAACCCGGGCCCTATATATGGATCCAATTGCAATGATCATCAT

GACAGATCTGCGCGCGATCGATATCAGCGCTTTAAATTTGCGCATGC

TAGCTATAGTTCTAGAGGGCCCTATTCTATAGTGTCACCTAAATGCT

AGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATC

TGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCA

CTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT

CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACA

GCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATG

CGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGGTAGATAAG

TAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGG

AGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG

CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG

TGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGT

CGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTA

ATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAA

GAGGCCCGCACCGATCGCGCGCAGATCTGTCATGTGAGCAAAAGGC

CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT

TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC

AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC

GTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC

CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG

CTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT

TCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC

GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA

CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA

GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCC

TAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGC

TGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGG

CAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGC

AGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT

TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG

ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTT

CACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAAT GTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAG

AGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACT

GGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGC

GCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCT

TTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATC

AAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATT

GCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTAT

GACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCC

GGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTG

TCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGT

GGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTC

ACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGC

AGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATC

ATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCT

GCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTAC

TCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAG

CATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGA

GCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTG

CTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCG

ACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTT

GGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGAC

CGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCAT

CGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTTAAAGCCCAA

TACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAA

SEQ ID NO: 12 (pMDLg/pRRE)

GGATCCCCTGAGGGGGCCCCCATGGGCTAGAGGATCCGGCCTCGGC

CTCTGCATAAATAAAAAAAATTAGTCAGCCATGAGCTTGGCCCATT

GCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCAT

GTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAA

TAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT

CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC

AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT

AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA

CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAA

GTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCA

TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACA

TCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAG

TACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA

GTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAAT

CAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGC

AAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGC

TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTT TTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAA

GCTTACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAG

TCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAG

TTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACC

AAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTT

TTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCT

TTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCA

TTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAAT

ATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAG

AGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCT

TTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTA

GGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCT

CCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAG

CACGTGAGATCTGAATTCGAGATCTGCCGCCGCCATGGGTGCGAGA

GCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTC

GGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG

TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCT

GTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAA

CCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATA

CAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGA

CACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAG

TAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAA

TCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAA

ATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAA

AAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTT

TTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATG

CTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAG

AGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGT

GCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGG

AAGTGACATAGCAGGAACTACTAGTACTAGTACCCTTCAGGAACAA

ATAGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCT

ATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTA

TAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCC

TTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGC

AAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGT

CCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGA

CCAGGAGCGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTG

GGGGGACCCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCC

AAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAG

GAACCAAAGAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGG

CACATAGCCAAAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGA

AATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGAC

AGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCC

AGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAA GAGAGCTTCAGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGC

AGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATC

ACTCTTTGGCAGCGACCCCTCGTCACAATAAAGATAGGGGGGCAAT

TAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGA

AGAAATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGG

AATTGGAGGTTTTATCAAAGTAGGACAGTATGATCAGATACTCATA

GAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTA

CACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGC

ACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATT

AAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACA

GAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAA

AAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATA

CTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAA

ATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGG

GAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAA

AATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCC

TTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTAT

AAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCA

CAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAA

AAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTA

TCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGG

CAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGT

GGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATT

CCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGC

CTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACA

GAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGG

ATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCAC

TAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGG

CAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTA

TGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCA

AGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTG

AAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGAT

GTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGC

ATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAA

AGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCT

GGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTA

TGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCT

ATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAG

GATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGA

CACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTG

CAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATG

CATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTT

AGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTAC

CTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAA GTAGATAAATTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAG

ATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTA

ATTGGAGAGCAATGGCTAGTGATTTTAACCTACCACCTGTAGTAGC

AAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGA

AGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTA

GATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGT

AGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGG

GCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCA

GTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTA

CAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGG

CATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAAT

AAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAA

CATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAA

AAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGT

AGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACA

AATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGA

GATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAG

GGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAG

AAGAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGG

TGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAACACATGGAA

TTCCGGAGCGGCCGCAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAG

CAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGC

CAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTG

AGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGG

GCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCT

AAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTC

ATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATC

TCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGA

GAAATTAACAATTACACAAGCTTCCGCGGAATTCACCCCACCAGTG

CAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGG

CCCACAAGTTTCACTAAGCTCGCTTCCTTGCTGTCCAATTTCTATTAA

AGGTTCCTTGGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTAT

GAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTT

TCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAA

GGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGA

GCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAA

AGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCTG

ATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCT

TGATTTGGAGGTTAAAGTTTGGCTATGCTGTATTTTACATTACTTATT

GTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCC

TGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATAC

CACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTT

CTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAG

GTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTG TGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCC

TCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCG

CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATC

AGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGAT

AACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGG

AACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCC

CCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA

AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT

CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG

TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACG

CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT

GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT

AACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT

GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC

GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTA

GAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTC

GGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG

GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA

AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG

CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATT

ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT

TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAC

CAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCG

TTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC

GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGA

CCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCC

GGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA

TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCA

GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT

GTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAAC

GATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGT

TAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAG

TGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTC

ATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA

GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGG

CGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT

GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATC

TTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAA

CTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA

AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACAC

GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGC

ATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT

TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAA GTGCCACCTGACGT SEQ ID NO: 13 (pMDLg/pRRintD64V)

GGATCCCCTGAGGGGGCCCCCATGGGCTAGAGGATCCGGCCTCGGC

CTCTGCATAAATAAAAAAAATTAGTCAGCCATGAGCTTGGCCCATT

GCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCAT

GTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAA

TAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT

CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC

AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT

AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA

CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAA

GTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCA

TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACA

TCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAG

TACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA

GTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAAT

CAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGC

AAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGC

TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTT

TTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAA

GCTTACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAG

TCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAG

TTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACC

AAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTT

TTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCT

TTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCA

TTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAAT

ATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAG

AGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCT

TTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTA

GGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCT

CCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAG

CACGTGAGATCTGAATTCGAGATCTGCCGCCGCCATGGGTGCGAGA

GCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTC

GGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG

TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCT

GTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAA

CCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATA

CAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGA

CACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAG

TAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAA

TCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAA

ATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAA AAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTT

TTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATG

CTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAG

AGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGT

GCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGG

AAGTGACATAGCAGGAACTACTAGTACTAGTACCCTTCAGGAACAA

ATAGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCT

ATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTA

TAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCC

TTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGC

AAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGT

CCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGA

CCAGGAGCGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTG

GGGGGACCCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCC

AAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAG

GAACCAAAGAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGG

CACATAGCCAAAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGA

AATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGAC

AGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCC

AGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAA

GAGAGCTTCAGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGC

AGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATC

ACTCTTTGGCAGCGACCCCTCGTCACAATAAAGATAGGGGGGCAAT

TAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGA

AGAAATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGG

AATTGGAGGTTTTATCAAAGTAGGACAGTATGATCAGATACTCATA

GAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTA

CACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGC

ACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATT

AAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACA

GAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAA

AAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATA

CTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAA

ATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGG

GAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAA

AATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCC

TTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTAT

AAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCA

CAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAA

AAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTA

TCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGG

CAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGT

GGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATT

CCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGC CTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACA GAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGG ATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCAC TAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGG CAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTA TGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCA AGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTG

AAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGAT GTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGC

ATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAA AGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCT GGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTA TGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCT ATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAG GATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGA CACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTG

CAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATG CATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTT AGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTAC CTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAA GTAGATAAATTAGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAG ATGGAATCGATAAGGCTCAAGAAGAACACGAAAAGTACCACTCTAA TTGGAGAGCCATGGCAAGTGATTTTAACCTGCCACCTGTAGTAGCA

AAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAA GCCATGCATGGACAAGTAGACTGTAGTCCAGGAATATGGCAACTAG

TTTGTACACATCTAGAAGGAAAAATTATCCTGGTAGCAGTTCATGTA GCCAGTGGATATATAGAAGCAGAAGTTATTCCAGCAGAGACAGGGC AGGAAACAGCATATTTTCTCTTAAAATTAGCAGGAAGATGGCCAGT AAAAACAATACATACAGACAATGGCAGCAATTTCACCAGTACTACG GTTAAGGCCGCCTGTTGGTGGGCAGGGATCAAGCAGGAATTTGGCA TTCCCTACAATCCCCAAAGCCAAGGAGTAGTAGAATCTATGAATAA TGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACAC

CTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAA GAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAG ACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAA TTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGA TCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGG GCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAA GAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGGTG

ATGATTGTGTGGCAAGTAGACAGGATGAGGATTAACACATGGAATT CCGGAGCGGCCGCAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGC AGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCC AGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGA GGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGG CATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTA AAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCA TTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCT CTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAG AAATTAACAATTACACAAGCTTCCGCGGAATTCACCCCACCAGTGC AGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGC CCACAAGTTTCACTAAGCTCGCTTCCTTGCTGTCCAATTTCTATTAA AGGTTCCTTGGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTAT GAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTT TCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAA GGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGA GCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAA AGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCTG ATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCT TGATTTGGAGGTTAAAGTTTGGCTATGCTGTATTTTACATTACTTATT GTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCC TGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATAC CACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTT CTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAG GTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTG TGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCC

TCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCG CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATC AGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGAT AACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGG AACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCC CCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACG CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT AACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTA GAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTC GGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATT ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT

TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAC CAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCG TTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGA

CCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCC

GGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA

TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCA

GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT

GTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAAC

GATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGT

TAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAG

TGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTC

ATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA

GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGG

CGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT

GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATC

TTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAA

CTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA

AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACAC

GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGC

ATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT

TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAA

GTGCCACCTGACGT

SEQ ID NO: 14 (pRSV-rev)

AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT

TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTG

AGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCA

GGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGA

GCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGA

ATTCGATGTACGGGCCAGATATACGCGTATCTGAGGGGACTAGGTG

TGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCC

CCTCAGGATTAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTA

GTCTTATGCAATACACTTGTAGTCTTGCAACATGGTAACGATGAGTT

AGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATT

GGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAG

ACAGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAG

AGATAATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTG

ACCATTCACCACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTAGT

GAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC

ATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTAGTCGA

TTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGA

CCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACC

CACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAA

GAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTG

AACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCT CTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGA TTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTG GTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGTGCT GTTAGCTTGCTCAATGCCACAGCTATAGCAGTAGCTGAGGGGACAG ATAGGGTTATAGAAGTAGTACAAGAAGCTTGGCACTGGCCGTCGTT TTACATGATCTGAGCCTGGGAGATCTCTGGCTAACTAGGGAACCCA CTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTG TGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCAGGAAAACCCT GGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAG TTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCT TACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATA GTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTC CTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCT TTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACG TAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGG AGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACA CTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCG ATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAA CGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTC TCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACAC CCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGC ATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTC AGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCT CGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTT CTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCC TATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCA TTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAAC GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTA

TTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATAC ACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAA GCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCC ATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGA TCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCC ATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAA CAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCC CGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGAC CACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA

TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGG

GGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG

GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATA

GGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTC

ATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGA

TCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAA

CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCA

AAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC

AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA

AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG

CAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCA

CTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCC

TGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGG

TTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCT

GAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTA

CACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGG

GTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC

TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCG

TCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC

GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTT

GCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGT

ATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGA

CCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG

SEQ ID NO: 15 (pMD2. VSVG)

GGATCCCCTGAGGGGGCCCCCATGGGCTAGAGGATCCGGCCTCGGC

CTCTGCATAAATAAAAAAAATTAGTCAGCCATGAGCTTGGCCCATT

GCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCAT

GTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAA

TAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT

CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC

AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT

AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA

CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAA

GTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCA

TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACA

TCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAG

TACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA

GTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAAT

CAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGC

AAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGC TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTT

TTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAA

GCTTACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAG

TCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAG

TTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACC

AAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTT

TTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCT

TTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCA

TTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAAT

ATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAG

AGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCT

TTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTA

GGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCT

CCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAG

CACGTGAGATCTGAATTCAACAGAGATCGATCTGTTTCCTTGACACT

ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGC

AAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAA

ATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATT

GGCATAATGACTTAATAGGCACAGCCATACAAGTCAAAATGCCCAA

GAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCC

AAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATAT

AACACAGTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAG

GAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCT

TCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGC

AGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACA

CAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAA

TTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACT

ATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATC

ACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGG

GCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAA

GGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCA

TCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGC

CAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTC

AGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTT

GGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGT

CTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCC

AGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACT

TTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCA

AGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGT

GGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGG

AGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTG

GACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAG

GTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGA

TGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAA TCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCC

TCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTC

CGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAA

GACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGTAACT

CAAATCCTGCACAACAGATTCTTCATGTTTGGACCAAATCAACTTGT

GATACCATGCTCAAAGAGGCCTCAATTATATTTGAGTTTTTAATTTT

TATGGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTG

GCTGGTGTGGCTAATGCCCTGGCCCACAAGTTTCACTAAGCTCGCTT

CCTTGCTGTCCAATTTCTATTAAAGGTTCCTTGGTTCCCTAAGTCCAA

CTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCT

GCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTA

TTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTT

AAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATAC

ACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTA

ATGCACATTGGCAACAGCCCTGATGCCTATGCCTTATTCATCCCTCA

GAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTGGCT

ATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTC

TTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCAC

TCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTC

CATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTT

AGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACA

GGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCC

CACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAG

TGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT

CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA

CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA

GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG

CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAA

TCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGA

TACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCC

GACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAA

GCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTG

TAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCA

GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACC

CGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG

GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG

TGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCT

GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC

TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT

GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC

CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA

CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTA

GATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATAT

ATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGC ACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT

CCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC

CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAG

ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAA

GTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCC

GGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT

TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA

TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA

TCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT

CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGG

CAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT

TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTAT

GCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACC

GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTT

CTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGT

TCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTAC

TTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCC

GCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATA

CTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC

ATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAG

GGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGT

Claims

1. A nucleic acid molecule comprising a codon optimised nucleotide sequence encoding for a functional human survival motor neuron 1 protein, the nucleotide sequence having at least 96% identity with SEQ ID NO:1.

2. A nucleic acid molecule as claimed in claim 1, wherein the nucleotide sequence has at least 98%, or at least 99% identity with SEQ ID NO:1.

3. A nucleic acid molecule as claimed in claim 2, wherein the nucleotide sequence has 100% identity with SEQ ID NO:1.

4. A vector for expressing survival motor neuron protein 1, the vector comprising the nucleic acid molecule of any of claims 1 to 3.

5. A vector as claimed in claim 4, wherein the vector further comprises a promoter.

6. A vector as claimed in claim 5, wherein the vector promoter is independently selected from the group comprising: cytomegalovirus; phosphoglycerate kinase, preferably human phosphoglycerate kinase; CAG; synapsin, preferably human synapsin; and combinations thereof.

7. A vector as claimed in claim 6, wherein the vector promoter is independently selected from the group comprising: cytomegalovirus, human synapsin, human phosphoglycerate kinase, and combinations thereof.

8. A vector as claimed in any one of claims 4 to 7, wherein the vector comprises at least one vector independently selected from the group comprising: an integrating lentivector, an integration-deficient lentiviral vector, and an adeno- associated viral vector.

9. A vector as claimed in any one of claims 4 to 8, wherein the vector is a singlestranded vector.

10. A vector as claimed in any one of claims 4 to 9, wherein the vector comprises at least one expression cassette.

11. A vector as claimed in claim 10, wherein the expression cassette comprises at least one expression control sequence.

12. A host cell comprising the nucleic acid molecule of any one of claims 1 to 3 or the vector of any one of claims 4 to 11.

13. A non-human transgenic animal comprising cells comprising the nucleic acid molecule of any one of claims 1 to 3 or the vector of any one of claims 4 to 11.

14. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 3 or the vector of any one of claims 4 to 11, and one or more pharmaceutically acceptable excipients.

15. A pharmaceutical composition as claimed in claim 14, wherein the nucleic acid molecule or vector is suspended in solution, preferably in an aqueous solution.

16. A pharmaceutical composition as claimed in claim 14 or 15, wherein the composition comprises between 1 x 10¹³ vg/mL and 1 x 10¹⁵ vg/mL of the nucleic acid molecule or vector.

17. A pharmaceutical composition as claimed in any one of claims 14 to 16, wherein the composition has a pH of between 4-11.

18. The nucleic acid molecule of any one of claims 1 to 3 or the vector of any one of claims 4 to 11 for use in therapy.

19. The nucleic acid molecule of any one of claims 1 to 3 or the vector of any one of claims 4 to 11 for use in the treatment of a neuromuscular disorder.

20. The nucleic acid molecule of any one of claims 1 to 3 or the vector of any one of claims 4 to 11 for use in a method of treating a neuromuscular disorder comprising administering a therapeutically effective amount of the nucleic acid molecule or the vector to a patient suffering from the neuromuscular disorder.

21. The nucleic acid molecule or vector for use as claimed in claim 19 or 20, wherein the neuromuscular disorder is independently selected from the group comprising: spinal bulbar muscular atrophy, spinal cerebellar ataxia, traumatic spinal cord injury, spinal muscular atrophy, and combinations thereof.

22. The nucleic acid molecule or vector for use as claimed in claim 21, wherein the neuromuscular disorder is spinal muscular atrophy.

23. The nucleic acid molecule or vector for use as claimed in claim 22, wherein the neuromuscular disorder is Type II or Type III spinal muscular atrophy.

24. A method for treating a neuromuscular disorder in a subject, said method comprising administering the nucleic acid molecule of any one of claims 1 to

3 or the vector of any one of claims 4 to 11 to a subject in need thereof.