OA21075A - Codon-optimized nucleic acid that encodes SMN1 protein, and use thereof - Google Patents

Abstract

The present application relates to the fields of genetics, gene therapy, and molecular biology. More specifically, the present invention relates to an isolated codon-optimized nucleic acid that encodes the SMN1 protein (survival motor neuron protein), an expression cassette and a vector based thereon, as well as an AAV9 (adenoassociated virus serotype 9)-based recombinant virus for increasing the expression of the SMN1 gene in target cells, and use thereof.

Description

Codon-optimized nucleic acid that encodes SMN1 protein, and use thereof

FIELD OF INVENTION

The present application relates to the fields of genetics, gene therapy, and molecular biology. More specifically, the present invention relates to an isolated codon-optimized nucleic acid that encodes the SMN l protein (survival motor neuron protein), an expression cassette and a vector based thereon, as well as an AAV9 (adeno-associated virus serotype 9)-based recombinant virus for increasing the expression of the SMN l gene in target cells, and use thereof.

BACKGROUND OF INVENTION

Spinal muscular atrophy (SMA) is an autosomal récessive neuroinuscular disorder caused by mutations în the survival motor neuron l (SMN i) gene and loss of encoded SMN protein (Lefebvre et al., Cell (1995) 80:155-165). The lack of SMN results in motor neuron degeneration in the ventral (anterior) horn of the spinal cord, which leads to weakness of the proximal muscles responsible for crawling, walking, neck movement and swallowing, and the involuntary muscles that control breathing and coughing (Suinner C.J., NeuroRx (2006) 3:235-245). Consequently, SMA patients are susceptible to pneumonia and other pulmonary problems such as restrictive lung disease.

Gene therapy is a promising approach to treating spinal muscular atrophy (SMA).

Adeno-associated virus (AAV) vectors are considered effective in CNS gene therapy because they hâve suitable toxicity and immunogenicity profiles, they may be used in nerve cell transduction, and they are able to médiate long-term expression in the CNS.

Adeno-associated virus (AAV) is a small (20 nm), independent replicationdefective, nonenveloped virus. Many different AAV serotypes hâve been described in human and primates. The adeno-associated virus genome îs composed of (+ or -) single-stranded DNA (ssDNA) being about 4,700 nucléotides long. The genomic DNA has terminal inverted repeats (ITRs) at the ends. The genome comprises two open reading fiâmes (ORFs), Rep and Cap comprising several alternative reading frames encoding various protein products. The rep products are essential for AAV réplication, whereas three capsid proteins (VP1, VP2, and VP3), along with other alternative products, are encoded by the Cap gene. VP1, VP2, and VP3 are present at 1:1:10 ratio to form an icosahedral capsid (Xie Q. et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Scî USA, 2002; 99:10405-10410). During recombinant AAV (rAAV) vector production, an expression cassette flanked by ITR is packaged into an AAV capsid. The genes required for AAV réplication are not included in the cassette. Recombinant AAV is considered to be one of the safest and most widely used viral vectors for in vivo gene transfer. Vectors can infect cells of multiple tissue types to provide strong and sustained transgene expression. They are also non-pathogenic, and hâve a low immunogenicity profile (High KA et al., rAAV human triai expérience Methods Mol Biol. 2011; 807:429-57).

One of the urgent purposes of research in the area of development of effective gene therapy is codon optimization of genes of interest in vectors to achieve the maximum level of expression of the genes of interest, which, in tum, will allow using lower doses of the vector to achieve a significant effect.

One of the properties of the genetic code is degeneracy, i.e. the ability of different codons (trinucleotides) to encode the same amino acid. Such codons that are translated to the same amino acid are called synonymous codons. In naturel sequences, one of the synonymous codons is selected randomly in the course of évolution, but the frequencies of usage of synonymous codons are different: each amino acid has more and less preferred ones. Codon optimization is a widely used technique to amplify the production of protein molécules, which provides a rational mapping of one of suitable synonymous codons to each amino acid in a protein sequence. One of the common principles of codon optimization invoives the usage of the most frequent codons, whereas other approaches were introduced later, such as hannonization (reproduction of distribution of codon usage frequencies), but they do not always increase productivity. In addition to codon frequencies, the sequence GC content (ratio of guanine and cytosine to the total length of the sequence) may affect the production efficiency, in particular, it was shown that high GC content is associated with increased mRNA levels in mammalian cells Grzegorz Kudla ET AL., High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells, June 2006, Volume 4, Issue 6, el80, pp. 933-942). It is further worth noting that stable secondary structure éléments of mRNA, i.e. those having a low free folding energy, may reduce the efficiency.

Different variants of codon-optimization of the sequence of a gene of interest may lead to the following (as compared to a wild-type gene):

a) expression levels of the genes of interest will be slightly increased;

b) expression levels of the genes of interest will be significantly increased;

c) expression levels of the genes of interest will remain approximately at the same level;

d) expression levels of the genes of interest will be lowered.

Thus, there is a need for a codon-optimized sequence of the SMN1 gene to increase the expression of the SMN 1 gene in target cells.

It was found that the codon-optimized sequence of SMN1 (SMN 1-GeneBeam (or abbreviated as SMN1-GB)), which has the nucléotide sequence of SEQ ID NO: 2, surprisingly increases the transcription of the SMN1 gene by more than 3 times, that is, surprisingly increases the mRNA copy number of SMNl-GeneBeam by more than 3 times as compared to SMN1-WT (wild type), which, în turn, leads to a significant increase in the expression of the SMN1 gene and, accordingly, the SMN protein.

Brief description of invention

In one aspect, the present invention relates to an isolated codon-optimized nucleic acid that encodes the SMN 1 protein (survival motor neuron protein) with SEQ ID NO: 1, and includes the nucleic acid sequence of SEQ ID NO: 2.

In one aspect, the present invention relates to an expression cassette that includes the above codon-optimized nucleic acid.

In some embodiments, the expression cassette includes the following éléments in the 5'-end to 3 end direction:

a left (first) 1TR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an întron ofthe hBGl gene (hemoglobin subunit gamma l gene) the above codon-optimized nucleic acid of the SMN1 gene;

an hGHl polyadénylation signal (human growth hormone gene polyadenylation signal) a l ight (second) ITR.

In some embodiments, the expression cassette includes a nucleic acid with the sequence of SEQ IDNO: 4.

In one aspect, the present invention relates to an expression vector that includes the above codonoptimized nucleic acid or the above cassette.

In one aspect, the present invention relates to an AAV9 (adeno-associated virus serotype 9)-based recombinant virus for increasing the expression ofthe SMN1 gene in target cells, which includes a capsid and the above expression cassette.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP].

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5 with one or more point mutations.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, and the expression cassette includes the following éléments in the 5'-end to 3'-end direction:

a CMV enhancer;

a CMV promoter;

an intron of the hBG 1 gene;

the above codon-optimized nucleic acid of the SMN 1 gene;

an hGHl polyadenylation signal;

a right ITR.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP] having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, and the expression cassette comprises a nucleic acid with SEQ ID NO: 4.

[n one aspect, the present invention relates to a pharmaceutical composition for delivering the SMNl gene to target cells, which includes the above AAV9-based recombinant virus in combination with one or more pharmaceutically acceptable excipients.

In one aspect, the present invention relates to the use of the above AAV9-based recombinant virus or the above composition to deliver the SMN1 gene to target cells.

Brief description of drawings

Figure 1. SMNl expression atthe mRNA level foilowing transfection. HEK293 cells and HSMCs were transfected with 5 pg of plasmids pAAV-SMNl-WT and pAAV-SMNl-GB (encoding the SMNl gene without codon optimization and with codon optimization according to the GeneBeam algorithm). After 72 hours, the copy number of the SMNl gene in each sample was determined by quantitative PCR (n = 3). The copy number of the GAPDH household gene was also determined. AU obtained levels for SMNl were normalized to 10,000 copies of the GAPDH gene in each sample. Provided is the data on the normalized average copy number of SMN1-WT, SMN-GB for both cell lines, with the indication of a standard déviation. Further provided is the ratio of the normalized copy number of SMN1-GB and SMN1-WT in each line.

Figure 2. SMNl expression at the protein level foilowing transfection. HSMCs were transfected with 5 pg of plasmids pAAV-SMNl-WT and pAAV-SMNl-GB (encoding the SMNl gene without codon optimization and with codon optimization according to the GeneBeam algorithm). After 72 h, the cells were stained with prîmary antibodies to the SMNl protein and secondary antibodies labeled with Alexa Fluor 488 in each sample (n = 3). Shown is the average intensity of the fluorescent signal for lîving cells in the samples after subtracting the background signal obtained on cells stained with secondary antibodies without primary antibodies, with the indication of a standard déviation.

Figure 3. The ratio of SMNl expression at the mRNA and protein levels foilowing transduction. HSMCs were transduced by AAV9-SMN1-WT and AAV9-SMN1-GB viruses in 3 independent experiments, in each of which the transduction efficiency was at least 50% for the control GFP-containing virus. SMNl expression was determined at the mRNA and protein levels (see above), foilowing which the ratio of SMNCGB and SMN1-WT expression was calculated. The figure illustrâtes average ratios along with standard déviations.

Définitions and general methods

Unless defined otherwise, ail technical and scientific terms used herein hâve the same meaning as is commonly understood by one of ordinary skill in the art.

Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Typically, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetîcs, analytîcal chemistry, organic synthesis chemistry, medical and pharmaceutical chemistry, as well as hybridization and chemistry of protein and nucleic acids described herein are well known and wîdely used by those skilied in the art. Enzyme reactions and purification methods are performed according to the manufacturer’® guidelines, as is common in the art. or as described herein.

“Isolated” means altered or removed from the natural State. For example, a nucleic acid or a peptide naturally present in an animal is not “isolated”, but the same nucleic acid or peptide parti al ly 01 completely separated from the coexisting materials of its natural State is “isolated . An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a genetically modified cell.

The terms “naturally occurring,” “native,” or “wild-type” is used to describe an object that can be found in nature as distinct from being artificially produced. For example, a protein or nucléotide sequence present in an organism (including a virus), which can be isolated from a source in nature and that has not been întentionally modified by a person in the laboratory, is naturally occurring.

The tenn “genome” refers to the complété genetic material of an organism.

As used in the present description and daims that follow, unless otherwise dictated by the context, the words include and comprise, or variations thereof such as having, includes, including, comprises, or comprising, will be understood to imply the inclusion of a stated integeror group of integers but not the exclusion of any other integer or group of integers.

Protein (Peptide)

As used in the present description, the terms peptide, “polypeptide” and “protein” are used interchangeably, and they refer to a compound consisting of amino acid residues that are covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used in the present description, the term refers to both short chai ns, which also commonly are referred to in the art, for example, as peptides, oligopeptides and oligomers, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, inter alia, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

Nucleic acid molécules

The terms nucleic acid, nucleic sequence, nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence and nucléotide sequence, used interchangeably in the present description, mean a précisé sequence of nucléotides, modified or not, determîning a fragment or a région of a nucleic acid, containing unnatural nucléotides or not, and being either a double-stranded DNA or RNA, a single-stranded DNA or RNA, or transcription products of said DNAs.

One skilled in the art has the general knowledge that nucleic acids are polynucleotides that can be hydrolyzed to monomeric “nucléotides”. Monomeric nucléotides can be hydrolyzed into nucleosides. As used in the present description, polynucleotides include, as non-limiting examples, ail nucleic acid sequences which are obtained by any means available in the art, including, as non-limiting examples, recombinant means, i.e. the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR and the like, and by synthetic means.

It should also be noted here that the present invention does not relate to nucléotide sequences in their natural chromosomal environment, i.e. în a natural state. The sequences of the present invention hâve been isolated and/or purified, i.e. they were sampled directly or indirectly, for example by a copy, their environment having been at least partially modified. Thus, isolated nucleic acids obtained by recombinant genetics, by means, for example, of host cells, or obtained by Chemical synthesis should also be mentioned here.

An isolated nucleic acid molécule is one which is identified and separated from at least one nucleic acid molecule-impurity, which the former is typically bound to in the natural source of nuclease nucleic acid. An isolated nucleic acid molécule is different from the form or set in which it îs found under natural conditions. Thus, an isolated nucleic acid molécule is different from a nucleic acid molécule that exists in cells under natural conditions. An isolated nucleic acid molécule however includes a nucleic acid molécule located in cells in which the nuclease is normally expressed, for example, if the nucleic acid molécule has a chromosomal localization that is different from its localization in cells under natural conditions.

Unless otherwise indicated, the term nucléotide sequence encompasses its complément. Thus, a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.

The terms “transformation,” “transfection,” and “transduction” refer to any method or means by which a nucleic acid is introduced into a cell or host organism, and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, infection, PEG-fusion, and the like.

Adeno-associated virus (AA V)

Viruses of the Parvoviridae family are small DNA-containing animal viruses. The Parvoviridae family may be divîded into two subfamilîes: the Parvovirinae, which members infect vertebrates, and the Densovirinae, which members infect insects. By 2006, there hâve been 11 serotypes of adeno-associated virus described (Mori, S. ET AL., 2004, «Two navel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein», Virology, T. 330 (2): 375-83). Ail of the known serotypes can infect cells from multiple tissue types. Tissue specificity is determined by the capsid protein serotype; therefore, the adeno-associated virus-based vectors are constructed by assigning the desired serotype. Further information on parvoviruses and other members ofthe Parvoviridae is described in the literature (Kenneth 1. Bénis, «Parvoviridae: The Viruses and Their Réplication», Chapter 69 in Fields Virology (3 d Ed. 1996)).

The genomîc organization of ail known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molécule that is less than about 5000 nucléotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucléotide sequences of réplication of non-structural proteins (Rep) and structural proteins (Cap). The Cap gene encodes the VP proteins (VP1, VP2, and VP3) which form the capsid. The terminal 145 nucléotides are self-coinplementary and are organized such that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. Such hairpin structures function as an origin for vira! DNA réplication, serving as primers for the cellular DNA polymerase complex. Following wild-type AAV (wtAAV) infection în mammalian cells, the Rep genes (e.g. Rep78 and Rep52) are expressed using the P5 promoter and the Pl9 promoter, respectively, and the both Rep proteins hâve a certain function in the réplication of the viral genome. A splîcîng event în the Rep open reading frame (Rep ORF) results in the expression of actually four Rep proteins (e.g. Rep78, Rep68, Rep52, and Rep40). However, it has been shown that the unspliced mRNA encoding Rep78 and Rep52 proteins is sufficient for AAV vector production in mammalian cells.

Vector

The term vector as used herein means a nucleic acid molécule capable of transporting another nucleic acid to which it has been linked.

The terms “infection unit (iu),” “infectious particle,” or “réplication unit,” as used în reference to a viral titer, refer to the number of infectious recombinant AAV vector particles as measured by the infectious center assay, also known as réplication center assay, as described, for example, in McLaughlin et al., J. Virol. (1988) 62:1963-1973.

The term “heterologous” as it relates to nucleic acid sequences such as coding sequences and regulatory sequences, dénotés sequences that are not normally joined together, and/or are not normally associated with a particular cell. Thus, a “heterologous” région of a nucleic acid construct or a vector is a fragment of nucleic acid within or attached to another nucleic acid molécule that is not found in association with the other molécule in nature. For example, a heterologous région of a nucleic acid construct may include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g. synthetic sequences having codons different from the native gene).

As used in the present description, the term “expression” is defined as the transcription and/or translation of a particular nucléotide sequence driven by its promoter.

Use

Gene therapy is the insertion of genes into subject’s cells and/or tissues to treat a disease, typically hereditary diseases, in which a defective mutant allele is replaced with a functional one.

Treat, treatment and therapy refer to a method of alleviating or abrogating a biological disorder and/or at least one of attendant symptoms thereof. As used herein, to alleviate a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of a disease, disorder, or condition. Further, references herein to treatment include references to curative, palliative and prophylactic treatment.

In one aspect, the subject of treatment, or patient, is a mammal, preferably a human subject. Said subject may be either male or female, of any âge.

The term disorder means any condition that would benefit from treatment according to the present invention. This includes chronic and acute disorders or diseases including those pathological conditions that prédisposé the mammal to the disorder in question.

“Disease” is a State of health of an animal where the animal cannot maintain homeostasis, and where if the disease is not amelîorated then the animal's health continues to deteriorate.

The terms “subject,” “patient,” “individual,” and the like are used interchangeably in the present description, and they refer to any animal amenable to the methods described in the present description. In certain non-limiting embodiments, the subject, patient or individual is a human.

Therapeutically effective amount refers to that amount of the therapeutic agent being administered during treatment which will relieve to some extent one or more of the symptoms of the disease being treated.

Detailed description of invention

Codon-optimized nucleic acid

In one aspect, the present invention relates to an isolated codon-optimized nucleic acid that encodes the SMN 1 protein (survival motor neuron protein) with SEQ ID NO: 1, and includes the nucleic acid sequence of SEQ ID NO: 2.

The corresponding amino acid sequence of the SMN_HUMAN protein was used as a basis to produce the codon-optimized SMN1 gene:

MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSD1WDDTAL1KAYDKAVASFKHALKNGD1CETS GKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGC1YPATIASIDFKRETCVVVY TGYGN REEQN LS DLLSP1CE V ANNIEQN AQENEN ESQV STDESENS RS PGN KS DN1KPKS AP WN SF LPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPPICPDSLDDADALGS MLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN (SEQ ID NO: 1 )

This amino acid sequence of SEQ ID NO:1 was translated into a nucléotide sequence by sequentially matching each amino acid starting from the N-end of one of the synonymous codons encoding same.

Detailed information on the codon-optimized SMN 1 gene and the sélection of a final sequence is provided in Example ].

The final codon-optimized sequence of SMNI (SMNl-GeneBeam) has <he following nucieotide sequence:

atggccatgagcagcggcggcagcggcggcggcgtgcctgagcaagaggacagcgt GCTGTTCAGAAGAGGCACCGGCCAGAGCGACGACAGCGACATCTGGGACGACACCGCCCTG ATCAAGGCCTACGACAAGGCCGTGGCCAGCTTCAAGCACGCCCTGAAGAACGGCGACATCT GCGAGACCAGCGGCAAGCCCAAGACCACCCCCAAGAGAAAGCCCGCCAAGAAGAACAAGA GCCAGAAGAAGAACACCGCCGCCAGCCTGCAGCAGTGGAAGGTGGGCGACAAGTGCAGCG CCATCTGGAGCGAGGACGGCTGCATCTACCCCGCCACCATCGCCAGCATCGACTTCAAGAGA GAGACCTGCGTGGTGGTGTACACCGGCTACGGCAACAGAGAGGAGCAGAACCTGAGCGACC TGCTGAGCCCCATCTGCGAGGTGGCCAACAACATCGAGCAGAACGCCCAAGAGAACGAGAA CGAGAGCCAAGTGAGCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCAACAAGAGCGA CAACATCAAGCCCAAGAGCGCCCCCTGGAACAGCTTCCTGCCCCCTCCCCCCCCTATGCCCG GCCCTAGACTGGGCCCTGGCAAGCCTGGCCTGAAGTTCAACGGCCCCCCCCCCCCTCCTCCT CCTCCTCCTCCTCACCTGCTGAGCTGCTGGCTGCCCCCCTTCCCCAGCGGCCCTCCTATCATC CCTCCTCCCCCCCCCATCTGCCCCGACAGCCTGG ACG ACGCCG ACGCCCT GGGC AGC ATGCT GATCAGCTGGTACATGAGCGGCTACCACACCGGCTACTACATGGGCTTCAGACAGAACCAG AAGGAGGGCCGGTGCAGCCACAGCCTGAACTAG (SEQ ID NO:2).

This final codon-optimized nucieotide sequence of SMNI (SMNl-GeneBeam) has an increased codon adaptation index (a standard measure for evaluating a sequence for codon frequencies) as compared to the coding sequence of the wild-type SMN gene (SMN1-WT): ATGGCGATGAGCAGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAGGAGGATTCCGTGCTGT TCCGGCGCGGCACAGGCCAGAGCGATGATTCTGACATTTGGGATGATACAGCACTGATAAA AGCATATGATAAAGCTGTGGCTTCATTTAAGCATGCTCTAAAGAATGGTGACATTTGTGAAA CTTCGGGTAAACCAAAAACCACACCTAAAAGAAAACCTGCTAAGAAGAATAAAAGCCAAAA GAAGAATACTGCAGCTTCCTTACAACAGTGGAAAGTTGGGGACAAATGTTCTGCCATTTGGT CAGAAGACGGTTGCATTTACCCAGCTACCATTGCTTCAATTGATTTTAAGAGAGAAACCTGT GTTGTGGTTTACACTGGATATGGAAATAGAGAGGAGCAAAATCTGTCCGATCTACTTTCCCC AATCTGTGAAGTAGCTAATAATATAGAACAAAATGCTCAAGAGAATGAAAATGAAAGCCAA GTTTCAACAGATGAAAGTGAGAACTCCAGGTCTCCTGGAAATAAATCAGATAACATCAAGC CCAAATCTGCTCCATGGAACTCTTTTCTCCCTCCACCACCCCCCATGCCAGGGCCAAGACTG GGACCAGGAAAGCCAGGTCTAAAATTCAATGGCCCACCACCGCCACCGCCACCACCACCAC CCCACTTACTATCATGCTGGCTGCCTCCATTTCCTTCTGGACCACCAATAATTCCCCCACCAC CTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGAAGTATGTTAATTTCATGGT ACATGAGTGGCTATCATACTGGCTATTATATGGGTTTCAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCITAAATTAA (SEQ ID NO:3).

The codon adaptation index for the final codon-optimized nucieotide sequence ofthe SMNI gene (SEQ ID NO:2) is 98% for the subject sequence, and that is 75% for the wild-type sequence.

The GC content ofthe wild-type sequence is 45%, i.e. it differs from the target value by 15%, and the GC content ofthe final codon-optimized nucléotide sequence of the SMN1 gene (SEQ ID NO; 2) for the optimized sequence is 64%, i.e. it differs from the target value by 4%.

The final codon-optimized nucléotide sequence of the SMN ! gene (SEQ ID N0:2) and the nucléotide sequence ofthe wild-type SMN1 gene (SEQ ID N 0:3) are identical by 71%.

Expression cassette. Expression vector.

In one aspect, the present invention relates to an expression cassette that includes the above codon-optimized nucleic acid.

The term expression cassette, as used herein, refers in particular to a DNA fragment that is capable, in an approprîate setting, of inducing the expression of a polynucleotide encoding the polypeptide of interest that is included in said expression cassette. When introduced into a host cell, the expression cassette is, inter alia, capable of engaging cellular mechanisms to transcribe the polynucleotide encoding the polypeptide of interest into RNA that is then typically further processed and eventually translated into the polypeptide of interest. The expression cassette may be contained in an expression vector.

The expression cassette of the present invention comprises a promoter as an element. The term promoter as used herein refers in particular to a DNA element that promûtes the transcription of a polynucleotide to which the promoter is operably linked. The promoter may further form part of a promoter/enhancer element. Although the physical boundaries between the promoter and enhancer éléments are not always clear, the term promoter typically refers to a site on the nucleic acid molécule to which an RNA polymerase and/or any associated factors binds and at which transcription is initiated. Enhancers potentiate promoter activity teinporally as well as spatially. Many promoters are known in the art to be transcriptionally active in a wide range of cell types. Promoters can be divided into two classes, those that function constitutively and those that are regulated by induction or derepression. The both classes are suitable for protein expression. Promoters that are used for high-level production of polypeptides in eukaryotic cells and, in particular, in mammalian cells, should be strong and preferably active in a wide range of cell types. Strong constitutive promoters which are capable of driving expression in many cell types are well known in the art and, therefore, it is not herein necessary to describe them in detail. In accordance with the idea of the present invention, it is préférable to use the cytomégalovirus (CMV) promoter. A promoter or promoter/enhancer derived from the immédiate early (1E) région of human cytomégalovirus (hCMV) is particularly suitable as a promoter in the expression cassette of the present invention. The immédiate early (1E) région of human cytomégalovirus (hCMV) and obtained therefrom functional expression-inducing fragments and/or functional expression-augmenting fragments, for example, are described in EP0173177 and EP0323997 and are also well known in the art. Thus, several fragments of the immédiate early (LE) région of hCMV may be used as a promoter and/or promoter/enhancer. According to one embodiment of the invention, the human CMV promoter is used in the expression cassette of the present invention.

In some embodiments, the expression cassette includes the following éléments in the 5'-end to 3'end direction:

a left (first) ITR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an intron of the hBGl gene (hemoglobin subunit gamma 1 gene) the above codon-optimized nucleic acid of the SMN1 gene;

an hGHl polyadenylation signal (human growth hormone gene polyadenylation signal) a rîght (second) ITR.

In some embodiments, the left (first) ITR (inverted terminal repeats) has the following nucleic _acid sequence:

Cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcag agagggagtggccaactccatcactaggggttcct (SEQ ID NO: 8).

In sonie embodiments, the CMV (cytomégalovirus) enhancer has the following nucleic acid sequence: cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgCcaata gggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtca atgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg (SEQ ID NO: 9).

In some embodiments, the CMV (cytomégalovirus) promoter has the following nucleic acid sequence: gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgtlttgG caccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcaga get (SEQ ID NO: 10).

In some embodiments, the intron of the hBGl (hemoglobin subunit gamma 1) gene has the following nucleic acid sequence:

cgaatcccggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctt tcttcttttaatatacttttttgtttatcttatttctaatactticcctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattctaaagaat aacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcaiataaattgtaactgatgtaagaggtttcatattgcraatagcagct acaatccagctaccattctgcttttattttatggttgggataaggctggattattctgagiccaagctaggcccttttgciaatcatgttcatacctcttatcncctc ccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggat (SEQ ID NO: 11 ).

In some embodiments, the hGHl (human growth hormone 1 gene) polyadenylation signal has the following nucleic acid sequence:

Acgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagigcccaccagccttgtcctaataaaaitaa gttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcc tgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcc cgagttgttgggattccaggcatgcaigaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcct aatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtcctt (SEQ ID NO: 12).

In some embodiments, the right (second) ITR has the foîlowing nucleic acid sequence: aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgeccgacgcccgggctttgcc cgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg (SEQ IDNO: 13).

In some embodiments, the expression cassette has the foîlowing nucleic acid sequence: cctgcaggcagctgcgcgctegctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagigagcgagcgagcgcgcaga gagggagtggccaactccatcactaggggttcctgcggccgcacgcgtctagttattaatagtaatcaattacggggtcattagttcatagcccatatatgg agttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacg Ccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtaegccccctatt gacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattac caiggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgaetcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttt tgGcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagc agagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggattc gaatcccggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgcttt cttcttttaatatacttttttgtttatcttatttetaatactticcctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattctaaagaata acagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagcta caatccagctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcatgttcatacctcttatcttcctcc cacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggattegaacatCGATTGTAATTCATGAGCC ACCATGGCCATGAGCAGCGGCGGCAGCGGCGGCGGCGTGCCTGAGCAAGAGGACAGCGTGC TGTTCAGAAGAGGCACCGGCCAGAGCGACGACAGCGACATCTGGGACGACACCGCCCTGAT CAAGGCCTACGACAAGGCCGTGGCCAGCTTCAAGCACGCCCTGAAGAACGGCGACATCTGC GAGACCAGCGGCAAGCCCAAGACCACCCCCAAGAGAAAGCCCGCCAAGAAGAACAAGAGC CAGAAGAAGAACACCGCCGCCAGCCTGCAGCAGTGGAAGGTGGGCGACAAGTGCAGCGCC ATCTGGAGCGAGGACGGCTGCATCTACCCCGCCACCATCGCCAGCATCGACTTCAAGAGAG AGACCTGCGTGGTGGTGTACACCGGCTACGGCAACAGAGAGGAGCAGAACCTGAGCGACCT GCTGAGCCCCATCTGCGAGGTGGCCAACAACATCGAGCAGAACGCCCAAGAGAACGAGAAC GAGAGCCAAGTGAGCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCAACAAGAGCGAC AACATCAAGCCCAAGAGCGCCCCCTGGAACAGCTTCCTGCCCCCTCCCCCCCCTATGCCCGG CCCTAGACTGGGCCCTGGCAAGCCTGGCCTGAAGTTCAACGGCCCCCCCCCCCCTCCTCCTC CTCCTCCTCCTCACCTGCTGAGCTGCTGGCTGCCCCCCTTCCCCAGCGGCCCTCCTATCATCC CTCCTCCCCCCCCCATCTGCCCCGACAGCCTGGACGACGCCGACGCCCTGGGCAGCATGCTG ATCAGCTGGTACATGAGCGGCTACCACACCGGCTACTACATGGGCTTCAGACAGAACCAGA AGGAGGGCCGGTGCAGCCACAGCCTGAACTGATctagagtcgacctgcagaagcttgccîcgagcagcgctgctcgag agatctacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagtt gcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgc ggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccga gttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatc tcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgiccttctgattttgtaggtaaccacgtgcgga ccgagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacg cccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg (SEQ ID NO: 4).

in one aspect, the present invention relates to an expression vector that includes the above codonoptimized nucleic acid or the above expression cassette.

In some embodiments, the vector is a plasmid, i.e., a circular double stranded pîece of DNA into which additional DNA segments may be ligated.

In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.

In some embodiments, vectors are capable of autonomous réplication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin site of réplication and episomal mammalian vectors). In further embodiments, vectors (e.g. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into a host cell, and thereby are replicated along with the host gene. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as recombinant expression vectors (or sîmply, expression vectors).

The expression vectors include piasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses, such as caulîflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. DNA molécules may be ligated into the vector such that transcriptional and translational control sequences within the vector serve their întended function of regulatîng the transcription and translation of the DNA. The expression vector and expression control sequences may be selected to be compatible with the expression host cell used. DNA molécules may be introduced into the expression vector by standard methods (e.g. ligation of complementary restriction sites, or blunt end ligation if no restriction sites are present).

The recombinant expression vector may also encode a signal peptide that facilitâtes the sécrétion of the protein of interest from a host cell. The gene of the protein of interest may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the protein of interest. The signal peptide may be an immunoglobulîn signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non-immunoglobulin protein).

In addition to the SMN1-GB gene of the present invention, the recombinant expression ofthe vectors ofthe present invention may carry regulatory sequences that control the expression ofthe SMN1GB gene in a host cell. It will be understood by those skilled in the art that the design of an expression vector, including the sélection of regulatory sequences, may dépend on such factors as the choice of a host cell to be transformed, the level of expression of a desired protein, and so forth. Preferred control sequences for an expression host cell in mammals include viral éléments that ensure high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomégalovirus (CMV) (such as a CMV promoter/enhancer), simian virus 40 (SV40) (such as a SV40 promoter/enhancer), adenovirus, (e.g. the major late promoter adenovirus (AdMLP)), polyo ma virus and strong mammalian promoters such as native immunoglobulîn promoter or actin promoter.

The term control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences thaï are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signais, and enhancers.

As used in the present description, the term “promoter” or “transcription regulatory sequence” or “regulatory sequence” refers to a nucleic acid fragment that Controls the transcription of one or more coding sequences, and that is located upstream with respect to the direction of reading relative to the direction of transcription from the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucléotides known to one of skill in the art that directly or indirectly regulate the level of transcription with said promoter. A “constitutive” promoter is a promoter that is active in most tissues under typical physiological and developinental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. under the influence of a Chemical inducer. A “tissue spécifie” promoter is only active in spécifie types of tissues or cells.

The terms “enhancers” or “enhancer” as used herein may refer to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product. Enhancer éléments are typically located in a 5' direction from a promoter element or can be located downstream of or within a coding DNA sequence (e.g. a DNA sequence transcribed or translated into a recombinant product or products). Hence, an enhancer element can be located 1Û0 base pairs, 200 base pairs, or 300 or more base pairs upstream of a DNA sequence that encodes a recombinant product, or downstream of said sequence. Enhancer éléments may increase the amount of a recombinant product being expressed from a DNA sequence above the level of expression associated with a single promoter element. Multiple enhancer éléments are readily available to those of ordinary skill in the art.

In addition to the above genes and regulatory sequences, recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate réplication of a vector in host cells (e.g. origins of réplication) and selectable marker genes. The selectable marker gene facilitâtes the sélection of host cells into which a vector has been introduced (see e.g. U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017). For example, the selectable marker gene typically confers résistance to médicinal agents, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. For example, selectable marker genes include a dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells during methotrexate selection/amplification), a neo gene (for G418 sélection), and a glutamate synthetase gene.

The term expression control sequence as used in the present description refers to polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signais such as splicing and polyadenylation signais; sequences that stabilîze cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein sécrétion. The nature of such control sequences differs depending upon the host organisai; in prokaryotes, such control sequences generally include the promoter of ribosome binding site, and transcription termination sequences; in eukaryotes, typically, such control sequences include promoters and transcription termination sequences. The term control sequences is intended to include at least ail components, the presence of which is essential for expression and processing, and can also include additional components, the presence of which is advantageous, for example, leader sequences and fusion partner sequences.

As used herein, the term “operably linked” refers to a linkage of polynucleotide (or polypeptide) éléments in a functional relationship. A nucleic acid is “operably linked” when it îs present în functional relationship conditions with another nucleic acid sequence. For example, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of said coding sequence. The term operably linked means that the DNA sequences being linked are typically contiguous and. where it is necessary to join two protein coding régions, are also contiguous and are present in the reading frame.

In one embodiment of the present invention, expression vector relates to a vector comprising one or more polynucleotide sequences of interest, genes of interest, or transgenes that are flanked by parvoviral sequences or inverted terminal repeat (ITR) sequences.

Neither the cassette nor the vector of the invention comprises nucléotide sequences of genes encodîng non-structural proteins (Rep) and structural proteins (Cap) ofthe adeno-associated virus.

AA V9 (adeno-associated virus serotype 9)-based recombinant virus

In one aspect, the present invention relates to an AAV9 (adeno-associated virus serotype 9)-based recombinant virus for increasing the expression of the SMN 1 gene in target cells, which includes a capsid and the above expression cassette.

The term AAV-based recombinant virus (or AAV-based virus-like particle, or AAV recombinant virus strain, or AAV recombinant vector, or rAAV vector) as used in this description refers to the above expression cassette (or the above expression vector), which is enclosed within the AAV capsid.

The Cap gene, among other alternative products, encodes 3 capsid proteins (VP1, VP2, and VP3). VP1, VP2, and VP3 are present at 1:1:10 ratio to form an icosahedral capsid (Xie Q. et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002; 99:10405-10410). Transcription of these genes starts from one promoter, p4û. The molecular weights of the corresponding proteins (VP1, VP2 u VP3) are 87, 72, and 62 kDa, respectively. Ail of the three proteins are translatée! from a single mRNA. Following transcription, pre-mRNA may be spliced in two different manners, where either longer or shorter intron is excised to form mRNAs of various nucléotide lengths.

During the production of the AAV (rAAV)-based recombinant virus, an expression cassette flanked by ITR is packaged into an AAV capsid. The genes required for AAV réplication, as mentioned above, are not included in the cassette.

The expression cassette DNA is packaged into a viral capsid in the form of a single stranded DNA molécule (ssDNA) being approximately 3000 nucléotides long. Once a cell is infected with the virus, the single-stranded DNA is converted to the form of double-stranded DNA (dsDNA). The dsDNA can only be used by the cell's proteins, which transcribe the present gene or genes into RNA.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VPI.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VPI having the foilowing amino acid sequence MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDK GEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLK.EDTSFGGNLGRAVFQAKK RLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPI GEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPT YNNHLYKQ1SNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRL1NNNWGFRPKRLNF KLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYL TLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLY YLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSW ALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEE1KTTNPVA TESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLM GGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQY TSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 5).

In some embodiments, the AAV9-based reconibinant virus has a capsid that includes the AAV9 protein VPI having the amino acid sequence of SEQ ID NO: 5 with one or more point mutations.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP2.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP2 having the foilowing amino acid sequence: TAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLT MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTS GGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDN NGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRS SFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKT1NGSGQN QQTLKFSV AGPSN MAVQGRN YIPG PS Y RQQRV STTVTQNNNSEF A WPG AS S W A LNGRN SLMN P GPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEE1KTTNPVATESYGQVATNH QSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQ1 likntpvpadpptafnkdklnsfitqystgqvsveiewelqkenskrwnpeiqytsnyyksnnve

FAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 6).

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP2 having the amino acid sequence of SEQ ID NO: 6 with one or more point mutations.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP3.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP3 having the following amino acid sequence MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTS GGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVK.EVTDN NGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFM1PQYGYLTLNDGSQAVGRS SFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKFSVAGPSNMAVQGRNY1PGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNP GPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNH QSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQI LIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVE FAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 7).

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP3 having the amino acid sequence of SEQ ID NO: 7 with one or more point mutations.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 proteins VPl, VP2, and VP3.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the proteins VPl with the amino acid sequence of SEQ ID NO: 5, VP2 with the amino acid sequence of SEQ ID NO:

6, and VP3 with the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the proteins VPl with the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, VP2 with the amino acid sequence of SEQ ID NO: 6 with one or more point mutations, and VP3 with the amino acid sequence of SEQ ID NO: 7 with one or more point mutations.

The phrase more point mutations refers to two, three, four, five, six, seven, eight, nine, or ten point substitutions.

Particularly preferred embodiments include substitutions (mutations) that are conservative in nature, i.e. substitutions that take place within a family of amino acids that are joined in their side chains. In particular, amino acids are typically divided into four families: (!) acidic amino acids are aspartate and glutamate; (2) basic amino acids are lysine, arginine, histîdine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, méthionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated substitution of leucine for isoleucine or valine, an aspartate for a glutamate, a threonine for a serine, or a similar conservative substitution of an amino acid for a structurally related amino acid, will not hâve a major effect on the biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, so long as the desired function of the molécule remains intact.

An embodiment with point mutations in the sequences of AAV9 proteins VPl, VP2, or VP3 using amino acid substitutions is a substitution of at least one amino acid residue in the AAV9 protein VPl, VP2, or VP3 with another amino acid residue.

Conservative substitutions are shown in Table A under preferred substitutions.

Table A
Original 1 residue	Exemplary substitutions	Preferred substitutions
Ala (A)	Val; Leu; lie	Val
Arg(R)	Lys; Gin; Asn	Lys
Asn(N)	Gin; His; Asp, Lys; Arg	Gin
Asp (D)	Glu; Asn	Glu
Cys (C)	Ser; Ala	Ser
Gln(Q)	Asn; Glu	Asn
Glu (E)	Asp; Gin	Asp
Gly(G)	Ala	Ala
His (H)	Asn; Gin; Lys; Arg	Arg
Ile (1)	Leu; Val; Met; Ala; Phe; Norleucine	Leu
Leu (L)	Norleucine; Ile; Val; Met; Ala; Phe	Ile
Lys (K)	Arg; Gin; Asn	Arg
Met (M)	Leu; Phe; lie	Leu
Phe(F)	Trp; Leu; Val; lie; Ala; Tyr	Tyr
Pro (P)	Ala	Ala
Ser(S)	Thr	Thr
Thr (T)	Val; Ser	Ser
Trp(W)	Tyr; Phe	Tyr
Tyr(Y)	Tip; Phe; Thr; Ser	Phe
Val (V)	Ile; Leu; Met; Phe; Ala; Norleucine	Leu

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VPl having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, and the expression cassette includes the following éléments in the 5'-end to 3’-end direction;

a CMV enhancer;

a CMV promoter;

an intron ofthe hBGl gene;

the above codon-optimized nucleic acid of the SMN l gene;

an hGHl polyadenylation signal;

a right ITR.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the proteins VPl with the amino acid sequence of SEQ ID NO; 5, VP2 with the amino acid sequence of SEQ ID NO: 6, and VP3 with the amino acid sequence of SEQ ID NO: 7, and the expression cassette includes the following éléments in the 5'-end to 3'-end direction:

a CMV enhancer;

a CMV promoter;

an intron of the hBGl gene;

the above codon-optimized nucleic acid of the SMN l gene;

an hGHl polyadenylation signal;

a right ITR.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the proteins VPl with the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, VP2 with the amino acid sequence of SEQ ID NO: 6 with one or more point mutations, and VP3 with the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, and the expression cassette includes the following éléments in the 5’-end to 3'-end direction:

a CMV enhancer;

a CMV promoter;

an intron of the hBG l gene;

the above codon-optimized nucleic acid ofthe SMNl gene;

an hGHl polyadenylation signal;

a right ITR.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VPl having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, and the expression cassette comprises a nucleic acid with SEQ IDNO: 4.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the proteins VPl with the amino acid sequence of SEQ ID NO: 5, VP2 with the amino acid sequence of SEQ ID NO:

6, and VP3 with the amino acid sequence of SEQ ID NO: 7, and the expression cassette comprises a nucleic acid with SEQ ID NO: 4.

in some embodiments, the AAV9-based recombinant virus has a capsid that includes the proteins VPl with the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, VP2 with the amino acid sequence of SEQ ID NO: 6 with one or more point mutations, and VP3 with the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, and the expression cassette comprises a nucleic acid with SEQ ID NO: 4.

Pharmaceutical composition

In one aspect, the présent invention relates to a pharmaceutical composition for delivering the SMN l gene to target cells, which includes the above AAV9-based recombinant virus in combination with one or more pharmaceutically acceptable excipients.

In particular embodiments, the present invention relates to a pharmaceutical composition comprising the AAV9-based recombinant virus of the invention in a pharmaceutically acceptable carrier or in other pharmaceutical agents, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid carrier. For other methods of administration, the carrier may be either solid or liquid, such as stérile pyrogen-free water or stérile pyrogen-free phosphate-buffered saline solution. For inhalation administration, the carrier is respirable, and preferably is in a solid or liquid particulate form. As an injection medium, it is preferred to use water that contains the additives that are common for injection solutions, such as stabilizing agents, salts or saline, and/or buffers.

Pharmaceutical composition means a composition comprising the above AAV9-based recombinant virus of the invention and at least one of components selected from the group consisting of phannaceutically acceptable and pharmacologically compatible excipients, such as fdlers, solvents, diluents, carriers, auxiliary, distributing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspendîng agents, thickeners, prolonged delivery controllers, the choice and proportions of which dépend on the type and route of administration and dosage. Pharmaceutical compositions of the present invention and methods for préparation thereof will be undoubtedly apparent to those skilled in the art. Pharmaceutical compositions should preferably be manufactured in compliance with the G MP (Good Manufacturing Practice) requirements. A composition may comprise a buffer composition, tonicity agents, stabilizers and solubilizers.

Pharmaceutically acceptable means a material that does not hâve biological or other négative side effects, for example, the material can be administered to a subject without causing any undesirable biological effects. Thus, such pharmaceutical compositions may be used, for example, in transfection of a cell ex vivo or in administration in vivo of the AAV9-based recombinant virus of the invention directly to a subject.

The term excipient is used herein to describe any ingrédient other than the above ingrédients of the invention. These are substances of inorganic or organic nature which are used in the pharmaceutical manufacturing in order to give drug products the necessary physicochemical properties.

Stabiliser refers to an excipient or a mixture of two or more excipients that provide the physical and/or Chemical stability of the active agent.

The term buffer, buffer composition, buffering agent refers to a solution, which is capable of resisting changes in pH by the action of its acid-base conjugale components, which allows the rAAV5 vector product to resist changes in pH. Generally, the phannaceutical composition preferably has a pH in the range from 4,0 to 8.0. Examples of buffers that can be used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, etc. buffer solutions.

A pharmaceutical composition is stable if the active agent retains physical stability and/or Chemical stability and/or biological activity thereof during the specified shelf life at storage température, for example, of 2-8 °C. Preferably, the active agent retains both physical and Chemical stability, as well as biological activity. Storage period is adjusted based on the results of stability test in accelerated or natural aging conditions.

A pharmaceutical composition of the invention can be manufactured, packaged, or widely sold in the form of a single unit dose or a plurality of single unit doses in the form of a ready formulation. The terni single unit dose as used herein refers to a discrète quantity of a pharmaceutical composition containing a predetermined quantity of an active ingrédient. The quantity of the active ingrédient typically equals the dose of the active ingrédient to be administered in a subject, or a convenient portion of such dose, for example, half or a third of such dose.

Use

In one aspect, the present invention relates to the use of the above AAV9-based recombinant virus or the above composition to deliver the SMN I gene to target cells.

Any method for administering the AAV9-based recombinant virus, which is recognîzed in the art, can be suitably used for the above AAV9-based recombinant virus of the present invention.

The AAV9-based recombinant virus is preferably administered to a cell in a biologically-effective amount. A “biologically-effective” amount of the recombinant virus is an amount that îs sufficient to cause infection (or transduction) and expression of the heterologous nucleic acid sequence in the cell. If the virus is administered to a cell in vivo (e.g. the virus is administered to a subject, as described below), a “biologically-effective” amount of the viral vector is an amount that is sufficient to cause the transduction and expression of the heterologous nucleic acid sequence in the target cell.

The cell for administering the above AAV9-based recombinant virus of the invention may be a cell of any type, including but not limited to neural cells (including cells of the peripheral and central nervous Systems, in particular, brain cells), lung cells, épithélial cells (e.g. gut and respiratory epitheliai cells), muscle cells, pancreatic cells (including islet cells), hepatic cells, myocardial cells, bone cells (e.g. bone marrow stem cells), hematopoietic stem cells, spleen cells, kératinocytes, fibroblasts, endothélial cells, prostate cells, germ cells, and the like. Altematîvely, the cell for administering the above AAV9based recombinant virus may be any progenitor cell. As a further alternative, the cells may be stem cells (e.g. neural stem cells, liver stem cells). Furthermore, the cells may be from any species of origin, as specified above.

The above AAV9-based recombinant virus is not used to modify the genetic integrity of human germ line cells.

Examples

The following exemples are provided for a better understanding ofthe invention. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

Ail publications, patents, and patent applications cited in this spécification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of ciarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments.

Materials and general methods

Recombinant DNA techniques

DNA manipulations were carried out by standard techniques as described by Sambrook J. et al, Molecular cloning: A laboratory manuai; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer instructions. Briefly, plastnid DNA was produced for further manipulation in E. eoli cells grown under sélective antibiotic pressure so that the plasmids were not lost in the cell population. We isolated the plasmid DNA from cells using commercial kits, measured the concentration, and used it for cloning by restriction endonuclease treatment or PCR amplification. The DNA fragments were ligated to each other using ligases and transformed into bacterial cells for the sélection of clones and further production. Ail resulting genetic constructs were confirmed by restriction patterns and complété Sanger sequencing.

Gene synthesis

Desired gene segments were prepared from oligonucleotides made by Chemical synthesis. Gene segments of 300 to 1000 bp long, which were flanked by unique restriction sites, were collected by renaturing oligonucleotides on top of each other, followed by PCR amplification from border primers. As a resuit, a mixture of fragments was produced, including the desired one. The fragments were cloned at restriction sites into intermediate vectors, following which the DNA sequences of the subcloned fragments were confirmed by DNA sequencing.

DNA sequence détermination

DNA sequences were determined by Sanger sequencing. DNA and protein sequences were analyzed and sequence data was processed in SnapGene Viewer 4.2 or higher for sequence création, mapping, analysis, annotation and illustration.

Culturing cell cultures

The experiments used HEK293 (Human Embryonic Kidney clone 293) and HSMC (Human Skeletal Muscle Cells) cell lines. The cells were cultured under standard conditions at 37 C and 5%CO2, on a DMEM complété culture medium supplemented with 10% FBS and an antibiotic. To culture HSMCs, the culture plastic was pre-coated with collagen (Gibco). Cells were subcultured upon reaching 80-90% confluence. Cell viability was assessed using either Trypan Blue stain and a hemocytometer or Pl stain and flow cytometry.

Cell transfection

Cell lines were inoculated the day before transfection into 6-well plates such that they reached 7080% confluence by the time of transfection. Transfection was performed using commercial lipofection kits according to the manufacturées protocol. After 72 h, the cells were treated with trypsin solutions or similar, removed from the substrate, washed in a phosphate buffer, and collected for further analysis of expression of target genes and proteins. For each transfection, a control plasmid expressing GFP was used to control the transfection efficiency (percentage of GFP-positive cells). Further analysis was performed only if the transfection efficiency was at least 50%.

Ah measurements were carried out în 3 independent experiments.

Gene expression analysis

SMN1 expression at the mRNA level was assessed by quantitative PCR. Briefly, primers and a sample spécifie for the wild-type SMN l sequence or GeneBeam were used. Primers and a sample spécifie for the GAPDH housekeeping gene were used to control the initial RNA levels. Calibration curves were plotted for each set of primers and samples using a known copy number of linearized plasmid DNA comprising the amplified sequence of the corresponding gene. Expression was analyzed by determining, using the calibration curves, the copy number of SMNl-GeneBeam, SMNl-WT, and GAPDH in each sample, following which we normalized the copy number of SMNl per 10,000 copies of GAPDH. The resulting values were compared for different samples within the same experiment.

Détermination of SMNl protein expression by flow cytometry

The SMN 1 protein content in the cells was assessed by intracellular staining, followed by analysis using flow cytometry. Briefly, the cells were removed from culture plates using TrypLE, washed ïn PBS, fixed in a 4% paraformaldéhyde solution, permeabilîzed using a 0.5% Triton X-100 solution in PBS, incubated in a blocking buffer supplemented with 1-5% BSA, and stained in two stages using primary antibodies to SMNl and secondary antibodies labeled with Alexa Fluor 488. Following staining, the cells were washed once in PBS and analyzed on a flow cytometer. The average intensity of the signal was assessed after subtracting the signal stained with secondary antibodies without adding the primary antibodies.

Assembly and purification of viral particles of recombinant AAV vectors

To assemble AAV particles containing the SMNl gene or GFP control gene, we used HEK293 packaging cells, into which 3 plasmids were transfected as foliows:

A plasmid comprising the AAV genome with a transgene (SNMl or GFP) expression cassette;

A plasmid for expression of the AAV9 serotype Cap gene and the AAV2 serotype Rep gene. Each gene, using alternative reading frames, encodes several protein products;

A plasmid for expression of Ad5 (adenovirus serotype 5) genes that are required for assembly and packaging of AAV capsids.

After 72 hours, the cells were lysed and the viral particles were purified and concentrated using filtration and chromatography methods. The tîter of the viral particles was determined by quantitative PCR with primers and a sample spécifie for the site of the recombinant viral genome and expressed as the copy number of viral genomes per l ml.

Transducing cell cultures

Cell lines were inoculated similarly to transfection experiments, following which the product with viral particles was added and the cells were analyzed after 72 hours. Transduction efficiency was estîmated by measuring the percentage of GFP+ cells.

The cultures being used were pre-tested with the check of the transduction efficiency. Briefly, the AAV9-GFP viral product was transduced into the cell lines în different ratios of cells and viral particles. The ratio of viral particle number to cell number is referred to as multiplicity of infection (MOI). The MOI ofthe AAV9-GFP virus ranged from 50,000 to 1,000,000. As a result, MOI ranges, within which the transduction efficiency varied linearly depending on MOI, were determined for each line. Further transduction of cell lines was carried out within their linear ranges.

Following transduction, gene and protein expression was analysed as described above.

AH measurements were carried out in 3 independent experiments.

Example 1. Method for producing a codon-optimized SMNl gene

The corresponding amino acid sequence of the SMN_HUM AN protein (SEQ ID NO: 1 ) was used as a basis to produce the codon-optimized SMNl gene.

This amino acid sequence of SEQ ID NO:1 was translated into a nucléotide sequence by sequentially matching each amino acid starting from the N-end of one of the synonymous codons encodîng same with the considération of one or a combination of the following features.

1) frequency of codon usage (Yasukazu Nakamura ET AL., Codon usage tabulated from the international DNA sequence daiabases; its status 1999, Nucleic Acids Research, 1999, Vol. 27, No. 1, doi: 10.1093/nar/27.1.292k

2) GC content at the terminal région of lhe resulting nucléotide sequence (the target value of GC content was 60% as following from the article by Grzegorz Kudla ET AL., High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells, PLoS Biol, June 2006, Volume 4, Issue 6, el80, doi: 10.1371/ioumal.pbio.0040180, so the smaller was the différence between the current GC content and the target one, the more préférable was the codon);

3) free energy of folding of the terminal région of the resulting nucléotide sequence (secondary structures were determined using the Zuker algorithm, Michael Zuker ET AL., Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information, Nucleic Acids Research, Volume 9, Issue 1,10 January 1981, Pages 133-148, doi: 10.1093/nar/9.1.133).

The construction process further avoided the génération of semantic nucléotide sequences, such as restriction sites, internai ribosome entry sites, and splicing sites.

As a resuit of translating the amino acid sequence of SEQ ID NO: 1 to a nucléotide sequence, an array ofcodon-optimized nucléotide sequences ofthe SMN] gene was produced.

Several sequences out of the above array of codon-optimized nucléotide sequences of SMN1 did not show an increase in SMN1 gene transcription in further studies, that is, there was no significant increase in the mRNA copy number of SMNl-opt as compared to SMN1-WT on any of the cell lines used, or this increase was insignifîcant.

Most of the codon-optimized nucléotide sequences of the SMNl gene showed a L.5-2-fold increase in the SMNl gene transcription in further studies, that is, significantly increasing the mRNA copy number of SMNl-opt as compared to SMN1-WT on ail the cell lines used.

One sequence out of the above array of codon-optimized nucléotide sequences of the SMN 1 gene surprisingly showed more than 3-fold increase in the SMN 1 gene transcription in further studies, that is, surprisingly increasing in the mRNA copy number of SMNl-opt by more than 3 times as compared to SMN 1-WT on al! the cell lines used (see Examples 3-4). This final codon-optimized nucléotide sequence ofthe SMNl gene is conventionally called SMNl-GeneBeam (or abbreviated as SMN1-GB).

The final codon-optimized sequence of SMNl (SMNl-GeneBeam) has a nucléotide sequence represented by SEQ ID NO: 2.

This final codon-optimized nucléotide sequence of SMNl (SMNl-GeneBeam) is characterized by an increased codon adaptation index (Paul M. Sharp ET AL., The codon adaptation index-a measure of 25 directional synonymous codon usage bias, and its potential applications, Nucleic Acids Research, Volume 15, Issue 3, 11 February 1987, Pages 128 l-l295, doi: 10.1093/nar/l 5.3.1281 - a standard measure for evaluating a sequence for codon usage frequencies) as compared to the coding sequence ot the wild-type SMN gene (SMN 1 -WT with SEQ ID NO: 3).

The codon adaptation index for the final codon-optimized nucléotide sequence ofthe SMN1 gene (SEQ ID NO: 2) is 98%, and that for the wild-type sequence is 75%.

The GC content ofthe wild-type sequence is 45%, i.e. it differs from the target value by 15%, and that ofthe final codon-optimized nucléotide sequence ofthe SMN1 gene (SEQ ID NO: 2) îs 64%, i.e. it differs from the target value by 4%.

The final codon-optimized nucléotide sequence of the SMN1 gene (SEQ ID NO:2) and the nucléotide sequence of the wild-type SMN l gene (SEQ ID NO:3) are identical by 71 %.

Example 2. Assembly of genetic constructs carrying recombinant AAV genome and encoding SMN1 gene.

A wild-type SMN1 gene sequence was produced by amplification with spécifie primers with cDNA synthesized based on the total RNA of HEK293 cells. During the amplification process, the Kozak sequence and Clal restriction site were added from the 5’-end of the gene, and the Xbal restriction site was added from the 3’-end. The sequence of the SMN1 gene was thereafter cloned by the restrictionligase method at the Clal and Xbal sites into A commercial construct pAAV-GFP Control plasmid (VPK402) from CellBiolab (USA), with substitution of the GFP gene with SMN1, thereby producing the pAAV-SMNl-WT construct.

The SMNl-GeneBeam sequence was assembled as described above. In view of sequence complexity, despite its relatively small size, we performed a sériés of subcloning of gene fragments in intermediate vectors pGEMT, with sequence vérification for each vector. Next, a full-length version of the gene was assembled from several intermediate vectors by PCR and cloned into the intermediate vector pGEMT. The construct pAAV-SMNl-WT was used as the final genetic construct, with substitution ofthe wild-type SMN1 with SMNl-GeneBeam at the Clal and Xbal sites added to the ends of the SMNlGeneBeam sequence by PCR.

The final vector contains ail the necessary éléments for expression and assembly of the gene as part of the recombinant AAV genome:

) ITRs at the ends of the sequence that is encapsidated into a viral capsid;

2) Eléments for expression of the target gene (promoter, enhancer, intron, Kozak sequence, transgene, polyadenylation site);

3) The bacterial réplication origin and antibiotic résistance gene to produce plasmid DNA in bacterial cells.

It îs important to note that the genetic constructs containing the SMN1-WT and SMNlGeneBeam genes differ only in the SMN 1 gene sequences, and are otherwise completely identical.

Example 3. Vérification of SMNl expression from genetic constructs.

The genetic constructs pAAV-GFP, pAAV-SMNl-WT, and pAAV-SMNl-GB were transfected into HEK293 cells and HSMCs as described above. We used 5 pg of DNA per l well. After 72 h, the cells were collected and the expression of SMNl (normalized to GAPDH) was analyzed as described above.

It was found that the codon optimization of the SMNl gene has an effect on the transcription of SMNl, reiiably increasing by several times the mRNA copy number of SMN l-GB as compared to that of SMN l-WT on the both cell fines used (Fig. I). in particular, for HEK293 cells, the normalized expression ratio of SMNl-GB to SMNl-WT was 3.9, whereas, for HSMCs, that was 12.8.

This property of SMN l-GeneBeam, as shown by the data obtained, is not cell-specific, and further provides a several-fold increased expression of the target gene in cells, which can be an important advantage in the development of gene-therapy drugs. Further, this property is not due to any différences in the gene expression cassette and to the properties of appropriate viral capsids carrying the genome from the SMN l-GeneBeam genes, since this analysis was performed on genetic constructs that differ only in the codon optimization of the SMN l genes, and are otherwise completely identical.

HSMCs were selected to check the expression of SMN l at the protein level by flow cytometry, as described above. It was shown that the signal from SMNl-specific antibodies in cells transfected with pAAV-SMNl-GB is 12.2 times higher as compared to those transfected with pAAV-SMNl-WT at 5 pg of DNA used per l well (Fig. 2). This observation suggests that SMNl-GB has no advantages in translation, but the increased transcription further increases the final protein levels in the cells.

Example 4. Creatiing viral products expressing SMNl

The plasmids pAAV-SMNl-WT and pAAV-SMNl-GB, along with other plasinids required to produce recombinant AAV viral particles (see above), were used for the bioprocess of AAV production. The serotype used was the wild-type AAV9 serotype or that with one or more point mutations.

In ail cases, the properties of the wiid-type SMNl and SMNl-GeneBeam were compared only as long as the serotype used and capsid mutations, if any, were identical. Ail serotypes based on AAV9, either that of wild type or with mutations, are hereinafter referred to as AAV9 without specifying mutations.

The bioprocess produced recombinant viral particles designated as AAV9-SMNI-WT and AAV9-SMN1-GB, as well as control particles AAV9-GFP. After determinîng the titers of viral particles, ail the 3 products with the same MOI (MO! values varied between experiments from 50,000 to 200,000) were used to transduce permissive cells, i.e. primary human myocytes HSMCs. Further analysis was performed only as long as the transduction efficiency was at least 50%.

Following successful transduction, the cells were removed from the substrate, washed in a phosphate buffer, and the expression of SMNl was analyzed at the mRNA and protein levels as described above. The increased transcriptional activity of SMN ]-GeneBeam was shown to remain consistent; thus, the mRNA of SMNl-GeneBeam was detected to be 7.3 times more than that of wild-type SMNl. A similar increase was further observed at the protein level (6.8 times) (Fig. 3), which shows that there are no advantages of SMNl-GB at the translation level; however, the détectable increase in transcription efficiency provîdes, using the AAV9-SMN1-GB product, a higher level of SMNl expression in target cells, which is an important advantage in the treatment of, for example, spinal muscular atrophy, where the level of SMN i protein expression defines the type of disease from 0 (embryonic lethality) to 4 (no spécial treatment required).

Claims (13)

1. l. A codon-optimized nucleic acid that encodes the SMNl protein (survival motor neuron protein) with SEQ ID NO: l, including the nucleic acid sequence of SEQ ID NO: 2.
2. 2. An expression cassette that include the codon-optimized nucleic acid of Claim 1.
3. 3. The expression cassette of Claim 2, including the foîlowing éléments in the 5'-end to 3'-end direction:

   a left (first) ITR (inverted terminal repeats);

   a CMV (cytomégalovirus) enhancer;

   a CMV (cytomégalovirus) promoter;

   an intron of the hBGl gene (hemoglobin subunît gamma 1 gene) the codon-optimized nucleic acid of Claim 1;

   an hGHl polyadenylation signal (human growth hormone gene polyadenylation signal) a right (second) ITR.
4. 4. The expression cassette of Claim 3 that includes a nucleic acid with SEQ ID NO: 4.
5. 5. An expression vector that includes the codon-optimized nucleic acid of Claim l or the cassette of Claims 2-4.
6. 6. An AAV9 (adeno-associated virus serotype 9)-based recombinant virus for increasing the expression of the SMN 1 gene in target cells, which includes a capsid and the expression cassette of any of Claims 2-4.
7. 7. The AAV9-based recombinant virus of Claim 6, wherein the capsid comprises the AAV9 protein VP1.
8. 8. The AAV9-based recombinant virus of Claim 7, wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5.
9. 9. The AAV9-based recombinant virus of Claim 7, wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5 with one or more point mutations.
10. 10. The AAV9-based recombinant virus of Claims 6-9, wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, and the expression cassette includes the foîlowing éléments in the 5'-end to 3'-end direction:

    a CMV enhancer;

    a CMV promoter;

    an intron ofthe hBGl gene;

    the codon-optimized nucleic acid of Claim 1;

    an hGHl polyadenylation signal;

    a right ITR.
11. 11. The AAV9-based recombinant virus of Claim 6, wherein the capsid includes the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID

    NO: 5 with one or more point mutations, and the expression cassette includes a nucleic acid with SEQ ID NO: 4.
12. 12. A pharmaceutical composition for delivering the SMN1 gene to target cells, including the AAV9-based recombinant virus of Claims 6-11 in combination with one or more pharmaceutically acceptable excipients.
13. 13. Use of the AAV9-based recombinant virus of Claims 6-11 or the composition of Claim 12 for delivering the SMN1 gene to target cells.