EA045749B1 - CODON-OPTIMIZED NUCLEIC ACID ENCODING THE SMN1 PROTEIN AND ITS APPLICATION - Google Patents

Description

Field of technology

The present invention 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 (motor neuron survival protein) protein, an expression cassette and a vector based thereon, as well as a recombinant virus based on AAV9 (adeno-associated virus serotype 9) to increase the expression of the SMN1 gene in target cells and their application.

State of the art

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN1) gene and loss of the SMN-encoded protein (Lefebvre et al., Cell (1995). 80:155-165) . The absence of SMN leads to degeneration of motor neurons 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 (Sumner C.J., NeuroRx ( 2006), 3:235-245). Thus, patients with SMA are predisposed to pneumonia and other pulmonary problems such as restrictive pulmonary disease.

Gene therapy represents a promising treatment option for spinal muscular atrophy (SMA).

Adeno-associated viral (AAV) vectors are considered effective for CNS gene therapy because they have a suitable toxicity and immunogenicity profile, can be used in neural cell transduction, and are capable of mediating long-lasting expression in the CNS.

Adeno-associated virus (AAV) is a small (20 nm), non-replicating, non-enveloped virus. Many different serotypes of AAV have been described in humans and primates. The genome of the adeno-associated virus contains (+ or -) single-stranded DNA (ssDNA) about 4.7 thousand nucleotides in length. At the ends of the genomic DNA molecule there are inverted terminal repeats (ITRs). The genome contains two open reading frames (ORFs): Rep and Car, which contain several alternative reading frames encoding various protein products. Rep products are essential for AAV replication, with the Cap gene encoding 3 capsid proteins (VP1, VP2, and VP3) among other alternative products. Proteins VP1, VP2 and VP3 are in a ratio of 1:1:10, forming an icosahedral capsid (Xie Q. et al., The atomic structure of adeno-associated vims (AAV-2), a vector for human gene therapy, Proc. Natl Acad. Sci., USA, 2002, 99:10405-10410). During the formation of a recombinant AAV vector (rAAV), an ITR-flanked expression cassette is packaged into the AAV capsid. Genes required for AAV replication are not included in the cassette. Recombinant AAV is considered the safest and one of the most widely used viral vectors for in vivo gene transfer. Vectors can infect cells from a variety of tissue types, providing potent and sustained transgene expression. They are also non-pathogenic and have a low immunogenicity profile (High K.A. et al., rAAV human trial experience, Methods Mol. Biol., 2011, 807:429-57).

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

One of the properties of the genetic code is degeneracy - the ability of different codons (trinucleotides) to encode the same amino acid. Such codons that give the same amino acid during translation are called synonymous. In natural sequences, the choice of one of the synonymous codons is carried out randomly in the process of evolution, but the frequencies of use of synonymous codons differ: for each amino acid there are more and less preferable ones. Codon optimization is a technique widely used in the world aimed at increasing the productivity of protein molecules, which consists in rationally matching each amino acid in the protein sequence with one of the suitable synonymous codons. One of the common principles of codon optimization involves the use of the most frequent codons; subsequently, other approaches were proposed, such as harmonization (reproducing the distribution of frequencies of used codons), but they do not always increase productivity. In addition to codon frequencies, the production efficiency can be influenced by the GC composition of the sequence (the ratio of the number of guanines and cytosines to the total length of the sequence); in particular, it has been shown that an increased GC composition is associated with an increase in the amount of mRNA in mammalian cells Grzegorz Kudla ET AL., High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells, June 2006, volume 4, issue 6, e180, p. 933-942). It is also worth noting that stable elements of the secondary structure of mRNA, i.e. having low free energy of folding may reduce efficiency.

Various options for codon optimization of the sequence of the gene of interest can lead to the following (compared to the wild type gene):

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

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

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

d) the level of expression of genes of interest will be reduced.

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

It was found that the codon-optimized sequence of SMN1 (SMN1-GeneBeam (or SMN1-GB for short)), which has the nucleotide sequence SEQ ID NO: 2, unexpectedly increases the transcription of the SMN1 gene by more than 3-fold, i.e. unexpectedly increases the number of copies of SMN1-GeneBeam mRNA by more than 3 times compared to SMN1-WT (wild type), which in turn leads to a significant increase in the expression of the SMN1 gene and, accordingly, SMN protein.

Brief description of the invention

In one aspect, the present invention provides an isolated codon-optimized nucleic acid that encodes the SMN1 (survival motor neuron protein) protein of SEQ ID NO: 1, and includes the nucleic acid sequence of SEQ ID NO: 2.

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

In some embodiments, the expression cassette includes the following elements in a 5' to 3' direction:

left (first) ITR (inverted terminal repeats);

CMV (cytomegalovirus) enhancer;

CMV (cytomegalovirus) promoter;

intron of the hBG1 gene (hemoglobin subunit gamma-1 gene);

the above codon-optimized nucleic acid of the SMN1 gene;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal); right (second) ITR.

In some embodiments, the expression cassette includes a nucleic acid having the sequence of SEQ ID NO: 4.

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

In one aspect, the present invention relates to a recombinant virus based on AAV9 (adeno-associated virus serotype 9) for increasing the expression of the 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 VP1 protein.

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

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

In some embodiments, the recombinant AAV9 virus has a capsid that includes an AAV9 VP1 protein 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 elements in the direction from 5 '-end to 3'-end:

CMV enhancer;

CMV promoter;

hBG1 gene intron;

the above codon-optimized nucleic acid of the SMN1 gene;

hGH1 polyadenylation signal;

right ITR.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes an AAV9 VP1 protein 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 nucleic acid of SEQ ID NO: : 4.

In one aspect, the present invention relates to a pharmaceutical composition for delivering the SMN1 gene to target cells, which includes the above-mentioned 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 recombinant AAV9 virus or the above composition for delivering the SMN1 gene to target cells.

Brief description of drawings

In fig. Figure 1 shows SMN1 expression at the mRNA level after transfection. HEK293 and HSMC cells

- 2 045749 were transfected with 5 μg of plasmids pAAV-SMN1-WT and pAAV-SMN1-GB (encoding the SMN1 gene without codon optimization and with codon optimization using the GeneBeam algorithm). After 72 h, the copy number of the SMN1 gene in each sample was determined using quantitative PCR (n=3). The copy number of the housekeeping gene GAPDH was also determined. All obtained quantities for SMN1 were normalized to 10,000 copies of the GAPDH gene in each sample. Data are presented on the normalized mean copy number of SMN1-WT, SMN-GB for both cell lines, with standard deviation indicated. The ratio of normalized copy numbers of SMN1-GB and SMN1-WT in each line is also presented.

In fig. Figure 2 shows the expression of SMN1 at the protein level after transfection. HSMC cells were transfected with 5 μg of plasmids pAAV-SMN1-WT and pAAV-SMN1-GB (encoding the SMN1 gene without codon optimization and with codon optimization using the GeneBeam algorithm). After 72 h, cells were stained with primary antibodies to the SMN1 protein and secondary antibodies labeled with Alexa Fluor 488 in each sample (n=3). The average fluorescent signal intensity for live cells in the samples minus the background signal obtained from cells stained with secondary antibodies without primary antibodies is presented, with the standard deviation indicated.

In fig. Figure 3 shows the ratio of SMN1 expression at the mRNA and protein levels after transduction. HSMC cells were transduced with AAV9-SMN1-WT and AAV9-SMN1-GB viruses in 3 independent experiments, in each of which the transduction efficiency was at least 50% of the control GFP-containing virus. SMN1 expression was determined at the mRNA and protein levels (see above), after which the ratio of SMN1-GB and SMN1-WT expression was calculated. The average ratios along with standard deviations are presented in the figure.

Definitions and general methods

Unless otherwise defined in the present invention, scientific and technical terms used in connection with the present invention will have the meanings that are commonly understood by those skilled in the art.

In addition, unless the context otherwise requires, terms in the singular include plural terms, and plural terms include singular terms. In general, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described in the present invention are well known to those skilled in the art. widely used in this field. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is usually carried out in the art, or as described in the present invention.

Separated means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in an animal is not isolated, but the same nucleic acid or peptide, partially or completely separated from the materials accompanying it in its natural state, is isolated. The isolated nucleic acid or protein may exist in substantially purified form or may exist in a non-natural environment, such as, for example, a genetically modified cell.

The terms naturally occurring, native, or wild type are used to describe an entity that can be found in nature to be different from that produced artificially. For example, a protein or nucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature, and that has not been intentionally modified by a person skilled in the laboratory, is naturally occurring.

The term genome refers to the complete genetic material of an organism.

In the present specification and in the following claims, unless the context otherwise requires, the words include and contain, or variations thereof such as having, comprises, including, contains or containing, are to be understood as including the specified whole or group of wholes, but not excluding any other whole or group of wholes.

Protein (peptide).

As used herein, the terms peptide, polypeptide and protein are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence can contain. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art, such as peptides, oligopeptides and oligomers, and longer chains, generally referred to in the art as proteins, of which there are many types. Polypeptides include, but are not limited to, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives,

- 3 045749 analogues, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

Nucleic acid molecules.

The terms nucleic acid, nucleic sequence or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence and nucleotide sequence, as used interchangeably herein, refer to a distinct sequence of nucleotides, modified or unmodified, defining a fragment or region of nucleic acid, whether or not containing unnatural nucleotides. and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of these DNAs.

One skilled in the art will have general knowledge that nucleic acids are polynucleotides that can be hydrolyzed to monomeric nucleotides. Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences produced by any methods available in the art, including, but not limited to, recombinant methods, i.e. cloning nucleic acid sequences from a recombinant library or cell genome, using conventional cloning and PCR technology, etc., and synthesis methods.

It should also be mentioned here that this invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in its natural state. The sequences of the present invention have been isolated and/or purified, i.e. were taken directly or indirectly, for example by copying, and their environment was at least partially modified. Thus, it should also be understood here as isolated nucleic acids obtained by genetic recombination, for example by host cells, or obtained by chemical synthesis.

An isolated nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one nucleic acid molecule contaminant with which it is typically associated in a natural nucleic acid source. The isolated nucleic acid molecule differs from the form or set in which it occurs naturally. Thus, the isolated nucleic acid molecule is different from the nucleic acid molecule that naturally exists in cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule found in cells that normally express the nuclease, for example, if the nucleic acid molecule has a chromosomal location that is different from its location in the cells under natural conditions.

The term nucleotide sequence covers its complement unless otherwise specified. Thus, a nucleic acid having a specific sequence should be understood as comprising its complementary strand with its complementary sequence.

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

Adeno-associated virus (AAV).

Viruses of the Parvoviridae family are small DNA-containing animal viruses. The family Parvoviridae can be divided into two subfamilies: Parvovirinae, whose members infect vertebrates, and Densovirinae, whose members infect insects. By 2006, 11 serotypes of adeno-associated virus had been described (Mori, S. et al., 2004, Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein, Virology, T., 330(2):375-83 ). All known serotypes can infect cells of many tissue types. Tissue specificity is determined by the serotype of the capsid proteins, so vectors based on adeno-associated virus are constructed by specifying the required serotype. Additional information on parvoviruses and other members of the Parvoviridae is described in the literature (Kenneth I. Berns, Parvoviridae: The Viruses and Their Replication, chapter 69 in Fields Virology (3d ed. 1996)).

The genomic organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule that is less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique replication nucleotide sequences of nonstructural proteins (Rep) and structural proteins (Sp). The Sar gene encodes the VP proteins (VP1, VP2 and VP3), which form the capsid. The terminal 145 nucleotides are self-complementary and are organized in such a way that an energetically stable intramolecular duplex can be formed, forming a T-shaped hairpin structure. Such hairpin structures function as origins of viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following infection of mammalian cells by wild-type AAV (wtAAV), Rep genes (e.g., Rep78 and Rep52) are expressed through the P5 promoter and

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The P19 promoter, respectively, and both Rep proteins perform a specific function in the replication of the viral genome. Splicing in the Rep open reading frame (Rep ORF) results in the expression of essentially four Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40). However, unspliced mRNA encoding the Rep78 and Rep52 proteins has been shown to be sufficient for AAV vector production in mammalian cells.

Vector.

The term vector as used in the present invention means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.

The terms infectious unit(s), infectious particle, or replication unit, as used in relation to viral titer, refer to the number of infectious particles of a recombinant AAV vector, which is measured by an infectious center assay, also known as a replication center assay, as described, for example, in McLaughlin et al., J. Virol. (1988), 62:1963-1973.

The term heterologous, when referring to nucleic acid sequences such as coding sequences and regulatory sequences, refers to sequences that are not typically linked together and/or are not typically associated with a particular cell. Thus, a heterologous region of a nucleic acid or vector construct is a fragment of a nucleic acid located within or attached to another nucleic acid molecule that is not naturally found together with another molecule. For example, a heterologous region of a nucleic acid construct may contain a coding sequence flanked by sequences that are not naturally found in association with the coding sequence. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (eg, synthetic sequences that contain codons different from the native gene).

As used herein, the term expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

Application.

Gene therapy is the insertion of genes into the cells and/or tissues of a subject to treat a disease, usually an inherited disease, whereby a defective mutant allele is replaced or supplemented with a functional allele.

Treat, cure, and therapy refer to a method of alleviating or eliminating a biological disorder and/or at least one of its accompanying symptoms. As used herein, the term ameliorate a disease, disorder, or condition means reducing the severity and/or frequency of symptoms of the disease, disorder, or condition. In addition, references to treatment contained herein include references to curative, palliative and prophylactic therapy.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The above subject may be male or female of any age.

The term disorder means any condition that can be improved by treatment according to the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the occurrence of a given disorder.

Disease is a state of animal health where the animal is unable to maintain homeostasis, and where, if the disease is not alleviated, the animal's health continues to deteriorate.

The term subject, patient, individual, etc. are used interchangeably herein and refer to any animal amenable to treatment by the methods presented herein. In specific non-limiting embodiments, the subject, patient, or individual is a human.

A therapeutically effective amount is the amount of therapeutic agent administered during treatment that will relieve, to a certain extent, one or more symptoms of the disease being treated.

Detailed Description of the Invention

Codon-optimized nucleic acid.

In one aspect, the present invention provides an isolated codon-optimized nucleic acid that encodes the SMN1 (survival motor neuron protein) protein of SEQ ID NO: 1 and includes the nucleic acid sequence of SEQ ID NO: 2.

To obtain the codon-optimized SMN1 gene, the corresponding amino acid sequence of the SMN_HUMAN protein was taken as a basis:

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MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNG DICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASI DFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENSRSP GNKSDNIKPKSAPWNSFLPP PPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPS GPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN (SEQ IDNO:1)

This amino acid sequence SEQ ID NO: 1 was translated into a nucleotide sequence by sequentially assigning each amino acid starting from the N-terminus of one of the synonymous codons encoding it.

The details of the codon-optimized SMN1 gene and the selection of the final sequence are described in example 1.

The final codon-optimized sequence of SMN1 (SMN1-GeneBeam) has the following nucleotide sequence: ATGGCCATGAGCAGCGGCGGCAGCGGCGGCGGCGTGCCTGAGCAAGAGGAC

AGCGTGCTGTTCAGAAGAGGCACCGGCCAGAGCGACGACAGCGACATCTGGGACGA CACCGCCCTGATCAAGGCCTACGACAAGGCCGTGGCCAGCTTCAAGCACGCCCTGA AGAACGGCGACATCTGCGAGACCAGCGCAAGCCCAAGACCACCCAAGAGAAA GCCCGCCAAGAAGAACAAGAGCCAGAAGAAGAACACCGCCGCCAGCCTGCAGCAG TGGAAGGTGGGCGACAAG TGCAGCGCCATCTGGAGCGAGGACGGCTGCATCTACCC CGCCACCATCGCCAGCATCGACTTCAAGAGAGACCTGCGTGGTGGTGTACACCG GCTACGGCAACAGAGAGGAGCAGAACCTGAGCGACCTGCTGAGCCCCATCTGCGAG GTGGCCAACAACATCGAGCAGAACGCCCAAGAGAACGAGAACGAGAGCCAAGTGA GCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCAACAA GAGCGACAACATCAA GCCCAAGAGCGCCCCCTGGAACAGCTTCCTGCCCCCTCCCCCCCCTATGCCCGGCCC TAGACTGGGCCCTGGCAAGCCTGGCCTGAAGTTCAACGGCCCCCCCCCCCCTCCTCC TCCTCCTCCTCCTCACCTGCTGAGCTGCTGGCTGCCCCCCTTCCCCAGCGGCCCTCCT ATCATCCCTCTCCCCCCCCCATCTGCCCCGACAGCCTGGACGACGCCGACGCCCTG G GCAGCATGCTGATCAGCTGGTACATGAGCGGCTACCACACCGGCTACTACATGGG CTTCAGACAGAACCAGAAGGAGGGCCGGTGCAGCCACAGCCTGAACTAG (SEQ Ш NO:2).

This final codon-optimized nucleotide sequence of SMN1 (SMN1GeneBeam) is characterized by an increased codon adaptation index (a standard metric for evaluating a sequence for the frequencies of codons used) compared to the coding sequence of the wild-type SMN gene (SMN1-WT):

ATGGCGATGAGCAGCGGCGGCAGTGGTGGCGGCGTCCCGGAGCAGGAGGATTCCGT GCTGTTCCGGCGCGGCACAGGCCAGAGCGATGATTCTGACATTTGGGATGATACAG CACTGATAAAAGCATATGATAAAGCTGTGGCTTCATTTAAGCATGCTCTAAAGAATG GTGACATTTGTGAAACTTCGGGTAAACCAAAAACCACACCTAAAAGAAAACCTGCT AAGAAGAATAAAAGCCAAAAGAA GAATACTGCAGCTTCCTTACAACAGTGGAAAGT TGGGGACAAATGTTCTGCCATTTGGTCAGAAGACGGTTGCATTTACCCAGCTACCAT TGCTTCAATTGATTTTAAGAGAGAAACCTGTGTTGTGGTTTACACTGGATATGGAAA TAGAGAGGAGCAAAATCTGTCCGATCTACTTTCCCCAATCTGTGAAGTAGCTAATAA TATAGAACAAAATGCTCAAGAGAATGAAAATGAAAGCCAAGTTTCAA CAGATGAAA GTGAGAACTCCAGGTCTCCTGGAAATAAATCAGATAACATCAAGCCCAAATCTGCT CCATGGAACTCTTTTCTCCCTCCACCACCCCCCATGCCAGGGCCAAGACTGGGACCA GGAAAGCCAGGTCTAAAATTCAATGGCCCACCACCGCCACCGCCACCACCACCACC CCACTTACTATGCTGGCTGCCTCCATTTCCTTCTGGACCACCAATAATTCCCCCA CCACCTCCCATATGTCCAGA TTCTCTTGATGATGCTGATGCTTTGGGAAGTATGTTAA TTTCATGGTACATGAGTGGCTATCATACTGGCTATTATGGGTTTCAGACAAAATC AAAAGAAGGAAGGTGCTCACATTCCTTAAAATTAA (SEQ ID NO:3).

The codon adaptation index for the final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) of our sequence is 98%, for the wild-type sequence - 75%.

The GC composition of the wild type sequence is 45%, i.e. differs from the target value by 15%, the GC composition of the final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) of the optimized sequence is 64%, i.e. differs from the target value by 4%.

Final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) and nucleotide sequence of the wild-type SMN1 gene (SEQ ID NO: 3). The sequences are 71% identical.

Expression cassette. Expression vector.

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

The term expression cassette as used herein specifically refers to a fragment of DNA that is capable, under appropriate circumstances, of driving the expression of a polynucleotide encoding a polypeptide of interest that is included in said expression cassette. When introduced into a host cell, the expression cassette is, among other things, capable of engaging cellular machinery to transcribe the polynucleotide encoding the polypeptide of interest into RNA, which is then typically further processed and finally translated into the polypeptide of interest. The expression cassette may be contained in an expression vector.

The expression cassette of the present invention contains a promoter element. The term promoter as used in the present invention specifically refers to a DNA element that promotes transcription of the polynucleotide to which the promoter is operably linked. A promoter may also form part of a promoter/enhancer element. Although the physical boundaries between promoter and enhancer elements are not always clear, the term promoter generally refers to the location on a nucleic acid molecule to which RNA polymerase and/or associated factors bind and from which transcription is initiated. Enhancers enhance promoter activity temporally as well as spatially. There are many promoters known in the art that are 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 release of repression. Both classes are suitable for protein expression. Promoters that are used to produce high levels of polypeptides in eukaryotic cells and, in particular, mammalian cells must be strong and preferably must be active in a wide range of cell types. Strong constitutive promoters that are capable of driving expression in many cell types are well known in the art and therefore need not be described in detail herein. In accordance with the idea of the present invention, it is preferable to use a cytomegalovirus (CMV) promoter. A promoter or promoter/enhancer derived from the immediate early (IE) region of human cytomegalovirus (hCMV) is particularly suitable as a promoter in the expression cassette of the present invention. The human cytomegalovirus (hCMV) immediate early (IE) region and functional expression-triggering fragments and/or functional expression-enhancing fragments derived therefrom are, for example, described in EP 0173177 and EP 0323997 and are also well known in the art. Thus, several fragments of the hCMV immediate early (IE) region can be used as a promoter and/or promoter/enhancer. In one embodiment, a human CMV promoter is used in an expression cassette of the present invention.

In some embodiments, the expression cassette includes the following elements in a 5' to 3' direction:

left (first) ITR (inverted terminal repeats);

CMV (cytomegalovirus) enhancer;

CMV (cytomegalovirus) promoter;

intron of the hBG1 gene (hemoglobin subunit gamma-1 gene);

the above codon-optimized nucleic acid of the SMN1 gene;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal); right (second) ITR.

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

(SEQ ID NO: 8).

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In some embodiments, the CMV (cytomegalovirus) enhancer has the following nucleic acid sequence: cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaac gCcaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatca (SEQ ID NO: 9).

In some embodiments, the CMV (cytomegalovirus) promoter has the following nucleic acid sequence: gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagt ttgttttgGcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacg caaatgggcggtaggcgtgtacggtggga ggtctatataagcagagct (SEQ ID NO: 10).

In some embodiments, the intron of the hBG1 gene (hemoglobin subunit gamma-1 gene) has the following nucleic acid sequence: cgaatcccggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaa aaaatgctttcttcttttaatatactttttttgtttatcttatttctaatactt tccctaatctctttctttcagggcaataatgatacaatgtatcatgcctcttt gcaccattctaaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaa gaggtttcatattgctaatagcagctacaatccagctaccattctgcttttattttatggttgggataa ggctggattattctgagtccaagctagg cccttttgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaatt gggat (SEQ ID NO: 11).

In some embodiments, the hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) has the following nucleic acid sequence:

Acgggtggcatccctgtgacccctccccagtgcctctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaat aaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaag acaacctgtagggcctgcggggtctatt gggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttca agcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggttt caccatattggccaggctggtctccaactcctaatctcaggtgatctacccacc ttggcctcccaaattgctgggattacaggcgtgaaccact gctcccttccctgtcctt (SEQ ID NO: 12).

In some embodiments, the right (second) ITR has the following nucleic acid sequence: aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccg ggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg (SEQ ID NO: 1 3).

In some embodiments, the expression cassette has the following nucleic acid sequence: cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgag cgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcgtctagttatta atcaag tgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttg actcacggggatttccaagtctccaccccattga cgtcaatgggagtttgttttgGcaccaaaatcaacgggactttccaaaatgtcgtaaca actccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctg gagacgccatccacgctgttttgacctccatagaagacaccgggaccga tccagcctccgcggattcgaatcccggccgggaacggtgc attggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctttcttcttttaatatactttt ttgtttatcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatg cctctttgcaccattctaaagaataacagtg ataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagc tacaatccagctaccattctgcttttattttatggttgggataaggctggattattctctgagtccaagctaggcccttttgctaatcatgtt catacctc ttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggattcgaacatCGATTGT

- 8 045749

AATTCATGAGCCACCATGGCCATGAGCAGCGGCGGCAGCGGCGGCGGCGTGCCTGA GCAAGAGGACAGCGTGCTGTTCAGAAGAGGCACCGGCCAGAGCGACGACAGCGAC ATCTGGGACGACACCGCCCTGATCAAGGCCTACGACAAGGCCGTGGCCAGCTTCAA GCACGCCCTGAAGAACGGCGACATCTGCGAGACCAGCGGCAAGCCCAAGACCACCC CCAAGAGAAAGCCCGCCA AGAAGAAACAAGAGCCAGAAGAAGAACACCGCCGCCAG CCTGCAGCAGTGGAAGGTGGGCGACAAGTGCAGCGCCATCTGGAGCGAGGACGGCT GCATCTACCCCGCCACCATCGCCAGCATCGACTTCAAGAGAGAGACCTGCGTGGTG GTGTACACCGGCTACGGCAACAGAGAGGAGCAGAACCTGAGCGACCTGCTGAGCCC CATCTGCGAGGTGGCCAACAACATCGAGCAGAACGCCCAAGA GAACGAGAACGAG AGCCAAGTGAGCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCAACAAGAGCG ACAACATCAAGCCCAAGAGCGCCCCCTGGAACAGCTTCCTGCCCCCTCCCCCCCCTA TGCCCGGCCCTAGACTGGGCCCTGGCAAGCCTGGCCTGAAGTTCAACGGCCCCCCCC CCCCTCCTCCTCCTCCTCCTCCTCACCTGCTGAGCTGCTGGCTGCCCCCCTTCCCCAG CGGCCC TCCTATCATCCCTCTCCCCCCCCCATCTGCCCCGACAGCCTGGACGACGC CGACGCCCTGGGCAGCATGCTGATCAGCTGGTACATGAGCGGCTACCACACCGGCT ACTACATGGGCTTCAGACAGAACCAGAAGGAGGGCCGGTGCAGCCACAGCCTGAAC TGATctagagtcgacctgcagaagcttgcctcgagcagctgctgctcgagagatctacgggtggcatccct gtgacccctccccagtgc ctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataa tattatggggtggaggggggtggtatggagcaaggggcaagtttgggaagacaacctgtagggcctgcggggtctattggaaccaagct ggag tgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattcca ggcatgcatgaccaggctcagctaatttttgttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggt gatctacccaccttggcctcccaa attgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaaccacgtgcg gaccgagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaagg tcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcagctgcctgcagg (SEQ ID NO: 4).

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

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

In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.

In some embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In other embodiments, vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, and thus replicate along with the host gene. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to in this invention as recombinant expression vectors (or simply expression vectors (expression vector or expression vector)).

Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YAC, EBV-derived episomes, and the like. DNA molecules can be ligated into a vector such that the transcription and translation control sequences in the vector perform the intended function of regulating DNA transcription and translation. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules can be introduced into an expression vector by standard methods (eg, ligation of complementary restriction sites or blunt-end ligation if restriction sites are missing).

The recombinant expression vector may also encode a signal peptide that facilitates production of the protein of interest by the host cell. The gene for the protein of interest can be cloned into a vector such that the signal peptide is linked to the amino terminus of the protein of interest. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a non-immunoglobulin protein signal peptide).

In addition to the SMN1-GB gene according to this invention, recombinant expression of vectors according to this

- 9045749 of the invention may carry regulatory sequences that control the expression of the SMN1-GB gene in a host cell. Those skilled in the art will appreciate that the design of an expression vector, including the choice of regulatory sequences, may depend on factors such as the selection of the host cell for transformation, the level of expression of the desired protein, etc. Preferred regulatory sequences for a mammalian host expression cell include viral elements that provide high level expression of proteins in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomegalovirus (CMV) (eg, CMV promoter/enhancer), simian virus 40 (SV40) (eg, SV40 promoter/enhancer), adenovirus (eg, adenovirus large late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the native immunoglobulin promoter or the actin promoter.

The expression control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes are, for example, a promoter, optionally an operator and a ribosome binding site. As is known, promoters, polyadenylation signals and enhancers are present in eukaryotic cells.

As used herein, the term promoter or transcriptional control 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 of the transcriptional start site of the coding sequence, and that is structurally identifiable by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and other DNA sequences, including, without limitation, transcription factor binding sites, repressor and protein activator binding sites, as well as any other nucleotide sequences known to those skilled in the art, which directly or indirectly regulate the level of transcription with a given promoter. A constitutive promoter is one that is active in most tissues under normal physiological and developmental conditions. An inducible promoter is a promoter that is subject to physiological or developmental regulation, such as upon exposure to a chemical inducer. A tissue-specific promoter is active only in specific types of tissues or cells.

The terms enhancers or enhancer as used in the invention may refer to a DNA sequence that is located adjacent to the DNA sequence encoding the recombinant product. Enhancer elements are typically located 5' of a promoter element or may be located downstream of or within a coding DNA sequence (eg, the DNA sequence transcribed or translated into the recombinant product or products). Thus, the enhancer element may be located 100 base pairs, 200 base pairs, or 300 base pairs or more before or after the DNA sequence that encodes the recombinant product. Enhancer elements can increase the amount of recombinant product expressed from a DNA sequence beyond the expression driven by a single promoter element. A variety of enhancer elements are available to those skilled in the art.

In addition to the above genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate the replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, for example, US Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically a selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, to the host cell into which the vector is introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells for methotrexate selection/amplification), the neo gene (for G418 selection) and the glutamate synthetase gene.

The term expression control sequence as used herein refers to polynucleotide sequences that are necessary to influence the expression and processing of the coding sequences to which they are ligated. Expression control sequences include the corresponding transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie, the Kozak consensus sequence); sequences that increase protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, ribosome binding site, and termination sequences

- 10 045749 transcriptions; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term control sequences includes, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion sequences.

As used herein, the term operably linked refers to the linking of polynucleotide (or polypeptide) elements into a functional link. A nucleic acid is operably linked if it is in a functional relationship with another nucleic acid sequence. For example, a transcription control sequence is operably linked to a coding sequence if it affects the transcription of said coding sequence. The term operably linked means that the linked DNA sequences are generally contiguous and, where appropriate, junctions of two protein coding regions are also contiguous and in frame.

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

Neither the cassette nor the vector according to the invention contains nucleotide sequences of genes encoding non-structural proteins (Rep) and structural proteins (Cap) of the adeno-associated virus.

Recombinant virus based on AAV9 (adeno-associated virus serotype 9).

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

The term recombinant AAV virus (or AAV virus-like particle, or recombinant AAV viral strain, or recombinant AAV vector, or rAAV vector) as used herein refers to the above expression cassette (or the above expression vector) that is contained within the AAV capsid .

The Sar gene, in addition to other alternative products, encodes 3 capsid proteins (VP1, VP2 and VP3). Proteins VP1, VP2 and VP3 are in a ratio of 1:1:10, forming 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 begins from a single promoter, p40. The molecular weights of the corresponding proteins (VP1, VP2 and VP3) are 87, 72 and 62 kDa, respectively. All three proteins are translated from the same mRNA. After transcription, pre-mRNA can be spliced in two different ways, cutting out a longer or shorter intron and producing mRNAs of different nucleotide lengths.

During the formation of an AAV-derived recombinant virus (rAAV), an ITR-flanked expression cassette is packaged into the AAV capsid. The genes required for AAV replication, as noted above, are not included in the cassette.

The expression cassette DNA is packaged within the viral capsid as a single-stranded DNA (ssDNA) molecule approximately 3000 nucleotides in length. Once a cell is infected with a virus, the single-stranded DNA is converted into double-stranded DNA (dsDNA) form. Only dsDNA can use cell proteins that transcribe the contained gene or genes into RNA.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 VP1 protein.

In some embodiments, the recombinant AAV9 virus has a capsid that includes an AAV9 VP1 protein having the following amino acid sequence:

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPI GEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS QAVGRSSFYCLEYFPSQML RTGNNFQF S YEFENVPFHS S YAHSQ SLDRLMNPLIDQYL YYLSKTINGSGQNQQTLKFS V AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGF

- 11 045749

GMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPE

IQYTSNYYKSNNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 5).

In some embodiments, the AAV9-based recombinant virus has a capsid that includes an AAV9 VP1 protein 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 VP2 protein.

In some embodiments, the recombinant AAV9 virus has a capsid that includes an AAV9 VP2 protein having the following amino acid sequence:

TAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSG VGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNN HLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRL NFKLFNIQVKEVTDNNGVKTI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVF MIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQS LDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGED RFFPLSGSLIFGKQGT GRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGM VWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEG VYSE PRPIGTRYLTRNL (SEQ ID NO: 6).

In some embodiments, the AAV9-based recombinant virus has a capsid that includes an AAV9 VP2 protein 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 VP3 protein.

In some embodiments, the recombinant AAV9 virus has a capsid that includes an AAV9 VP3 protein having the following amino acid sequence:

MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYK QISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKL FNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQ YGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRL MNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVT QNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDN VDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGW VQNQGILPGMVWQD RDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNS FITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGT RYLTRNL (SEQ ID NO: 7).

In some embodiments, the AAV9-based recombinant virus has a capsid that includes an AAV9 VP3 protein 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 VP1, VP2, and VP3 proteins.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes proteins VP1 having amino acid sequence SEQ ID NO: 5, VP2 having amino acid sequence SEQ ID NO: 6, and VP3 having amino acid sequence SEQ ID NO: 7.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes proteins VP1 of amino acid sequence SEQ ID NO: 5 with one or more point mutations, VP2 of amino acid sequence SEQ ID NO: 6 with one or more point mutations, and VP3 of amino acid sequence SEQ ID NO: 7 with one or more point mutations.

Multiple point mutations are defined as 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. those substitutions that occur in a family of amino acids that are combined along their side chains. Specifically, amino acids are generally divided into four families: (1) acidic aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine,

- 12 045749 isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar ones - glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predicted that a selected substitution of leucine for isoleucine or valine, aspartate for glutamate, threonine for serine, or a similar conservative amino acid substitution for a structurally related amino acid will not have an important effect on biological activity. For example, a 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 molecule remains unaffected.

A variant of point mutations in the AAV9 VP1, VP2 or VP3 protein sequences using amino acid substitutions is the replacement of at least one amino acid residue in the AAV9 VP1, VP2 or VP3 protein with another amino acid residue.

Conservative substitutions are shown in the table under the heading Preferred Substitutions.

Original remainder Replacement examples Preferred replacements Ala (A) Vai; Leu; He Vai 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 He(I) Leu; Vai; Met; Ala; Ph; Norleucine Leu Leu (L) Norleucine; He; Vai; Met; Ala; Phe lie Lys(K) Arg; Gin; Asn Arg Met(M) Leu; Ph; He Leu Phe(F) Trp; Leu; Vai; He; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Vai; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Ph; Thr; Ser Phe Vai (V) He; Leu; Met; Ph; Ala; Norleucine Leu

In some embodiments, the recombinant AAV9 virus has a capsid that includes an AAV9 VP1 protein 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 elements in the direction from 5 '-end to 3'-end:

CMV enhancer;

CMV promoter;

hBG1 gene intron;

the above codon-optimized nucleic acid of the SMN1 gene;

hGH1 polyadenylation signal;

right ITR.

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

- 13 045749 proteins VP1 with amino acid sequence SEQ ID NO: 5, VP2 with amino acid sequence SEQ ID NO: 6 and VP3 with amino acid sequence SEQ ID NO: 7, and the expression cassette includes the following elements in the direction from the 5' end to the 3' -end:

CMV enhancer;

CMV promoter;

hBG1 gene intron;

the above codon-optimized nucleic acid of the SMN1 gene;

hGH1 polyadenylation signal;

right ITR.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes proteins VP1 of amino acid sequence SEQ ID NO: 5 with one or more point mutations, VP2 of amino acid sequence SEQ ID NO: 6 with one or more point mutations, and VP3 of amino acid sequence SEQ ID NO: 7 with one or more point mutations, and the expression cassette includes the following elements in the direction from the 5' end to the 3' end:

CMV enhancer;

CMV promoter;

hBG1 gene intron;

the above codon-optimized nucleic acid of the SMN1 gene;

hGH1 polyadenylation signal;

right ITR.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes an AAV9 VP1 protein 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 nucleic acid of SEQ ID NO: : 4.

In some embodiments, the AAV9-based recombinant virus has a capsid that includes proteins VP1 having amino acid sequence SEQ ID NO: 5, VP2 having amino acid sequence SEQ ID NO: 6, and VP3 having amino acid sequence SEQ ID NO: 7, and an expression cassette comprising a nucleic acid with SEQ ID NO: 4.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes proteins VP1 of amino acid sequence SEQ ID NO: 5 with one or more point mutations, VP2 of amino acid sequence SEQ ID NO: 6 with one or more point mutations, and VP3 of amino acid sequence SEQ ID NO: 7 with one or more point mutations, and the expression cassette includes the nucleic acid of SEQ ID NO: 4.

Pharmaceutical composition.

In one aspect, the present invention relates to a pharmaceutical composition for delivering the SMN1 gene to target cells, which includes the above-mentioned AAV9-based recombinant virus in combination with one or more pharmaceutically acceptable excipients.

In specific embodiments, the present invention provides a pharmaceutical composition comprising a recombinant AAV9 virus of the invention in a pharmaceutically acceptable carrier or other pharmaceutical agents, adjuvants, diluents, etc. The injectable carrier is usually liquid. The carrier for other routes of administration may be either a solid or a liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline. For administration by inhalation, the carrier is inhalable and is preferably in solid or liquid dispersed form. As the injection medium, it is preferable to use water containing additives generally accepted for injection solutions, such as stabilizing agents, salts or saline solutions and/or buffers.

Pharmaceutical composition means a composition comprising the above recombinant AAV9 virus of the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients such as excipients, solvents, diluents, carriers, auxiliaries, dispensers , delivery vehicles, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, prolonged delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage. The pharmaceutical compositions of the present invention and methods for their manufacture will be readily apparent to those skilled in the art. The production of pharmaceutical compositions should preferably comply with GMP (Good Manufacturing Practices) requirements. The composition may include a buffer composition, tonic agents, stabilizers and solubilizers.

A pharmaceutically acceptable material is one that has no biological or other contraindications, e.g., the material can be administered to a subject without causing any undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, for ex vivo transfection of a cell or for in vivo administration of a recombinant AAV9-based virus

- 14 045749 according to the invention directly to the subject.

The term excipient or excipient is used in this invention to describe any component other than those previously described in this invention. These are substances of inorganic or organic origin, used in the production process of medicines to give them the necessary physical and chemical properties.

By stabilizer is meant an excipient or a mixture of two or more excipients that provide physical and/or chemical stability of the active agent.

The term buffer, buffer composition, buffer agent refers to a solution capable of maintaining the pH value due to the interaction of the acidic and alkaline components included in its composition, which allows the vector preparation based on rAAV5 to exhibit resistance to changes in pH. In general, pH values of the pharmaceutical composition from 4.0 to 8.0 are preferred. As buffering agents, for example, acetate, phosphate, citrate, histidine, succinate, etc. can be used. buffer solutions, but not limited to them.

A pharmaceutical composition is stable if the active agent maintains its physical stability and/or chemical stability and/or biological activity through its stated shelf life at storage temperature, for example at 2-8°C. It is preferred that the active agent retains both physical and chemical stability as well as biological activity. The storage period is selected based on the results of stability studies during accelerated and natural storage.

The pharmaceutical composition of this invention may be manufactured, packaged or widely sold as a single unit dose or a plurality of unit dose units in a finished dosage form. As used herein, the term unit dose means a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of active ingredient is typically equal to the dosage of the active ingredient to be administered to the subject, or a convenient portion of such dosage, such as one-half or one-third of such dosage.

Application.

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

Any method of administering an AAV9-based recombinant virus accepted in the art can be suitably used for the above-mentioned AAV9-based recombinant virus of the present invention.

The AAV9-based recombinant virus is preferably introduced into the cell in a biologically effective amount. A biologically effective amount of a recombinant virus is an amount that is sufficient to cause infection (or transduction) and expression of a heterologous nucleic acid sequence in a cell. If the virus is introduced into a cell in vivo (eg, 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 transduction and expression of a heterologous nucleic acid sequence in the target cell.

The cell for introducing the above AAV9-based recombinant virus of the invention may be any type of cell, including but not limited to neural cells (including cells of the peripheral and central nervous system, in particular brain cells), lung cells, epithelial cells (eg , intestinal and respiratory epithelial cells), muscle cells, pancreatic cells (including islet cells), liver cells, myocardial cells, bone cells (eg, bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts , endothelial cells, prostate cells, germ cells, etc. Alternatively, the cell for introducing the above AAV9-based recombinant virus can be any progenitor cell. As a further alternative, the cells may be stem cells (eg, neural stem cells, liver stem cells). Additionally, the cells can come from any species, as noted above.

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

Examples

For a better understanding of the invention, the following examples are provided. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents and patent applications referenced in this specification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example in order to avoid ambiguity, those skilled in the art will appreciate from the teachings disclosed herein that certain changes and modifications may be made without departing from the spirit. and the scope of the accompanying embodiments of the invention.

Materials and general methods.

Recombinant DNA methods.

For DNA manipulation, standard methods were used as described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions. Briefly, plasmid DNA was produced for further manipulation in E. coli cells grown under selective pressure with antibiotics to ensure that plasmids were not lost in the cell population. Plasmid DNA was isolated from cells using commercial kits, the concentration was measured and used for cloning using restriction endonuclease treatment or PCR amplification methods. DNA fragments were ligated with each other using ligases and transformed into bacterial cells for clone selection and further development. All obtained genetic constructs were confirmed by restriction patterns and full Sanger sequencing.

Gene synthesis.

The required gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments ranging from 300 to 1000 bp in length, which are flanked by unique restriction sites, were assembled by renaturation of oligonucleotides on each other, followed by PCR amplification from extreme primers. As a result, a mixture of fragments was obtained, including the desired one. The fragments were cloned at restriction sites into intermediate vectors, after which the DNA sequences of the subcloned fragments were confirmed by DNA sequencing.

Determination of DNA sequences.

DNA sequences were determined by Sanger sequencing. DNA and protein sequence analysis and sequence data processing were performed using SnapGene 4.2 and higher to generate, map, analyze, annotate, and illustrate sequences.

Cultivation of cell cultures.

Cell lines HEK293 (Human Embryonic Kidney clone 293) and HSMC (Human Skeletal Muscle Cells) were used in the experiments. Cells were cultured under standard conditions at 370°C and 5% CO ₂ in complete nutrient medium DMEM supplemented with 10% FBS and antibiotic. To culture HSMC, the culture plastic was precoated with collagen (Gibco). Cell reseeding was carried out when 80-90% confluency was achieved. Cell viability was assessed using Trypan Blue staining and a Goryaev chamber or using PI staining and flow cytometry.

Cell transfection.

Cell lines were seeded on the eve of transfection into 6-well plates so that they reached 70-80% confluency at the time of transfection. Transfection was performed using commercial lipofection kits according to the manufacturer's protocol. After 72 h, the cells were treated with trypsin or similar solutions, removed from the substrate, washed in phosphate buffer, and collected for further analysis of the expression of target genes and proteins. During each transfection, a control plasmid expressing GFP was used to control the transfection efficiency (the percentage of GFP-positive cells). Further analysis was performed only if the transfection efficiency was at least 50%.

All work was carried out in 3 independent experiments.

Gene expression analysis.

Expression of SMN1 at the mRNA level was tested using quantitative PCR. Briefly, primers and probe specific to the wild-type or GeneBeam SMN1 sequence were used. To control the amount of initial RNA, primers and a probe specific to the housekeeping gene GAPDH were used. For each set of primers and probes, calibration curves were constructed using a known number of copies of linearized plasmid DNA containing the amplifiable sequence of the corresponding gene. Expression analysis was performed by determining the copy numbers of SMN1-GeneBeam, SMN1-WT, and GAPDH in each sample from calibration curves, and then normalizing the copy number of SMN1 per 10,000 copies of GAPDH. The obtained values were compared with each other for different samples within the same experiment.

Determination of SMN1 protein expression by flow cytometry.

SMN1 protein content in cells was assessed by intracellular staining, followed by flow cytometry analysis. Briefly, cells were removed from culture plates using TrypLE, washed in PBS, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, incubated in blocking buffer with 1–5% BSA, and stained in two steps with using primary antibodies to SMN1 and secondary antibodies labeled with Alexa Fluor 488. After staining, cells were washed once in PBS and analyzed on a flow cytometer. The average signal intensity was assessed minus the signal stained with secondary antibodies without the addition of primary antibodies.

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Assembly and purification of viral particles from recombinant AAV vectors.

To assemble AAV viral particles containing the SMN1 gene or the control GFP gene, HEK293 packaging cells were used, into which the following 3 plasmids were transfected:

a plasmid containing the AAV genome with a transgene expression cassette (SNM1 or GFP);

plasmid for expression of the Sar gene of serotype AAV9 and the Rep gene of serotype AAV2. Each gene encodes multiple protein products using alternative reading frames;

plasmid for expression of Ad5 adenovirus genes required for assembly and packaging of AAV capsids.

After 72 h, the cells were lysed and the viral particles were purified and concentrated using filtration and chromatography methods. The titer of viral particles was determined using quantitative PCR with primers and a probe specific to a region of the recombinant viral genome and expressed as the number of copies of viral genomes per 1 ml.

Transduction of cell cultures.

Cell lines were seeded similarly to the transfection experiments, after which the drug with viral particles was added, and after 72 hours the cells were analyzed. Transduction efficiency was calculated by analyzing the percentage of GFP+ cells.

For the cultures used, experiments were previously carried out to test the efficiency of transduction. Briefly, the AAV9-GFP virus preparation was transduced into cell lines at various ratios of cells and viral particles. The ratio of the number of viral particles and cells is called multiplicity of infection (MOI). The MOI of the AAV9-GFP virus varied from 50,000 to 1,000,000. As a result, MOI ranges were determined for each line, within which the transduction efficiency varied linearly depending on the MOI. Further work with transduction of cell lines was carried out in their linear ranges.

After transduction, gene and protein expression analysis was performed as described above.

All work was carried out in 3 independent experiments.

Example 1. Method for obtaining a codon-optimized SMN1 gene.

To obtain the codon-optimized SMN1 gene, the corresponding amino acid sequence of the SMN_HUMAN protein (SEQ ID NO: 1) was taken as a basis.

This amino acid sequence SEQ ID NO: 1 was translated into a nucleotide sequence by sequentially matching each amino acid, starting from the N-terminus, with one of the synonymous codons encoding it, taking into account one or a combination of the following features:

1) frequency of use of a given codon (Yasukazu Nakamura et al., Codon usage tabulated from the international DNA sequence databases; its status 1999, Nucleic Acids Research, 1999, vol. 27, No. 1, DOI: 10.1093/nar/27.1.292) ;

2) GC composition of the terminal region of the resulting nucleotide sequence (the target value of GC composition was considered to be 60% based on 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, e180, DOI: 10.1371/joumal.pbio.0040180, therefore the codon was more preferable, the smaller the difference between the current GC composition and the target one);

3) free energy of folding of the terminal region of the resulting nucleotide 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).

During the construction process, the generation of sense nucleotide sequences, such as restriction sites, internal ribosome entry sites, splicing sites, etc., was also avoided.

As a result of translating the amino acid sequence of SEQ ID NO: 1 into a nucleotide sequence, a number of codon-optimized nucleotide sequences of the SMN1 gene were obtained.

Of the above series of codon-optimized SMN1 nucleotide sequences, several sequences did not show an increase in SMN1 gene transcription in further studies, i.e. there was no significant increase in SMN1-opt mRNA copy number compared to SMN1-WT in any of the cell lines used, or the increase was negligible.

Most codon-optimized nucleotide sequences of the SMN1 gene showed an increase in SMN1 gene transcription by 1.5-2 times in further studies, i.e. significantly increasing the number of SMN1-opt mRNA copies compared to SMN1-WT in all cell lines used.

One of the above series of codon-optimized nucleotide sequences of the SMN1 gene unexpectedly showed an increase in SMN1 gene transcription by more than 3 times in further studies, i.e. unexpectedly increasing the copy number of SMN1-opt mRNA by more than 3-fold compared to SMN1-WT in all cell lines used (see examples 3-4). This final codon-optimized nucleotide sequence of the SMN1 gene received the code name

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SMN1-GeneBeam (or SMN1-GB for short).

The final codon-optimized sequence of SMN1 (SMNl-GeneBeam) has the nucleotide sequence shown by SEQ ID NO: 2.

This final codon-optimized nucleotide sequence of SMN1 (SMN1GeneBeam) is characterized by an increased codon adaptation index (Paul M. Sharp et al., The codon adaptation index-a measure of directional synonymous codon usage bias, and its potential applications, Nucleic Acids Research, volume 15 , issue 3,11 February 1987, pages 1281-1295, DOI: 10.1093/nar/15.3.1281 - a standard metric for evaluating a sequence for the frequencies of codons used) compared to the coding sequence of the wild type SMN gene (SMN1-WT with SEQ ID NO: 3).

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

The GC composition of the wild type sequence is 45%, i.e. differs from the target value by 15%, the GC composition of the final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) is 64%, i.e. differs from the target value by 4%.

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

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

The wild-type SMN1 gene sequence was obtained by amplification with specific primers from cDNA synthesized from total RNA of HEK293 cells. During the amplification process, a Kozak sequence and a ClaI restriction site were added to the 5' end of the gene, and an XbaI restriction site to the 3' end. After this, the SMN1 gene sequence was cloned using the restriction enzyme ligase method at the ClaI and XbaI sites into the commercial construct pAAV-GFP Control plasmid (VPK-402) from CellBiolab (USA), with the GFP gene replaced by SMN1, obtaining the pAAV-SMN1-WT construct.

The SMN1-GeneBeam sequence was assembled as described above. Due to the complexity of the sequence, despite its relatively small size, a series of subcloning of gene fragments was carried out in intermediate pGEMT vectors with sequence verification for each vector. Next, the full-length version of the gene was assembled from several intermediate vectors using PCR and cloned into the intermediate vector pGEMT. The pAAV-SMN1-WT construct was used as the final genetic construct, replacing wild-type SMN1 with SMN1-GeneBeam at the ClaI and XbaI sites added to the ends of the SMN1-GeneBeam sequence using PCR.

The final vector contains all the necessary elements for gene expression and assembly into the recombinant AAV genome:

1) terminal ITR repeats at the ends of the sequence that is encapsidated into the viral capsid;

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

3) the origin of bacterial replication and the antibiotic resistance gene for the production of plasmid DNA in bacterial cells.

It is important to note that the genetic constructs containing the SMN1-WT and SMNl-GeneBeam genes differ only in the sequences of the SMN1 genes, and are otherwise completely identical.

Example 3. Testing SMN1 expression from genetic constructs.

Gene constructs pAAV-GFP, pAAV-SMN1-WT, and pAAV-SMN1-GB were transfected into HEK293 and HSMC cells as described above. 5 μg of DNA per well was used. After 72 h, cells were harvested and SMN1 expression was analyzed (normalized to GAPDH) as described above.

Codon optimization of the SMN1 gene was found to affect SMN1 transcription, significantly increasing the copy number of SMN1-GB mRNA severalfold compared to SMN1-WT in both cell lines used (Fig. 1). Specifically, for HEK293 cells the ratio of normalized expression of SMN1-GB to SMN1-WT was 3.9, and for HSMC cells it was 12.8.

This property of SMN1-GeneBeam, as the data obtained shows, is not cell-specific, but provides a several-fold increase in the expression of the target gene in cells, which can be an important advantage in the development of gene therapy drugs. Moreover, this property is not due to any differences in the gene expression cassette and the properties of possible viral capsids carrying the genome from the SMN1-GeneBeam genes, since this analysis was carried out on genetic constructs that differ only in the codon optimization of the SMN1 genes, and are otherwise completely identical.

HSMC cells were selected to examine SMN1 expression at the protein level using flow cytometry as described above. It was shown that the signal from SMN1-specific antibodies in cells transfected with pAAV-SMN1-GB was higher than in cells transfected with pAAV-SMN1-WT by 12.2 times when the amount of DNA used was 5 μg per well (Fig. 2). This result means that SMN1-GB does not have a translational advantage, but due to increased transcription, the amount of the final protein in cells also increases.

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Example 4. Creation of viral preparations expressing SMN1.

Plasmids pAAV-SMN1-WT and pAAV-SMN1-GB, along with the remaining plasmids necessary for the production of recombinant AAV viral particles (see above), were used for the bioprocess of producing AAV. The serotype chosen was AAV9 serotype wild type or with one or more point mutations.

In all cases, comparisons between the properties of wild-type SMN1 and SMN1-GeneBeam were made only in the case of identity of the serotype used and capsid mutations, if present. All AAV9-based serotypes, wild type or with mutations, are hereafter referred to as AAV9 without specifying mutations.

The bioprocess resulted in recombinant viral particles designated AAV9-SMN1-WT and AAV9-SMN1-GB, as well as control AAV9-GFP particles. After determining the titers of viral particles, all 3 drugs with the same MOI (MOI values varied between experiments from 50 to 200 thousand) were used for transduction of permissive cells - primary human myocytes HSMC. Further analysis was performed only if the transduction efficiency was at least 50%.

After successful transduction, cells were removed from the support, washed in phosphate buffer, and SMN1 expression was analyzed at the mRNA and protein levels as described above. The increase in SMN1-GeneBeam transcriptional activity was shown to be maintained, such that 7.3-fold more SMN1-GeneBeam mRNA was detected than for wild-type SMN1. A similar increase was observed at the protein level (6.8-fold) (Fig. 3), which shows that SMN1-GB does not benefit at the translation level, however, the detectable increase in transcription efficiency allows AAV9-SMN1-GB to provide a higher level of expression SMN1 in target cells, which is an important advantage in the treatment of, for example, spinal muscular atrophy, where the level of SMN1 protein expression determines the type of disease from 0 (fetal lethal) to 4 (does not require special treatment).

Claims (13)

1. A codon-optimized nucleic acid that encodes the SMN1 (survival motor neuron protein) protein of SEQ ID NO: 1, comprising the nucleic acid sequence of SEQ ID NO: 2. 2. An expression cassette that includes a codon-optimized nucleic acid according to claim 1. 3. Expression cassette according to claim 2, including the following elements in the direction from the 5' end to the 3' end: left (first) ITR (inverted terminal repeats); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; intron of the hBG1 gene (hemoglobin subunit gamma-1 gene); codon-optimized nucleic acid according to claim 1; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal); right (second) ITR. 4. The expression cassette according to claim 3, which includes the nucleic acid with SEQ ID NO: 4. 5. An expression vector that includes a codon-optimized nucleic acid according to claim 1 or a cassette according to claims 2-4. 6. A recombinant virus based on AAV9 (adeno-associated virus serotype 9) to increase the expression of the SMN1 gene in target cells, which includes a capsid and an expression cassette according to any one of claims 2-4. 7. The AAV9-based recombinant virus according to claim 6, wherein the capsid includes the AAV9 VP1 protein. 8. The AAV9-based recombinant virus according to claim 7, wherein the capsid includes an AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 5. 9. The AAV9-based recombinant virus of claim 7, wherein the capsid comprises an AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 5 with one or more point mutations. 10. The AAV9-based recombinant virus according to claims 6 to 9, wherein the capsid includes an AAV9 VP1 protein having the amino acid sequence SEQ ID NO: 5 or the amino acid sequence SEQ ID NO: 5 with one or more point mutations, and the expression cassette includes the following elements in the direction from the 5' end to the 3' end: CMV enhancer; CMV promoter; hBG1 gene intron; codon-optimized nucleic acid according to claim 1; hGH1 polyadenylation signal; right ITR. 11. A recombinant virus based on AAV9 according to claim 6, where the capsid includes the AAV9 VP1 protein, having - 19 045749 amino acid sequence SEQ ID NO: 5 or amino acid sequence SEQ ID NO: 5 with one or more point mutations, and the expression cassette includes the nucleic acid of SEQ ID NO: 4. 12. Pharmaceutical composition for delivery of the SMN1 gene to target cells, including a recombinant virus based on AAV9 according to claims 6-11 in combination with one or more pharmaceutically acceptable excipients. 13. Use of a recombinant virus based on AAV9 according to claims 6-11 or a composition according to claim 12 for delivering the SMN1 gene to target cells.