Codon-optimized nucleic acids encoding smn1 proteins

Abstract

The present application relates to the field of genetics, gene therapy and molecular biology. More specifically, the present invention relates to an isolated codon-optimized nucleic acid encoding a SMN1 protein (survival motor neuron protein), expression cassettes and vectors based thereon, and recombinant viruses based on AAV9 (adeno-associated virus serotype 9) for increasing the expression of the SMN1 gene in target cells, and uses thereof.

Description

Technical Field

[0001] This application relates to the fields of genetics, gene therapy, and molecular biology. More specifically, the present invention relates to isolated codon-optimized nucleic acids encoding the SMN1 protein (survival motor neuron protein), expression cassettes and vectors based thereon, and recombinant viruses based on AAV9 (adeno-associated virus serotype 9) for increasing the expression of the SMN1 gene in target cells, and their uses. Background Technology

[0002] Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN1) gene and the loss of the encoded SMN protein (Lefebvre et al., Cell (1995) 80:155-165). The lack of SMN leads to degeneration of motor neurons in the ventral horn (anterior horn) of the spinal cord, resulting in weakness of the proximal muscles responsible for crawling, walking, neck movement, and swallowing, as well as the involuntary muscles controlling breathing and coughing (Sumner CJ, NeuroRx (2006) 3:235-245). Therefore, SMA patients are highly susceptible to pneumonia and other lung problems, such as restrictive lung disease.

[0003] Gene therapy is a promising approach to treating spinal muscular atrophy (SMA).

[0004] Adeno-associated virus (AAV) vectors are considered effective in CNS gene therapy because they have suitable toxicity and immunogenicity profiles, can be used for neural cell transduction, and are able to mediate long-term expression in the CNS.

[0005] Adeno-associated virus (AAV) is a small (20 nm), independently replicating, non-enveloped virus. Many different AAV serotypes have been described in humans and primates. The AAV genome consists of a single-stranded DNA (ssDNA) of approximately 4,700 nucleotides (+ or -). The genomic DNA ends with inverted terminal repeats (ITRs). The genome contains two open reading frames (ORFs), Rep and Cap, which contain several alternative reading frames encoding various protein products. The Rep product is essential for AAV replication, while three capsid proteins (VP1, VP2, and VP3) and other alternative products are encoded by the Cap gene. VP1, VP2, and VP3 exist in a 1:1:10 ratio, 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). During the production of recombinant AAV (rAAV) vectors, an expression cassette flanked by an ITR is packaged into the AAV capsid. The genes required for AAV replication are not included in the cassette. Recombinant AAV is considered one of the safest and most widely used viral vectors for in vivo gene transfer. The vectors can infect cells of a variety of tissue types, providing robust and sustained transgene expression. They are also nonpathogenic and have a low immunogenicity profile (High KA et al., "rAAV human trial experience" Methods Mol Biol. 2011; 807:429-57).

[0006] In the field of developing effective gene therapies, one of the urgent goals of research is to codon optimize the target gene in the vector to achieve the maximum level of expression of the target gene, which in turn will allow for significant effects using lower doses of the vector.

[0007] One characteristic of genetic codons is degeneracy, the ability of different codons (trinucleotides) to encode the same amino acid. Codons that translate to the same amino acid are called synonymous codons. In a natural sequence, one of the synonymous codons is randomly selected during evolution, but the frequency of use of synonymous codons varies: each amino acid has more preferred and less preferred codons. Codon optimization is a widely used technique for expanding the production of protein molecules, providing a reasonable mapping of one of the suitable synonymous codons to each amino acid in the protein sequence. One of the common principles of codon optimization involves the most frequently used codons, while other methods, such as coordination (reproducing the distribution of codon usage frequencies), were introduced later, but they do not always improve productivity. Besides codon frequency, sequence GC content (the proportion of guanine and cytosine in the total sequence length) may also affect production efficiency, particularly showing 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, Vol. 4, No. 6, e180, pp. 933-942). It is worth noting further that stable secondary structural elements of mRNA, i.e., those with low free folding energies, may reduce efficiency.

[0008] Different variants of codon optimization of the target gene sequence may result in the following (compared to the wild-type gene): a) the expression level of the target gene will be slightly increased;

[0009] b) The expression level of the target gene will be significantly increased;

[0010] c) The expression level of the target gene will remain roughly the same;

[0011] d) The expression level of the target gene will decrease.

[0012] Therefore, codon optimization of the SMN1 gene is needed to increase SMN1 gene expression in target cells.

[0013] Compared with SMN1-WT (wild type), the codon-optimized sequence of SMN1 (SMN1-GeneBeam (or abbreviated as SMN1-GB)) (which has the nucleotide sequence of SEQ ID NO:2) significantly increased the transcription of the SMN1 gene by more than 3 times, that is, the mRNA copy number of SMN1-GeneBeam significantly increased by more than 3 times, which in turn led to a significant increase in the expression of the SMN1 gene and the corresponding SMN protein. Summary of the Invention

[0014] On one hand, the present invention relates to isolated codon-optimized nucleic acids that encode the SMN1 protein (survival motor neuron protein) having SEQ ID NO:1 and include the nucleic acid sequence of SEQ ID NO:2.

[0015] On one hand, the present invention relates to an expression cassette comprising the aforementioned codon-optimized nucleic acid.

[0016] In some implementations, the expression box includes the following elements in the 5'-to-3'-end direction:

[0017] Left-side (first) ITR (reverse terminal repeat);

[0018] CMV (cytomegalovirus) enhancer;

[0019] CMV (cytomegalovirus) promoter;

[0020] Introns of the hBG1 gene (hemoglobin subunit γ1 gene);

[0021] The above codon-optimized nucleic acid of the SMN1 gene;

[0022] hGH1 polyadenylation signaling (human growth hormone gene polyadenylation signaling)

[0023] The right side (second) ITR.

[0024] In some implementations, the expression cassette includes a nucleic acid having the sequence SEQ ID NO:4.

[0025] On the one hand, the present invention relates to expression vectors comprising the above-described codon-optimized nucleic acids or the above-described boxes.

[0026] On one hand, 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, comprising a capsid and the aforementioned expression cassette.

[0027] In some implementations, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP1.

[0028] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1, which has the amino acid sequence of SEQ ID NO:5.

[0029] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1, said VP1 having an amino acid sequence of SEQ ID NO:5 with one or more point mutations.

[0030] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1, said 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 elements in the 5'-to-3'-direction:

[0031] CMV enhancer;

[0032] CMV promoter;

[0033] Introns of the hBG1 gene;

[0034] The above codon-optimized nucleic acid of the SMN1 gene;

[0035] hGH1 polyadenylation signal;

[0036] Right side ITR.

[0037] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1, said 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 contains nucleic acid having SEQ ID NO:4.

[0038] On one hand, the present invention relates to a pharmaceutical composition for delivering the SMN1 gene to target cells, comprising the aforementioned AAV9-based recombinant virus in combination with one or more pharmaceutically acceptable excipients.

[0039] On one hand, the present invention relates to the use of the above-mentioned AAV9-based recombinant virus or the above-mentioned composition for delivering the SMN1 gene to target cells. Attached Figure Description

[0040] Figure 1 SMN1 expression at the mRNA level after transfection. HEK293 cells and HSMCs were transfected with 5 μg plasmids pAAV-SMN1-WT and pAAV-SMN1-GB (encoding codon-free and codon-optimized SMN1 genes, respectively, according to the GeneBeam algorithm). After 72 hours, the copy number of the SMN1 gene in each sample (n=3) was determined by quantitative PCR. The copy number of the GAPDH housekeeping gene was also determined. All obtained SMN1 levels were normalized relative to 10,000 copies of the GAPDH gene in each sample. Normalized mean copy numbers of SMN1-WT and SMN-GB for both cell lines are provided, with standard deviations indicated. Furthermore, the proportion of normalized copy numbers of SMN1-GB and SMN1-WT in each line is provided.

[0041] Figure 2 SMN1 protein expression after transfection. HSMCs were transfected with 5 μg plasmids pAAV-SMN1-WT and pAAV-SMN1-GB (encoding codon-free and codon-optimized SMN1 genes according to the GeneBeam algorithm). After 72 hours, cells in each sample were stained with a primary antibody against the SMN1 protein and a secondary antibody labeled with Alexa Fluor 488 (n=3). The mean fluorescence intensity of viable cells in the sample is shown after subtracting the background signal obtained on cells stained with secondary antibody but not primary antibody, with standard deviation indicated.

[0042] Figure 3 The proportions of SMN1 expression at the mRNA and protein levels after transduction. HSMCs were transduced with AAV9-SMN1-WT and AAV9-SMN1-GB viruses in three independent experiments, with a transduction efficiency of at least 50% of the control GFP-containing virus in each experiment. SMN1 expression was determined at the mRNA and protein levels (see above), and the proportions of SMN1-GB and SMN1-WT expression were then calculated. The mean proportions and standard deviations are shown in the figure.

[0043] Definition and general method

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0045] Furthermore, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include singular terms. Generally, the cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medical and medicinal chemistry, and the classification and methods of protein and nucleic acid hybridization and chemistry described herein are well known and widely used by those skilled in the art. As is common in the art, or as described herein, enzyme reactions and purification methods are performed according to the manufacturer's instructions.

[0046] "Separated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in animals are not "separated," but the same nucleic acid or peptide that is partially or completely separated from its natural coexisting material is "separated." Separated nucleic acids or proteins can exist in a substantially purified form or can exist in non-natural environments (such as, for example, genetically modified cells).

[0047] The terms “natural,” “nature,” or “wild-type” are used to describe objects that can be found in nature and are distinct from those produced artificially. For example, a protein or nucleotide sequence that can be isolated from natural sources and is present in an organism (including a virus) without intentional modification by laboratory personnel is considered naturally occurring.

[0048] The term "genome" refers to the complete genetic material of an organism.

[0049] As used in this description and the following claims, unless the context otherwise requires, the words “include” and “comprise” or variations thereof, such as “having,” “includes,” “including,” “comprises,” or “comprising,” shall be understood to mean including the said integer or group of integers, but not excluding any other integer or group of integers.

[0050] Proteins (peptides)

[0051] As used in this description, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and they refer to compounds composed 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 in the sequence constituting a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used in this description, the term refers to both short chains (also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (commonly referred to in the art as proteins, of which there are many types). “Polypeptide” specifically includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0052] Nucleic acid molecules

[0053] The terms “nucleic acid,” “nucleic acid sequence,” “nucleic acid sequence,” “polynucleotide,” “oligonucleotide,” “polynucleotide sequence,” and “nucleotide sequence” are used interchangeably in this description and refer to the exact sequence of modified or unmodified nucleotides that determines the segment or region of a nucleic acid, whether it contains or does not contain non-natural nucleotides, and whether it is a double-stranded DNA or RNA, a single-stranded DNA or RNA, or a transcript of said DNA.

[0054] It is widely known to those skilled in the art that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, by way of non-limiting example, polynucleotides include all nucleic acid sequences obtained by any means available in the art, including, by way of non-limiting example, recombinant means (i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR, etc.) and synthetic means.

[0055] It should also be noted that this invention does not relate to nucleotide sequences in their natural chromosomal environment (i.e., in their natural state). The sequences of this invention have been isolated and / or purified, i.e., they have been sampled directly or indirectly (e.g., by replication), and their environment has been at least partially modified. Therefore, isolated nucleic acids obtained through recombinant genetics (e.g., through host cells) or through chemical synthesis should also be mentioned herein.

[0056] "Isolated" nucleic acid molecules are those identified and isolated from at least one nucleic acid molecule impurity (the former typically binds to the nuclease in its natural source of nucleic acid). Isolated nucleic acid molecules differ from the form or set found under natural conditions. Therefore, isolated nucleic acid molecules differ from those present in cells under natural conditions. However, isolated nucleic acid molecules include those located in cells where nucleases are normally expressed, for example, if the chromosomal location of the nucleic acid molecule differs from its location in cells under natural conditions.

[0057] Unless otherwise stated, the term nucleotide sequence encompasses its complement. Therefore, nucleic acid having a specific sequence should be understood to encompass nucleic acid having its complementary strand.

[0058] The terms “transformation,” “transfection,” and “transduction” refer to any method or means of introducing nucleic acids into cells or host organisms and are used interchangeably to express the same meaning. These methods include, but are not limited to, transfection, electroporation, microinjection, infection, and PEG fusion.

[0059] Adeno-associated virus (AAV)

[0060] The Parvovirinae family comprises small, DNA-containing animal viruses. Parvovirinae is divided into two subfamilies: Parvovirinae (whose members infect vertebrates) and Densovirinae (whose members infect insects). By 2006, eleven adeno-associated virus serotypes had been described (Mori, S. et al., 2004, "Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein", Virology, Т.330(2):375-83). All known serotypes can infect cells from a wide variety of tissue types. Tissue specificity is determined by the capsid protein serotype; therefore, adeno-associated virus-based vectors are constructed by assigning the desired serotype. Further information on parvoviruses and other members of the Parvoviridae family is described in the literature (Kenneth I. Berns, Parvoviridae: The Viruses and Their Replication, Chapter 69 of Fields Virology (3rd edition, 1996)).

[0061] All known AAV serotypes share a very similar genome organization. The AAV genome is a linear, single-stranded DNA molecule less than approximately 5000 nucleotides (nt) in length. Unique coding nucleotide sequences for the replication of non-structural proteins (Rep) and structural proteins (Cap) are flanked by inverted terminal repeats (ITRs). The Cap genes encode the VP proteins (VP1, VP2, and VP3) that form the capsid. The terminal 145 nucleotides are self-complementary and organized to form an energy-stable intramolecular double helix that forms a T-hairpin. This hairpin structure functions as the initiation point for viral DNA replication, acting as a primer for the cellular DNA polymerase complex. In mammalian cells infected with wild-type AAV (wtAAV), Rep genes (e.g., Rep78 and Rep52) are expressed using the P5 and P19 promoters, respectively, and both Rep proteins have a function in viral genome replication. Splicing events in the Rep open reading frame (Rep ORF) result in the expression of virtually all four Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that unspliced ​​mRNAs encoding Rep78 and Rep52 proteins are sufficient to generate AAV vectors in mammalian cells.

[0062] carrier

[0063] As used in this article, the term "carrier" refers to a nucleic acid molecule that can transport another nucleic acid linked to it.

[0064] The terms “infectious unit (iu),” “infectious particle” or “replication unit” when used in relation to viral titers refer to the number of infectious recombinant AAV vector particles as measured by the infection center assay (also known as the replication center assay) (as described, for example, in McLaughlin et al., J. Virol. (1988) 62: 1963-1973).

[0065] The term "heterologous," when referring to nucleic acid sequences (such as coding and regulatory sequences), indicates sequences that are not typically linked together and / or not typically associated with a particular cell. Therefore, a "heterologous" region of a nucleic acid construct or vector is a nucleic acid fragment within or linked to another nucleic acid molecule that is not found in nature to be associated with that molecule. For example, a heterologous region of a nucleic acid construct may include a coding sequence flanked by a sequence not found in nature to be associated with that coding sequence. Another example of a heterologous coding sequence is a construct whose coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from those of a natural gene).

[0066] As used in this description, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0067] use

[0068] "Gene therapy" involves inserting genes into a subject's cells and / or tissues to treat diseases, often hereditary diseases, in which defective mutated alleles are replaced with functional alleles.

[0069] "Treatment" and "therapeutic methods" refer to methods that reduce or eliminate at least one of a biological condition and / or its accompanying symptoms. As used herein, "reducing" a disease, symptom, or condition means reducing the severity and / or frequency of the symptoms of the disease, symptom, or condition. Furthermore, references to "treatment" in this article include references to curative, palliative, and preventative treatments.

[0070] On the one hand, the subjects or patients undergoing treatment are mammals, preferably human subjects. The subjects can be males or females of any age.

[0071] The term "symptom" refers to any condition from which one may benefit from the treatment according to the present invention. This includes chronic and acute symptoms or diseases, including those pathological conditions that predispose mammals to developing symptoms.

[0072] "Disease" refers to a state of health in an animal in which the animal is unable to maintain homeostasis, and in which the animal's health will continue to deteriorate if the disease is not treated.

[0073] The terms “subject,” “patient,” “individual,” etc., are used interchangeably in this description and refer to any animal to which the methods described herein are applicable. In some non-limiting embodiments, the subject, patient, or individual is a human.

[0074] "Therapeutic effective dose" refers to the amount of therapeutic agent administered during treatment that will alleviate one or more symptoms of the disease being treated to a certain extent. Detailed Implementation

[0075] Codon-optimized nucleic acids

[0076] On one hand, the present invention relates to isolated codon-optimized nucleic acids encoding the SMN1 protein (survival motor neuron protein) having SEQ ID NO:1, and includes the nucleic acid sequence of SEQ ID NO:2.

[0077] Using the corresponding amino acid sequence of the SMN_HUMAN protein as a basis, a codon-optimized SMN1 gene was generated: MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN (SEQ ID NO:1).

[0078] The amino acid sequence of SEQ ID NO:1 is translated into a nucleotide sequence by sequentially matching each amino acid, starting from the N-terminus of one of the synonymous codons encoding SEQ ID NO:1.

[0079] Example 1 provides detailed information on the codon-optimized SMN1 gene and the selection of the final sequence.

[0080] The final codon-optimized sequence of SMN1 (SMN1-GeneBeam) has the following nucleotide sequence: ATGGCCATGAGCAGCGGCGGCAGCGGCGGCGGCGTGCCTGAGCAAGAGGACAGCGTGCTGTTCAGAAGAGGCACCGGCCAGAGCGACGACAGCGACATCTGGGACGACACCGCCCTGATCAAGGCCTACGACAAGGCCGTGGCCAGCTTCAAGCACGCCCTGAAGAACGGCGACATCTGCGAGACCAGCGGCAAGCCCAAGACCACCCCCAAGAGAAAGCCCGCCAAGAAGAACAAGAGCCAGAAGAAGAACACCGCCGCCAGCCTGCAGCAGTGGAAGGTGGGCGACAAGTGCAGCGCCATCTGGAGCGAGGACGGCTGCATCTACCCCGCCACCATCGCCAGCATCGACTTCAAGAGAGAGACCTGCGTGGTGGTGTACACCGGCTACGGCAACAGAGAGGAGCAGAACCTGAGCGACCTGCTGAGCCCCATCTGCGAGGTGGCCAACAACATCGAGCAGAACGCCCAAGAGAACGAGAACGAGAGCCAAGTGAGCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCAACAAGAGCGACAACATCAAGCCCAAGAGCGCCCCCTGGAACAGCTTCCTGCCCCCTCCCCCCCCTATGCCCGGCCCTAGACTGGGCCCTGGCAAGCCTGGCCTGAAGTTCAACGGCCCCCCCCCCCCTCCTCCTCCTCCTCCTCCTCACCTGCTGAGCTGCTGGCTGCCCCCCTTCCCCAGCGGCCCTCCTATCATCCCTCCTCCCCCCCCCATCTGCCCCGACAGCCTGGACGACGCCGACGCCCTGGGCAGCATGCTGATCAGCTGGTACATGAGCGGCTACCACACCGGCTACTACATGGGCTTCAGACAGAACCAGAAGGAGGGCCGGTGCAGCCACAGCCTGAACTAG (SEQ ID NO: 2).

[0081] Compared to the coding sequence of the wild-type SMN gene (SMN1-WT), the final codon-optimized nucleotide sequence of SMN1 (SMN1-GeneBeam) has an increased codon fitness index (a standard measure for assessing codon frequencies in a sequence): (SEQ ID NO:3).

[0082] The codon-fit index of the final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO:2) is 98% for the subject sequence and 75% for the wild-type sequence.

[0083] The wild-type sequence has a GC content of 45%, which is 15% lower than the target value. For the optimized sequence, the GC content of the optimized nucleotide sequence of the final codon of the SMN1 gene (SEQ ID NO:2) is 64%, which is 4% lower than the target value.

[0084] The optimized nucleotide sequence of the final codon of the SMN1 gene (SEQ ID NO:2) has 71% identity with the nucleotide sequence of the wild-type SMN1 gene (SEQ ID NO:3).

[0085] Expression box. Expression carrier.

[0086] On the one hand, the present invention relates to expression cassettes of nucleic acids including the above-described codon-optimized ones.

[0087] As used herein, the term "expression cassette" specifically refers to a DNA fragment capable of inducing the expression of a polynucleotide encoding a target polypeptide contained within it in a suitable environment. When introduced into a host cell, the expression cassette is particularly capable of engaging cellular mechanisms to transcribe the polynucleotide encoding the target polypeptide into RNA, which is then typically further processed and ultimately translated into the target polypeptide. The expression cassette may be contained within an expression vector.

[0088] The expression cassette of the present invention comprises a promoter as an element. As used herein, the term "promoter" specifically refers to a DNA element that promotes the transcription of a polynucleotide operatively linked to the promoter. The promoter may further form part of a promoter / enhancer element. Although the physical boundary between "promoter" and "enhancer" elements is not always clear, the term "promoter" generally refers to a site on a nucleic acid molecule to which RNA polymerase and / or any associated factors bind and initiate transcription. The enhancer enhances the activity of the promoter both temporally and spatially. Many promoters with transcriptional activity in a wide variety of cell types are known in the art. Promoters can be classified into two categories: those that function constitutively and those that are regulated by induction or disrepression. Both are suitable for protein expression. Promoters used for high-level polypeptide production in eukaryotic cells, and particularly mammalian cells, should be strong and preferably active in a wide variety of cell types. Strong constitutive promoters capable of driving expression in many cell types are well known in the art, and therefore need not be described in detail herein. According to the concept of the invention, cytomegalovirus (CMV) promoters are preferably used. Promoters or promoter / enhancers derived from the immediate early (IE) region of human cytomegalovirus (hCMV) are particularly suitable as promoters in the expression cassette of the present invention. The immediate early (IE) region of human cytomegalovirus (hCMV), and the resulting functional expression-inducing and / or functional expression-enhancing fragments, are described, for example, in EP0173177 and EP0323997, and are well known in the art. Therefore, several fragments of the immediate early (IE) region of hCMV can be used as promoters and / or promoter / enhancers. According to one embodiment of the invention, a human CMV promoter is used in the expression cassette of the present invention.

[0089] In some implementations, the expression box includes the following elements in the 5'-to-3'-end direction:

[0090] Left-side (first) ITR (reverse terminal repeat);

[0091] CMV (cytomegalovirus) enhancer;

[0092] CMV (cytomegalovirus) promoter;

[0093] Introns of the hBG1 gene (hemoglobin subunit γ1 gene);

[0094] The above codon-optimized nucleic acid of the SMN1 gene;

[0095] hGH1 polyadenylation signaling (human growth hormone gene polyadenylation signaling)

[0096] The right side (second) ITR.

[0097] In some implementations, the left (first) ITR (inverted terminal repeat) has the following nucleic acid sequence: Cctgcaggcagctgcgcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct (SEQ ID NO:8).

[0098] In some implementations, the CMV (cytomegalovirus) enhancer has the following nucleic acid sequence: cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgCcaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg (SEQ ID NO:9).

[0099] In some implementations, the CMV (cytomegalovirus) promoter has the following nucleic acid sequence: gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgtttgGcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagct (SEQ ID NO:10).

[0100] In some implementations, the introns of the hBG1 (hemoglobin subunit γ1) gene have the following nucleic acid sequence: cgaatcccggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctttcttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattctaaagaataacagtgataatttct gggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccagctaccattctgcttttattttatggttgggataa ggctggattattctgagtccaagctaggcccttttgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattggggat(SEQ ID NO:11).

[0101] In some implementations, the hGH1 (human growth hormone 1 gene) polyadenylation signal has the following nucleic acid sequence:

[0102] Acgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtcctt(SEQ ID NO:12).

[0103] In some embodiments, the right (second) ITR has the following nucleic acid sequence: aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg(SEQ ID NO:13).

[0104]

[0105] On the one hand, the present invention relates to expression vectors comprising the above-described codon-optimized nucleic acid or the above-described expression cassette.

[0106] In some implementations, the vector is a plasmid, which is a circular double-stranded DNA segment that can be linked to other DNA segments.

[0107] In some implementations, the vector is a viral vector in which additional DNA segments can be linked to the viral genome.

[0108] In some embodiments, the vector is capable of autonomous replication in the host cell to which it is introduced (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). In further embodiments, the vector (e.g., a non-free-living mammalian vector) can integrate into the host cell's genome after introduction and thus replicate along with the host gene. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0109] Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, colloids, YAC, and EBV-derived free organisms. DNA molecules can be ligated into the vector, allowing the transcriptional and translational control sequences within the vector to perform their intended functions of regulating DNA transcription and translation. Expression vectors and expression control sequences compatible with the host cells used can be selected. DNA molecules can be introduced into the expression vector using standard methods (e.g., ligation at complementary restriction sites, or blunt-end ligation if no restriction sites are present).

[0110] Recombinant expression vectors can also encode signal peptides that promote the secretion of target proteins by host cells. The gene for the target protein can be cloned into the vector, allowing the signal peptide to be linked within the N-terminal frame of the target protein. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0111] In addition to the SMN1-GB gene of the present invention, the recombinant expression of the vector of the present invention can carry regulatory sequences controlling the expression of the SMN1-GB gene in host cells. Those skilled in the art will understand that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the selection of the host cells to be transformed and the desired protein expression level. Preferred control sequences for expression in mammalian host cells include viral elements that ensure high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from: retroviral LTRs, cytomegalovirus (CMV) (such as CMV promoters / enhancers), simian virus 40 (SV40) (such as SV40 promoters / enhancers), adenoviruses (e.g., major late promoter adenovirus (AdMLP)), polyomaviruses, and strong mammalian promoters, such as innate immunoglobulin promoters or actin promoters.

[0112] The term "control sequence" refers to the DNA sequence necessary for the expression of an operablely linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0113] As used in this description, the terms "promoter," "transcriptional regulatory sequence," or "regulatory sequence" refer to a nucleic acid segment that controls the transcription of one or more coding sequences, and is located upstream of the reading direction relative to the transcriptional start site of the coding sequence, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence (including, but not limited to, transcription factor binding sites, repressor protein binding sites, and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that directly or indirectly regulate transcriptional levels together with said promoter). A "constitutive" promoter is a promoter that is active in most tissues under normal physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, under the influence of a chemical inducer. A "tissue-specific" promoter is active only in specific types of tissues or cells.

[0114] As used herein, the term "enhancers" or "enhancer" can refer to a DNA sequence located adjacent to the DNA sequence encoding a recombinant product. Enhancer elements are typically located at the 5' direction of a promoter element, or may be located downstream or internal to the DNA sequence encoding a recombinant product (e.g., a DNA sequence transcribed or translated into one or more recombinant products). Thus, enhancer elements may be located 100, 200, or 300 or more base pairs upstream or downstream of the DNA sequence encoding the recombinant product. Enhancer elements can increase the amount of recombinant product expressed by the DNA sequence to a level higher than that associated with a single promoter element. A variety of enhancer elements are readily available to those skilled in the art.

[0115] In addition to the genes and regulatory sequences described above, the recombinant expression vector of the present invention may carry additional sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate selection in host cells to which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, selection marker genes typically confer resistance in host cells to vectors to drugs such as G418, hygromycin, or methotrexate. Selection marker genes include, for example, the dihydrofolate reductase (DHFR) gene (for dhfr-host cells during methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthase gene.

[0116] As used in this description, the term "expression control sequence" refers to the polynucleotide sequence necessary for the expression and processing of the coding sequence to which it is linked. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when needed. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter with a ribosome-binding site and a transcription termination sequence; in eukaryotes, such control sequences typically include both a promoter and a transcription termination sequence. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion chaperone sequences.

[0117] As used herein, the term "operably ligated" refers to the ligation of polynucleotide (or polypeptide) elements in a functional relationship. It is "operably ligated" when one nucleic acid is present with another nucleic acid sequence under functional relationship conditions. For example, if a transcriptional regulatory sequence affects the transcription of a coding sequence, then it is operably ligated to said coding sequence. The term "operably ligated" means that the ligated DNA sequences are typically contiguous, and when ligation of two protein-coding regions is required, they are also contiguous and present within the reading frame.

[0118] In one embodiment of the invention, an "expression vector" refers to a vector containing one or more target polynucleotide sequences, target genes, or "transgenic" sequences that are side-joined parvovirus sequences or inverted terminal repeat (ITR) sequences.

[0119] Neither the cassette nor the vector of the present invention contains nucleotide sequences of genes encoding the non-structural protein (Rep) and structural protein (Cap) of adeno-associated virus.

[0120] Recombinant virus based on AAV9 (adeno-associated virus serotype 9)

[0121] On one hand, 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, comprising a capsid and the aforementioned expression cassette.

[0122] As used in this description, the term "AAV-based recombinant virus" (or "AAV-based virus-like particle", "AAV recombinant virus strain", "AAV recombinant vector", or "rAAV vector") refers to the above-mentioned expression cassette (or the above-mentioned expression vector) which is encapsulated in an AAV capsid.

[0123] Among other substitute products, the Cap gene encodes three capsid proteins (VP1, VP2, and VP3). VP1, VP2, and VP3 exist in a 1:1:10 ratio, 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 SciUSA, 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 a single mRNA. Posttranscriptionally, the pre-mRNA can be spliced ​​in two different ways, in which longer or shorter introns are cleaved, resulting in mRNAs of various nucleotide lengths.

[0124] During the production of AAV-based (rAAV) recombinant viruses, an expression cassette with a side-linked ITR is packaged into the AAV capsid. As mentioned above, the genes required for AAV replication are not included in the cassette.

[0125] The expression cassette DNA is packaged into the viral capsid as a single-stranded DNA molecule (ssDNA), approximately 3000 nucleotides in length. Once a cell is infected by the virus, the single-stranded DNA is converted into double-stranded DNA (dsDNA). dsDNA can only be used by cellular proteins that transcribe one or more genes of the invention into RNA.

[0126] In some implementations, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP1.

[0127] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1, which has the following amino acid sequence: (SEQ ID NO:5).

[0128] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1, said VP1 having an amino acid sequence of SEQ ID NO:5 with one or more point mutations.

[0129] In some implementations, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP2.

[0130] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP2, which has the following amino acid sequence: (SEQ ID NO:6).

[0131] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP2, said VP2 having an amino acid sequence of SEQ ID NO:6 with one or more point mutations.

[0132] In some implementations, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP3.

[0133] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP3, which has the following amino acid sequence: (SEQ ID NO:7).

[0134] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP3, said VP3 having an amino acid sequence of SEQ ID NO:7 with one or more point mutations.

[0135] In some implementations, the AAV9-based recombinant virus has a capsid comprising AAV9 proteins VP1, VP2, and VP3.

[0136] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the following proteins: VP1 having the amino acid sequence of SEQ ID NO:5, VP2 having the amino acid sequence of SEQ ID NO:6, and VP3 having the amino acid sequence of SEQ ID NO:7.

[0137] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the following proteins: VP1 having an amino acid sequence of SEQ ID NO:5 with one or more point mutations, VP2 having an amino acid sequence of SEQ ID NO:6 with one or more point mutations, and VP3 having an amino acid sequence of SEQ ID NO:7 with one or more point mutations.

[0138] The phrase "multiple point mutations" refers to the substitution of two, three, four, five, six, seven, eight, nine, or ten points.

[0139] Particularly preferred embodiments include inherently conserved substitutions (mutations), i.e., substitutions occurring within a family of amino acids added according to their side chains. Specifically, amino acids are generally classified into four families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonable to predict that independently substituting leucine for isoleucine or valine, substituting aspartic acid for glutamic acid, substituting threonine for serine, or similarly conserving amino acids for structurally related amino acids will not have a significant impact on biological activity. For example, a target polypeptide may include up to about 5-10 conserved or nonconserved amino acid substitutions, provided that the desired function of the molecule remains intact.

[0140] One implementation of point mutations using amino acid substitutions in the sequence of AAV9 protein VP1, VP2, or VP3 is to replace at least one amino acid residue in AAV9 protein VP1, VP2, or VP3 with another amino acid residue.

[0141] Conservative substitutions are shown in the "Preferred Substitutions" column of Table A.

[0142]

[0143] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1, said 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 elements in the 5'-to-3'-direction:

[0144] CMV enhancer;

[0145] CMV promoter;

[0146] Introns of the hBG1 gene;

[0147] The above codon-optimized nucleic acid of the SMN1 gene;

[0148] hGH1 polyadenylation signal;

[0149] Right side ITR.

[0150] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the following proteins: VP1 having the amino acid sequence of SEQ ID NO:5, VP2 having the amino acid sequence of SEQ ID NO:6, and VP3 having the amino acid sequence of SEQ ID NO:7, and the expression cassette includes the following elements in the 5'-to-3'-end direction:

[0151] CMV enhancer;

[0152] CMV promoter;

[0153] Introns of the hBG1 gene;

[0154] The above codon-optimized nucleic acid of the SMN1 gene;

[0155] hGH1 polyadenylation signal;

[0156] Right side ITR.

[0157] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the following proteins: VP1 having an amino acid sequence of SEQ ID NO:5 with one or more point mutations, VP2 having an amino acid sequence of SEQ ID NO:6 with one or more point mutations, and VP3 having an amino acid sequence of SEQ ID NO:7 with one or more point mutations, and the expression cassette includes the following elements in the 5'-to-3'-end direction:

[0158] CMV enhancer;

[0159] CMV promoter;

[0160] Introns of the hBG1 gene;

[0161] The above codon-optimized nucleic acid of the SMN1 gene;

[0162] hGH1 polyadenylation signal;

[0163] Right side ITR.

[0164] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1, said 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 contains nucleic acid having SEQ ID NO:4.

[0165] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the following proteins: VP1 having the amino acid sequence of SEQ ID NO:5, VP2 having the amino acid sequence of SEQ ID NO:6, and VP3 having the amino acid sequence of SEQ ID NO:7, and the expression cassette contains nucleic acid having SEQ ID NO:4.

[0166] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the following proteins: VP1 having an amino acid sequence of SEQ ID NO:5 with one or more point mutations, VP2 having an amino acid sequence of SEQ ID NO:6 with one or more point mutations, and VP3 having an amino acid sequence of SEQ ID NO:7 with one or more point mutations, and the expression cassette contains a nucleic acid having SEQ ID NO:4.

[0167] Pharmaceutical Composition

[0168] On one hand, the present invention relates to a pharmaceutical composition for delivering the SMN1 gene to target cells, comprising the aforementioned AAV9-based recombinant virus in combination with one or more pharmaceutically acceptable excipients.

[0169] In certain embodiments, the present invention relates to pharmaceutical compositions comprising the AAV9-based recombinant virus of the present 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 a solid or liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline solution. For inhalation administration, the carrier is inhalable and preferably in solid or liquid particulate form. As an injection medium, water containing additives commonly used in injection solutions (such as stabilizers, salts or saline solutions and / or buffers) is preferred.

[0170] "Pharmaceutical composition" refers to a composition comprising the above-described AAV9-based recombinant virus of the present invention and at least one component selected from the following: pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, adjuvants, distributors, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, and extended delivery control agents, the selection and proportion of which depend on the type, route, and dosage of administration. The pharmaceutical compositions of the present invention and their preparation methods will undoubtedly be apparent to those skilled in the art. The pharmaceutical compositions should preferably be manufactured in accordance with GMP (Good Manufacturing Practice) requirements. The compositions may contain buffering compositions, tension agents, stabilizers, and solubilizers.

[0171] "Pharmaceutical acceptable" means a material that does not have biological or other negative side effects; for example, a material that can be administered to a subject without causing any undesirable biological effects. Therefore, this pharmaceutical composition can be used, for example, to transfect cells in vitro or to administer directly to a subject the AAV9-based recombinant virus of the present invention.

[0172] The term "excipient" is used herein to describe any component other than those described above in this invention. These are substances of inorganic or organic nature that are used in pharmaceutical manufacturing to impart the necessary physicochemical properties to the pharmaceutical product.

[0173] "Stabilizer" refers to an excipient or a mixture of two or more excipients that provides physical and / or chemical stability to the active agent.

[0174] The terms "buffer solution," "buffer composition," and "buffer agent" refer to solutions capable of resisting changes in pH through the action of their acid-base conjugate components, thereby enabling the rAAV5 carrier product to resist pH changes. Generally, the pH of the pharmaceutical composition is preferably in the range of 4.0 to 8.0. Examples of buffer agents that can be used include, but are not limited to, buffer solutions containing acetate, phosphate, citrate, histidine, succinate, etc.

[0175] A pharmaceutical composition is considered "stable" if the active agent maintains its physical and / or chemical stability and / or biological activity during its specified shelf life at a storage temperature, for example, 2-8°C. Preferably, the active agent maintains both physical and chemical stability as well as biological activity. The shelf life is adjusted based on the results of stability tests under accelerated or natural aging conditions.

[0176] The pharmaceutical compositions of the present invention can be manufactured, packaged, or widely marketed as ready-to-use formulations, in single unit doses or in multiple single unit doses. As used herein, the term "single unit dose" refers to a pharmaceutical composition containing a discrete amount of a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject, or a convenient portion of such a dose, such as half or one-third of such a dose.

[0177] use

[0178] On one hand, the present invention relates to the use of the above-mentioned AAV9-based recombinant virus or the above-mentioned composition to deliver the SMN1 gene to target cells.

[0179] Any method recognized in the art for administering AAV9-based recombinant viruses can be appropriately used for the AAV9-based recombinant viruses described above in this invention.

[0180] AAV9-based recombinant viruses are preferably administered to cells in a bioeffective amount. A “bioeffective” amount of a recombinant virus is sufficient to induce infection (or transduction) and expression of a heterologous nucleic acid sequence in the cell. If the virus is administered to cells in vivo (e.g., to a subject as described below), the “bioeffective” amount of the viral vector is sufficient to induce transduction and expression of a heterologous nucleic acid sequence in the target cell.

[0181] The cells to which the above-described AAV9-based recombinant virus of the present invention is administered can be any type of cell, including but not limited to nerve cells (including cells of the peripheral and central nervous systems, particularly brain cells), lung cells, epithelial cells (e.g., intestinal and respiratory epithelial cells), muscle cells, pancreatic cells (including islet cells), hepatocytes, cardiomyocytes, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, etc. Alternatively, the cells to which the above-described AAV9-based recombinant virus is administered can be any progenitor cell. As a further alternative, the cells can be stem cells (e.g., neural stem cells, liver stem cells). Furthermore, as specified above, the cells can be of any species origin.

[0182] The aforementioned AAV9-based recombinant viruses are not used to modify the genetic integrity of human germline cells.

[0183] Example

[0184] To better understand the present invention, the following embodiments are provided. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0185] 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 for purposes of clear understanding, certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments, as will be apparent to those skilled in the art.

[0186] Materials and General Methods

[0187] Recombinant DNA technology

[0188] As described by Sambrook J. et al., *Molecular Cloning: A Laboratory Manual*; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, DNA manipulation was performed using standard techniques. Molecular biology reagents were used according to the manufacturer's instructions. In short, plasmid DNA was produced for further manipulation in *E. coli* cells grown under selective antibiotic stress (so that the plasmid would not be lost in the cell population). We isolated plasmid DNA from the cells using commercial kits, measured its concentration, and used it for cloning by restriction endonuclease treatment or PCR amplification. DNA fragments were ligated together using ligases and transformed into bacterial cells for clone selection and further production. All resulting genetic constructs were confirmed by restriction mapping and full Sanger sequencing.

[0189] Gene synthesis

[0190] The desired gene segment was prepared from oligonucleotides produced through chemical synthesis. Gene segments of 300 to 1000 bp in length with unique restriction sites were collected by annealing the oligonucleotides to each other at the top, followed by PCR amplification from boundary primers. As a result, a mixture of fragments was produced, including the desired fragment. The fragment was cloned into an intermediate vector at the restriction site, and the DNA sequence of the subcloned fragment was subsequently confirmed by DNA sequencing.

[0191] DNA sequencing

[0192] DNA sequences are determined using Sanger sequencing. DNA and protein sequences are analyzed and processed in SnapGene Viewer 4.2 or later for sequence creation, mapping, analysis, annotation, and description.

[0193] Cell cultures

[0194] The experiments used HEK293 (human embryonic kidney clone 293) and HSMC (human skeletal muscle cells) cell lines. Cells were cultured in DMEM complete medium supplemented with 10% FBS and antibiotics under standard conditions of 37°C and 5% CO2. For HSMC culture, culture plastic was pre-coated with collagen (Gibco). Cells were passaged when they reached 80-90% confluence. Cell viability was assessed using trypan blue staining and hemocytometer or PI staining and flow cytometry.

[0195] Cell transfection

[0196] The cell lines were seeded into 6-well plates one day prior to transfection, allowing them to reach 70-80% confluence at transfection time. Transfection was performed using a commercial lipid infection kit according to the manufacturer's protocol. After 72 hours, the cells were treated with trypsin solution or similar, removed from the matrix, washed in phosphate-buffered saline, and collected for further analysis of target gene and protein expression. For each transfection, a control plasmid expressing GFP was used to measure transfection efficiency (percentage of GFP-positive cells). Further analysis was only performed when the transfection efficiency was at least 50%.

[0197] All measurements were performed in three independent experiments.

[0198] Gene expression analysis

[0199] SMN1 expression at the mRNA level was assessed by quantitative PCR. In short, primers and samples specific to the wild-type SMN1 sequence or GeneBeam were used. Primers and samples specific to the GAPDH housekeeping gene were used as controls for initial RNA levels. Calibration curves were plotted for each primer and sample set using linearized plasmid DNA containing the amplified sequences of the corresponding genes at known copy numbers. Expression was analyzed by determining the copy numbers of SMN1-GeneBeam, SMN1-WT, and GAPDH in each sample using the calibration curves, and then the SMN1 copy number per 1000 GAPDH copies was normalized. Values ​​obtained from different samples were compared within the same experiment.

[0200] SMN1 protein expression was determined by flow cytometry.

[0201] SMN1 protein levels in cells were assessed by intracellular staining followed by flow cytometry analysis. In short, cells were removed from culture plates using TrypLE, washed in PBS, fixed in 4% paraformaldehyde solution, permeabilized with 0.5% Triton X-100 in PBS, incubated in blocking buffer supplemented with 1–5% BSA, and stained in two stages using primary antibody against SMN1 and secondary antibody labeled with Alexa Fluor 488. After staining, cells were washed once in PBS and analyzed by flow cytometry. The mean signal intensity was assessed after subtracting the signal from staining with secondary antibody without primary antibody.

[0202] Assembly and purification of viral particles from recombinant AAV vector

[0203] To assemble AAV particles containing the SMN1 gene or the GFP control gene, we packaged cells using HEK293, which were then transfected with the following three plasmids:

[0204] Plasmids containing AAV genomes with transgene (SNM1 or GFP) expression cassettes;

[0205] Plasmids for expressing the Cap gene of AAV9 serotype and the Rep gene of AAV2 serotype. Alternate reading frames are used; each gene encodes several protein products.

[0206] Plasmids used to express the Ad5 (adenovirus serotype 5) gene, which is required for the assembly and packaging of AAV capsids.

[0207] After 72 hours, cells were lysed, and viral particles were purified and concentrated using filtration and chromatography. The viral particle titer was determined by quantitative PCR using site-specific primers for the recombinant viral genome and the sample, and expressed as the number of viral genome copies per 1 ml.

[0208] Transduced cell cultures

[0209] Similar to transfection experiments, cell lines were seeded, then products containing viral particles were added, and the cells were analyzed after 72 hours. Transduction efficiency was estimated by measuring the percentage of GFP+ cells.

[0210] The cultures used were pre-tested to examine transduction efficiency. In short, AAV9-GFP viral products were transduced into cell lines at different ratios of cells to viral particles. The ratio of viral particles to cell numbers is called the multiple of infection (MOI). The MOI of AAV9-GFP virus ranges from 50,000 to 1,000,000. As a result, an MOI range was determined for each line, within which transduction efficiency changed linearly with MOI. Further transduction of the cell lines was carried out within this linear range.

[0211] After transduction, gene and protein expression were analyzed as described above.

[0212] All measurements were performed in three independent experiments.

[0213] Example 1. Method for generating a codon-optimized SMN1 gene

[0214] The corresponding amino acid sequence of the SMN_HUMAN protein (SEQ ID NO:1) is used as the basis for generating the codon-optimized SMN1 gene.

[0215] Considering one or a combination of the following features, the amino acid sequence of SEQ ID NO:1 is translated into a nucleotide sequence by sequentially matching each amino acid, starting from the N-terminus of one of the synonymous codons encoding SEQ ID NO:1:

[0216] 1) Codon usage frequency (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);

[0217] 2) GC content of the terminal region of the obtained nucleotide sequence (the target value of GC content is 60%, as shown in the article by Grzegorz Kudla et al., High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells, PLoS Biol, June 2006, Vol. 4, No. 6, e180, doi:10.1371 / journal.pbio.0040180. Therefore, the smaller the difference between the current GC content and the target GC content, the more preferred the codon).

[0218] 3) The folding free energy of the terminal region of the obtained nucleotide sequence (secondary structure was determined using the Zuker algorithm, Michael Zuker et al., Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information, Nucleic Acids Research, Vol. 9, No. 1, January 10, 1981, pp. 133-148, doi:10.1093 / nar / 9.1.133).

[0219] The construction process further avoids the generation of semantic nucleotide sequences, such as restriction sites, internal ribosome entry sites, and splicing sites.

[0220] As a result of translating the amino acid sequence of SEQ ID NO:1 into a nucleotide sequence, an array of codon-optimized nucleotide sequences of the SMN1 gene was generated.

[0221] In further studies, several sequences in the array of codon-optimized nucleotide sequences of SMN1 did not show an increase in SMN1 gene transcription; that is, the number of SMN1-opt mRNA copies was not significantly increased or was not significant compared to SMN1-WT on any cell line used.

[0222] In further studies, the nucleotide sequence of most codons of the SMN1 gene was optimized, showing an increase in SMN1 gene transcription of 1.5-2 times, i.e., a significant increase in the mRNA copy number of SMN1-opt compared to SMN1-WT on all cell lines used.

[0223] In further research, one sequence from the array of the codon-optimized nucleotide sequences of the SMN1 gene unexpectedly showed an increase in SMN1 gene transcription of more than 3-fold; that is, the mRNA copy number of SMN1-opt unexpectedly increased by more than 3-fold compared to SMN1-WT on all cell lines used (see Examples 3-4). The final codon-optimized nucleotide sequence of this SMN1 gene is commonly referred to as SMN1-GeneBeam (or abbreviated as SMN1-GB).

[0224] The final codon-optimized sequence of SMN1 (SMN1-GeneBeam) has a nucleotide sequence represented by SEQ ID NO:2.

[0225] The final codon-optimized nucleotide sequence of SMN1 (SMN1-GeneBeam) is characterized by an increased codon adaptation index compared to the coding sequence of the wild-type SMN gene (SMN1-WT with SEQ ID NO:3) (Paul M. Sharp et al., The codon adaptation index—a measure of directional synonymous codonusage bias, and its potential applications, Nucleic Acids Research, Vol. 15, No. 3, February 11, 1987, pp. 1281-1295, doi: 10.1093 / nar / 15.3.1281 - A standard measure used to evaluate the frequency of codon usage in a sequence.

[0226] The codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) has a codon fitness index of 98%, while the wild-type sequence has a codon fitness index of 75%.

[0227] The wild-type sequence has a GC content of 45%, which is 15% lower than the target value, while the GC content of the final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) is 64%, which is 4% lower than the target value.

[0228] The optimized nucleotide sequence of the final codon of the SMN1 gene (SEQ ID NO:2) shares 71% identity with the nucleotide sequence of the wild-type SMN1 gene (SEQ ID NO:3).

[0229] Example 2. Assembly of a genetic construct carrying a recombinant AAV genome and encoding the SMN1 gene.

[0230] The wild-type SMN1 gene sequence was generated by amplification using specific primers and cDNA synthesized from total RNA from HEK293 cells. During amplification, a Kozak sequence and a ClaI restriction site were added to the 5' end of the gene, and an XbaI restriction site was added to the 3' end. Subsequently, the SMN1 gene sequence was cloned into the commercial construct pAAV-GFP control plasmid (VPK-402) from CellBiolab (USA) at the ClaI and XbaI sites using restriction ligase, replacing the GFP gene with SMN1 to generate the pAAV-SMN1-WT construct.

[0231] The SMN1-GeneBeam sequence was assembled as described above. Given the sequence's complexity, despite its relatively small size, we performed a series of subclonings of the gene fragments in the intermediate vector pGEMT, with sequence validation performed on each vector. Next, the full-length version of the gene was assembled from several intermediate vectors by PCR and cloned into the intermediate vector pGEMT. The construct pAAV-SMN1-WT was used as the final genetic construct, in which wild-type SMN1 was replaced with SMN1-GeneBeam at the ClaI and XbaI sites added to the ends of the SMN1-GeneBeam sequence by PCR.

[0232] The final vector contains all the elements necessary for gene expression and assembly as part of the recombinant AAV genome:

[0233] 1) The ITR at the end of the sequence encapsulated in the viral capsid;

[0234] 2) Elements used to express the target gene (promoter, enhancer, intron, Kozak sequence, transgene, polyadenylation site);

[0235] 3) Bacterial replication origin and antibiotic resistance genes to produce plasmid DNA in bacterial cells.

[0236] It is worth noting that the genetic constructs containing the SMN1-WT and SMN1-GeneBeam genes differ only in the SMN1 gene sequence, while being completely identical in all other respects.

[0237] Example 3. Validation of SMN1 expression from genetic constructs.

[0238] As described above, the genetic constructs pAAV-GFP, pAAV-SMN1-WT, and pAAV-SMN1-GB were transfected into HEK293 cells and HSMCs. We used 5 μg of DNA per well. After 72 hours, cells were collected and SMN1 expression (normalized relative to GAPDH) was analyzed as described above.

[0239] Codon optimization of the SMN1 gene was found to affect SMN1 transcription, reliably increasing the mRNA copy number of SMN1-GB by several times in both cell lines used compared to the SMN1-WT mRNA copy number. Figure 1 Specifically, for HEK293 cells, the normalized expression ratio of SMN1-GB to SMN1-WT was 3.9, while for HSMC, this ratio was 12.8.

[0240] As the data obtained indicate, this property of SMN1-GeneBeam is not cell-specific and further increases the expression of the target gene in cells by several times, which could be a significant advantage in the development of gene therapy drugs. Furthermore, this property is not due to any differences in gene expression cassettes or the characteristics of the appropriate viral capsid carrying the genome from the SMN1-GeneBeam gene, because the analysis was performed on a genetic construct that differed only in codon optimization of the SMN1 gene, while being otherwise identical.

[0241] As described above, HSMCs were selected to examine SMN1 protein expression at the level by flow cytometry. The results showed that, with 5 μg DNA per well, cells transfected with pAAV-SMN1-GB exhibited a 12.2-fold higher signal from the SMN1-specific antibody compared to cells transfected with pAAV-SMN1-WT. Figure 2 This observation suggests that SMN1-GB does not offer an advantage in translation, but the increased transcription further enhances the final protein levels in the cell.

[0242] Example 4. Creating a viral product expressing SMN1

[0243] Plasmids pAAV-SMN1-WT and pAAV-SMN1-GB, along with other plasmids required for the production of recombinant AAV viral particles (see above), are used in the biological process of AAV production. The serotype used is either wild-type AAV9 or wild-type AAV9 with one or more point mutations.

[0244] In all cases, the characteristics of wild-type SMN1 and SMN1-GeneBeam are compared only when the serotype and capsid mutation (if any) used are the same. All AAV9-based serotypes, whether wild-type or mutated, are referred to as AAV9 below, without specifying the mutation.

[0245] The biological process produced recombinant viral particles named AAV9-SMN1-WT and AAV9-SMN1-GB, as well as a control particle, AAV9-GFP. After determining the viral particle titer, all three products were transduced into sensory cells, namely primary human myocytes (HSMCs), using primary human myocytes with the same MOI (MOI values ​​varied between 50,000 and 200,000 between experiments). Further analysis was only performed when the transduction efficiency was at least 50%.

[0246] After successful transduction, cells were removed from the matrix, washed in phosphate-buffered saline, and SMN1 expression was analyzed at both the mRNA and protein levels as described above. The increase in SMN1-GeneBeam transcriptional activity was consistent; therefore, SMN1-GeneBeam mRNA was detected at a 7.3-fold increase compared to wild-type SMN1 mRNA. A similar increase (6.8-fold) was further observed at the protein level. Figure 3 This shows that SMN1-GB has no advantage at the translational level; however, the detectable increase in transcriptional efficiency with the AAV9-SMN1-GB product provides higher levels of SMN1 expression in target cells, which is an important advantage in the treatment of, for example, spinal muscular atrophy, in which the level of SMN1 protein expression defines the disease type from 0 (embryonic lethal) to 4 (no specific treatment required). sequence list <110> LLC "ANABION" <120> Codon-optimized nucleic acid encoding SMN1 protein <160> 13 <170> BiSSAP 1.3.6 <210> 1 <211> 294 <212> PRT <213> native sequence <220> <223> The amino acid sequence of the SMN1 protein <400> 1 Met Ala Met Ser Ser Gly Gly Ser Gly Gly Gly Val Pro Glu Gln Glu 1 5 10 15 Asp Ser Val Leu Phe Arg Arg Gly Thr Gly Gln Ser Asp Asp Ser Asp 20 25 30 Ile Trp Asp Asp Thr Ala Leu Ile Lys Ala Tyr Asp Lys Ala Val Ala 35 40 45 Ser Phe Lys His Ala Leu Lys Asn Gly Asp Ile Cys Glu Thr Ser Gly 50 55 60 Lys Pro Lys Thr Thr Pro Lys Arg Lys Pro Ala Lys Lys Asn Lys Ser 65 70 75 80 Gln Lys Lys Asn Thr Ala Ala Ser Leu Gln Gln Trp Lys Val Gly Asp 85 90 95 Lys Cys Ser Ala Ile Trp Ser Glu Asp Gly Cys Ile Tyr Pro Ala Thr 100 105 110 Ile Ala Ser Ile Asp Phe Lys Arg Glu Thr Cys Val Val Val Tyr Thr 115 120 125 Gly Tyr Gly Asn Arg Glu Glu Gln Asn Leu Ser Asp Leu Leu Ser Pro 130 135 140 Ile Cys Glu Val Ala Asn Asn Ile Glu Gln Asn Ala Gln Glu Asn Glu 145 150 155 160 Asn Glu Ser Gln Val Ser Thr Asp Glu Ser Glu Asn Ser Arg Ser Pro 165 170 175 Gly Asn Lys Ser Asp Asn Ile Lys Pro Lys Ser Ala Pro Trp Asn Ser 180 185 190 Phe Leu Pro Pro Pro Pro Pro Met Pro Gly Pro Arg Leu Gly Pro Gly 195 200 205 Lys Pro Gly Leu Lys Phe Asn Gly Pro Pro Pro Pro Pro Pro Pro Pro 210 215 220 Pro Pro His Leu Leu Ser Cys Trp Leu Pro Pro Phe Pro Ser Gly Pro 225 230 235 240 Pro Ile Ile Pro Pro Pro Pro Pro Ile Cys Pro Asp Ser Leu Asp Asp 245 250 255 Ala Asp Ala Leu Gly Ser Met Leu Ile Ser Trp Tyr Met Ser Gly Tyr 260 265 270 His Thr Gly Tyr Tyr Met Gly Phe Arg Gln Asn Gln Lys Glu Gly Arg 275 280 285 Cys Ser His Ser Leu Asn 290 <210> 2 <211> 882 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized nucleic acid sequence encoding the SMN1 protein (SMN1 GeneBeam) <400> 2 atggccatga gcagcggcgg cagcggcggc ggcgtgcctg agcaagagga cagcgtgctg 60 ttcagaagag gcaccggcca gagcgacgac agcgacatct gggacgacac cgccctgatc 120 aaggcctacg acaaggccgt ggccagcttc aagcacgccc tgaagaacgg cgacatctgc 180 gagaccagcg gcaagcccaa gaccaccccc aagagaaagc ccgccaagaa gaacaagagc 240 cagaagaaga acaccgccgc cagcctgcag cagtggaagg tgggcgacaa gtgcagcgcc 300 atctggagcg aggacggctg catctacccc gccaccatcg ccagcatcga cttcaagaga 360 gagacctgcg tggtggtgta caccggctac ggcaacagag aggagcagaa cctgagcgac 420 ctgctgagcc ccatctgcga ggtggccaac aacatcgagc agaacgccca agagaacgag 480 aacgagagcc aagtgagcac cgacgagagc gagaacagca gaagccccgg caacaagagc 540 gacaacatca agcccaagag cgccccctgg aacagcttcc tgccccctcc cccccctatg 600 cccggcccta gactgggccc tggcaagcct ggcctgaagt tcaacggccc ccccccccct 660 cctcctcctc ctcctcctca cctgctgagc tgctggctgc cccccttccc cagcggccct 720 cctatcatcc ctcctccccc ccccatctgc cccgacagcc tggacgacgc cgacgccctg 780 ggcagcatgc tgatcagctg gtacatgagc ggctaccaca ccggctacta catgggcttc 840 agacagaacc agaaggaggg ccggtgcagc cacagcctga ac 882 <210> 3 <211> 885 <212> DNA <213> Native sequence <220> <223> SMN1-infected cells (SMN1-WT, SMN1-WT) <400> 3 atggcgatga gcagcggcgg cagtggtggc ggcgtcccgg agcaggagga ttccgtgctg 60 ttccggcgcg gcacaggcca gagcgatgat tctgacattt gggatgac agcactgata 120 aaagcatatg ataaagctgt ggcttcattt aagcatgctc taaagaatgg tgacatttgt 180 gaaacttcgg gtaaaccaaa aaccacacct aaaagaaaac ctgctaagaa gaataaaagc 240 caaaagaaga atactgcagc ttccttacaa cagtggaaag ttggggacaa atgttctgcc 300 atttggtcag aagacggttg cattaccca gctaccattg cttcaattga ttttaagaga 360 gaaacctgtg ttgtggttta cactggatat ggaatagag aggagcaaaa tctgtccgat 420 ctactttccc caatctgtga agtagctaat aatatagac aaaatgctca agagaatgaa 480 aatgaaagcc aagtttcaac agatgaaagt gagaactcca ggtctcctgg aaataaatca 540 gataacatca agcccaaatc tgctccatg aactcttttc tcctccacc accccccatg 600 ccagggccaa gactgggacc aggaaagcca ggtctaaaat tcaatggccc accaccgcca 660 ccgccaccac caccacccca cttactatca tgctggctgc ctccatttcc ttctggacca 720 ccaataattc ccccaccacc tcccatatgt ccagattctc ttgatgatgc tgatgctttg 780 ggaagtatgt taatttcatg gtacatgagt ggctatcata ctggctatta tatgggtttc 840 agacaaaatc aaaaagaagg aaggtgctca cattccttaa attaa 885 <210> 4 <211> 2924 DNA Artificial sequence <220> Nucleic acid sequence of expression cassette (complete) <400> 4 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct gcggccgcac gcgtctagtt attaatagta atcaattacg gggtcattag 180 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 240 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 300 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 360 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 420 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 480 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 540 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 600 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 660 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag 720 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc 780 gggaccgatc cagcctccgc ggattcgaat cccggccggg aacggtgcat tggaacgcgg 840 attccccgtg ccaagagtga cgtaagtacc gcctatagag tctataggcc cacaaaaaat 900 gctttcttct tttaatatac ttttttgttt atcttatttc tatactttc cctaatctct 960 ttctttcagg gcaataatga tacaatgtat catgcctctt tgcaccattc taaagaataa 1020 cagtgataat ttctgggtta aggcaatagc aatatttctg catataaata tttctgcata 1080 taaattgtaa ctgatgtaag aggtttcata ttgctaatag cagctacaat ccagctacca 1140 ttctgctttt attttatggt tgggataagg ctggattatt ctgagtccaa gctaggccct 1200 tttgctaatc atgttcatac ctcttatctt cctcccacag ctcctgggca acgtgctggt 1260 ctgtgtgctg gcccatcact ttggcaaaga attgggattc gaacatcgat tgtaattcat 1320 gagccaccat ggccatgagc agcggggcca gcggcgggg cgtgcctgag caagaggaca 1380 gcgtgctggt cagaagaggc accggccaga gcgacgacag cgacatctgg gacgacaccg 1440 ccctgatcaa ggcctacgac aaggccgtgg ccagcttcaa gcacgccctg aagaacggcg 1500 acatctgcga gaccagcggc aagcccaaga ccacccccaa gagaaagccc gccaagaaga 1560 acaagagcca gaagaaac accgccgcca gcctgcagca gtggaaggtg ggcgacaagt 1620 gcagcgccat ctggagcgag gacggctgca tctaccccgc caccatcgcc agcatcgact 1680 1740 tgagcgacct gctgagcccc atctgcgagg tggccaacaa catcgagcag aacgcccaag 1800 agaacgagaa cgagagccaa gtgagcaccg acgagagcga gaacagcaga agccccggca 1860 acaagagcga caacatcaag cccaagagcg ccccctggaa cagcttcctg ccccctcccc 1920 cccctatgcc cggccctaga ctgggccctg gcaagcctgg cctgaagttc aacggccccc 1980 ccccccctcc tcctcctcct cctctccacc tgctgagctg ctggctgccc cccttcccca 2040 gcggccctcc tatcatccct cctcccccc ccatctgccc cgacagcctg gacgacgccg 2100 acgccctggg cagcatgctg atcagctggt acatgagcgg ctaccacacc ggctactaca 2160 tgggcttcag acagaaccag aagggggcc ggtgcagcca cagcctgaac tgatctagag 2220 tcgacctgca gaagcttgcc tcgagcagcg ctgctcgaga gatctacggg tggcatccct 2280 gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc 2340 ttgtcctaat aaaaattaagt tgcatcattt tgtctgacta ggtgtccttt tataatatta 2400 tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct 2460 gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct 2520 ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg 2580 catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat 2640 tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat 2700 tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa 2760 ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct 2820 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 2880 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg cagg 2924 <210> 5 <211> 736 <212> PRT <213> Natural sequence <220> <223> Amino acid sequence of VP1 AAV9 protein <400> 5 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585 590 Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 6 <211> 599 <212> PRT <213> natural sequence <220> <223> Amino acid sequence of VP2 AAV9 protein <400> 6 Thr Ala Pro Gly Lys Lys Arg Pro Val Glu Gln Ser Pro Gln Glu Pro 1 5 10 15 Asp Ser Ser Ala Gly Ile Gly Lys Ser Gly Ala Gln Pro Ala Lys Lys 20 25 30 Arg Leu Asn Phe Gly Gln Thr Gly Asp Thr Glu Ser Val Pro Asp Pro 35 40 45 Gln Pro Ile Gly Glu Pro Pro Ala Ala Pro Ser Gly Val Gly Ser Leu 50 55 60 Thr Met Ala Ser Gly Gly Gly Ala Pro Val Ala Asp Asn Asn Glu Gly 65 70 75 80 Ala Asp Gly Val Gly Ser Ser Ser Gly Asn Trp His Cys Asp Ser Gln 85 90 95 Trp Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu 100 105 110 Pro Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Asn Ser Thr Ser 115 120 125 Gly Gly Ser Ser Asn Asp Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp 130 135 140 Gly Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp 145 150 155 160 Trp Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu 165 170 175 Asn Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Asp Asn Asn 180 185 190 Gly Val Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe 195 200 205 Thr Asp Ser Asp Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Glu 210 215 220 Gly Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr 225 230 235 240 Gly Tyr Leu Thr Leu Asn Asp Gly Ser Gln Ala Val Gly Arg Ser Ser 245 250 255 Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn 260 265 270 Asn Phe Gln Phe Ser Tyr Glu Phe Glu Asn Val Pro Phe His Ser Ser 275 280 285 Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp 290 295 300 Gln Tyr Leu Tyr Tyr Leu Ser Lys Thr Ile Asn Gly Ser Gly Gln Asn 305 310 315 320 Gln Gln Thr Leu Lys Phe Ser Val Ala Gly Pro Ser Asn Met Ala Val 325 330 335 Gln Gly Arg Asn Tyr Ile Pro Gly Pro Ser Tyr Arg Gln Gln Arg Val 340 345 350 Ser Thr Thr Val Thr Gln Asn Asn Asn Ser Glu Phe Ala Trp Pro Gly 355 360 365 Ala Ser Ser Trp Ala Leu Asn Gly Arg Asn Ser Leu Met Asn Pro Gly 370 375 380 Pro Ala Met Ala Ser His Lys Glu Gly Glu Asp Arg Phe Phe Pro Leu 385 390 395 400 Ser Gly Ser Leu Ile Phe Gly Lys Gln Gly Thr Gly Arg Asp Asn Val 405 410 415 Asp Ala Asp Lys Val Met Ile Thr Asn Glu Glu Glu Ile Lys Thr Thr 420 425 430 Asn Pro Val Ala Thr Glu Ser Tyr Gly Gln Val Ala Thr Asn His Gln 435 440 445 Ser Ala Gln Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile 450 455 460 Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro 465 470 475 480 Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro 485 490 495 Leu Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile 500 505 510 Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe Asn Lys Asp 515 520 525 Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val 530 535 540 Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro 545 550 555 560 Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe 565 570 575 Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr 580 585 590 Arg Tyr Leu Thr Arg Asn Leu 595 <210> 7 <211> 534 <212> PRT <213> Natural sequence <220> <223> Amino acid sequence of VP3 AAV9 protein <400> 7 Met Ala Ser Gly Gly Gly Ala Pro Val Ala Asp Asn Asn Glu Gly Ala 1 5 10 15 Asp Gly Val Gly Ser Ser Ser Gly Asn Trp His Cys Asp Ser Gln Trp 20 25 30 Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro 35 40 45 Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly 50 55 60 Gly Ser Ser Asn Asp Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 65 70 75 80 Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp 85 90 95 Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn 100 105 110 Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Asp Asn Asn Gly 115 120 125 Val Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr 130 135 140 Asp Ser Asp Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Glu Gly 145 150 155 160 Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly 165 170 175 Tyr Leu Thr Leu Asn Asp Gly Ser Gln Ala Val Gly Arg Ser Ser Phe 180 185 190 Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn 195 200 205 Phe Gln Phe Ser Tyr Glu Phe Glu Asn Val Pro Phe His Ser Ser Tyr 210 215 220 Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln 225 230 235 240 Tyr Leu Tyr Tyr Leu Ser Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln 245 250 255 Gln Thr Leu Lys Phe Ser Val Ala Gly Pro Ser Asn Met Ala Val Gln 260 265 270 Gly Arg Asn Tyr Ile Pro Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser 275 280 285 Thr Thr Val Thr Gln Asn Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala 290 295 300 Ser Ser Trp Ala Leu Asn Gly Arg Asn Ser Leu Met Asn Pro Gly Pro 305 310 315 320 Ala Met Ala Ser His Lys Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser 325 330 335 Gly Ser Leu Ile Phe Gly Lys Gln Gly Thr Gly Arg Asp Asn Val Asp 340 345 350 Ala Asp Lys Val Met Ile Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn 355 360 365 Pro Val Ala Thr Glu Ser Tyr Gly Gln Val Ala Thr Asn His Gln Ser 370 375 380 Ala Gln Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile Leu 385 390 395 400 Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile 405 410 415 Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu 420 425 430 Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile Lys 435 440 445 Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys 450 455 460 Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu 465 470 475 480 Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu 485 490 495 Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala 500 505 510 Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg 515 520 525 Tyr Leu Thr Arg Asn Leu 530 <210> 8 <211> 130 <212> DNA <213> native sequence <220> <223> Left-side (first) ITR (reverse terminal repeat) <400> 8 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct 130 <210> 9 <211> 304 <212> DNA <213> native sequence <220> <223> CMV (Cytomegalovirus) enhancer <400> 9 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300 catg 304 <210> 10 <211> 204 <212> DNA <213> native sequence <220> <223> CMV (cytomegalovirus) promoter <400> 10 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 60 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 180 tgggaggtct atataagcag agct 204 <210> 11 <211> 493 <212> DNA <213> native sequence <220> <223> Introns of the hBG1 gene (hemoglobin γ-1 subunit) <400> 11 cgaatcccgg ccgggaacgg tgcattggaa cgcggattcc ccgtgccaag agtgacgtaa 60 gtaccgccta tagagtctat aggcccacaa aaaatgcttt cttcttttaa tatacttttt 120 tgtttatctt atttctaata ctttccctaa tctctttctt tcagggcaat aatgatacaa 180 tgtatcatgc ctctttgcac cattctaaag aataacagtg ataatttctg ggttaaggca 240 atagcaatatttctgcatat aaatatttct gcatataaat tgtaactgat gtaagaggtt 300 tcatattgct aatagcagct acaatccagc taccattctg cttttatttt atggttggga 360 taaggctgga ttattctgag tccaagctag gcccttttgc taatcatgtt catacctctt 420 atcttcctcc cacagctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc 480 aaagaattgg gat 493 <210> 12 <211> 479 <212> DNA <213> native sequence <220> <223> hGH1 polyadenylation signaling (human growth hormone polyadenylation signaling (poly(A)))) <400> 12 acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc 60 cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt 120 ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga 180 caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt 240 ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt 300 tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac 360 ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac 420 cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtcctt 479 <210> 13 <211> 141 <212> DNA <213> Native sequence <220> <223> Right (second) ITR <400> 13 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120 gagcgcgcag ctgcctgcag g 141

Claims

1. A codon-optimized nucleic acid encoding the amino acid sequence of the SMN1 protein (survival protein of motor neurons) of SEQ ID NO: 1, said nucleic acid being composed of the nucleic acid sequence of SEQ ID NO:

2.

2. An expression cassette comprising the codon-optimized nucleic acid of claim 1.

3. The expression box according to claim 2, comprising the following elements in the direction from the 5' end to the 3' end: Left-side first ITR (reverse terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; Introns of the hBG1 gene (hemoglobin subunit γ1 gene); The codon-optimized nucleic acid as described in claim 1; hGH1 gene polyadenylation signal (human growth hormone 1 gene polyadenylation signal); The second ITR on the right.

4. The expression cassette of claim 3, comprising a nucleic acid having SEQ ID NO:

4.

5. An expression vector comprising the codon-optimized nucleic acid of claim 1 or the expression cassette of claims 2-4.

6. A recombinant virus based on AAV9 (adeno-associated virus serotype 9) for increasing the expression of the SMN1 gene in target cells, comprising a capsid and an expression cassette as described in any one of claims 2-4.

7. The AAV9-based recombinant virus of claim 6, wherein the capsid comprises the AAV9 protein VP1.

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. The AAV9-based recombinant virus of claim 6, wherein the capsid comprises the AAV9 protein VP1, the VP1 having the amino acid sequence of SEQ ID NO: 5, and the expression cassette includes the following elements in the 5'-to-3'-direction: CMV enhancer; CMV promoter; Introns of the hBG1 gene; The codon-optimized nucleic acid as described in claim 1; hGH1 polyadenylation signal; Right side ITR.

10. The AAV9-based recombinant virus of claim 6, wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5, and the expression cassette comprises a nucleic acid having SEQ ID NO:

4.

11. A pharmaceutical composition for delivering the SMN1 gene to target cells, comprising the AAV9-based recombinant virus of claims 6-10 in combination with one or more pharmaceutically acceptable excipients.

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