Codon-optimized nucleic acids encoding SMN1 proteins and uses thereof

Abstract

This application relates to the fields of genetics, gene therapy, and molecular biology. More specifically, the present invention relates to an isolated codon-optimized nucleic acid encoding the SMN1 protein (survival motor neuron protein), expression cassettes and vectors based thereon, and an AAV9 (adeno-associated virus serotype 9)-based recombinant virus 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 an isolated codon-optimized nucleic acid encoding the SMN1 protein (survival motor neuron protein), expression cassettes and vectors based thereon, and an AAV9 (adeno-associated virus serotype 9)-based recombinant virus for increasing the expression of the SMN1 gene in target cells, and uses thereof. [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 loss of the encoded SMN protein [Lefebvre et al., Cell (1995) 80:155-165]. SMN deficiency leads to motor neuron degeneration in the ventral (anterior) horn of the spinal cord, which leads to weakness of proximal muscles involved in crawling, walking, neck movement, and swallowing, as well as involuntary muscles controlling breathing and coughing [Sumner C.J. NeuroRx (2006) 3:235-245]. As a result, SMA patients are susceptible to other lung diseases, such as pneumonia and restrictive lung disease.

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

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

[0005] Adeno-associated virus (AAV) is a small (20 nm), non-enveloped virus that is replication-independently defective. Many different AAV serotypes have been described in humans and primates. The adeno-associated virus genome is composed of a single-stranded (+ or -) DNA (ssDNA) approximately 4700 nucleotides in length. The genomic DNA has inverted terminal repeats (ITRs) at its ends. 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, and three capsid proteins (VP1, VP2, and VP3), along with other alternative products, are encoded by the Cap gene. VP1, VP2, and VP3 exist in a ratio of 1:1:10 and form an icosahedral capsid [Xie Q. et al., The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002;99:10405-10410]. In the construction of recombinant AAV (rAAV) vectors, an expression cassette flanked by ITRs is packaged into the AAV capsid. 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 vector can infect cells of multiple tissue types, resulting in strong and persistent transgene expression. It is also non-pathogenic and has a low immunogenicity profile (High KA et al., "rAAV human trial", Methods Mol Biol. 2011;807:429-57).

[0006] One of the pressing research goals in the area of ​​developing effective gene therapy is to optimize the codons of the gene of interest in the vector to achieve maximum levels of expression of the gene of interest, thereby allowing for the use of lower doses of the vector to achieve significant efficacy.

[0007] One of the properties of the genetic code is degeneracy, the ability of different codons (trinucleotides) to encode the same amino acid. Such codons that translate to the same amino acid are called synonymous codons. In natural sequences, one of the synonymous codons is randomly selected during evolution, but the frequency of synonymous codon usage varies: each amino acid has a more favorable and a less favorable one. Codon optimization is a widely used technique to enhance the production of protein molecules and provides a rational mapping of one of the appropriate synonymous codons to each amino acid in a protein sequence. One of the common principles of codon optimization is to use the most frequent codon, while other methods such as harmonization (reproducing the codon usage distribution) were later introduced, but they do not necessarily increase productivity. In addition to codon frequency, the GC content of the sequence (ratio of guanine and cytosine to the total length of the sequence) may affect production efficiency, and it has been shown that a high GC content in mammalian cells is associated with increased mRNA levels (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 should further be noted that stable secondary structural elements of mRNA, i.e., secondary structural elements with low folding free energy, may decrease efficiency.

[0008] Different codon-optimized variants of the sequence of a gene of interest may result in (compared to the wild-type gene): a) the expression level of the gene of interest will be slightly increased; b) the expression level of the gene of interest will be significantly increased; c) the expression level of the gene of interest will remain at approximately the same level; d) The expression level of the gene of interest will be reduced.

[0009] Therefore, a codon-optimized sequence of the SMN1 gene is required to increase the expression of the SMN1 gene in target cells.

[0010] It was found that a codon-optimized sequence of SMN1 having the nucleotide sequence of SEQ ID NO: 2 [SMN1-GeneBeam (or abbreviated as SMN1-GB)] surprisingly increased the transcription of the SMN1 gene by more than three-fold, i.e., surprisingly increased the mRNA copy number of SMN1-GeneBeam by more than three-fold compared to SMN1-WT (wild type), resulting in a significant increase in the expression of the SMN1 gene and, accordingly, the SMN protein. Summary of the Invention [Means for solving the problem]

[0011] In one aspect, the present invention relates to an isolated codon-optimized nucleic acid encoding the SMN1 protein (survival motor neuron protein) of SEQ ID NO:1 and comprising the nucleic acid sequence of SEQ ID NO:2.

[0012] In one aspect, the present invention relates to an expression cassette comprising the above-described codon-optimized nucleic acid.

[0013] In some embodiments, the expression cassette comprises the following elements from the 5' to the 3' end: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; Introns of the hBG1 gene (hemoglobin subunit gamma 1 gene) the above codon-optimized nucleic acid of the SMN1 gene; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Right (second) ITR Includes.

[0014] In some embodiments, the expression cassette comprises a nucleic acid having the sequence of SEQ ID NO:4.

[0015] In one aspect, the present invention relates to an expression vector comprising the codon-optimized nucleic acid or the cassette.

[0016] In one aspect, the present invention relates to an AAV9 (adeno-associated virus serotype 9)-based recombinant virus for increasing the expression of the SMN1 gene in target cells, comprising a capsid and the above-described expression cassette.

[0017] In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP1.

[0018] 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.

[0019] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 containing one or more point mutations.

[0020] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:5 containing one or more point mutations, and the expression cassette comprises, from the 5' end to the 3' end, the following elements: CMV enhancer; CMV promoter; introns of the hBG1 gene; the above codon-optimized nucleic acid of the SMN1 gene; hGH1 polyadenylation signal; right ITR Includes.

[0021] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:5 containing one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO:4.

[0022] In one aspect, the present invention relates to a pharmaceutical composition for delivering the SMN1 gene to a target cell, the pharmaceutical composition comprising the above-described AAV9-based recombinant virus in combination with one or more pharmaceutically acceptable excipients.

[0023] In one aspect, the present invention relates to the use of the AAV9-based recombinant virus or the composition for delivering the SMN1 gene to target cells. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows SMN1 expression at the mRNA level after transfection. HEK293 cells and HSMCs were transfected with 5 μg of plasmids pAAV-SMN1-WT and pAAV-SMN1-GB (encoding the SMN1 gene without codon optimization and with codon optimization using the GeneBeam algorithm). 72 hours later, the copy number of the SMN1 gene in each sample was determined by quantitative PCR (n=3). The copy number of the GAPDH household gene was also determined. All obtained levels of SMN1 were normalized to 10,000 copies of the GAPDH gene in each sample. Data on the normalized mean copy numbers of SMN1-WT and SMN1-GB for both cell lines are provided, along with indications of standard deviations. The ratio of the normalized copy numbers of SMN1-GB to SMN1-WT in each line is also provided. [Figure 2]Figure 1 shows SMN1 expression at the protein level after transfection. HSMCs were transfected with 5 μg of plasmids pAAV-SMN1-WT and pAAV-SMN1-GB (encoding the SMN1 gene without codon optimization and with codon optimization by the GeneBeam algorithm). 72 hours later, cells in each sample were stained with a primary antibody against SMN1 protein and a secondary antibody labeled with Alexa Fluor 488 (n=3). The average intensity of the fluorescent signal for live cells in the sample after subtraction of the background signal obtained in cells stained with the secondary antibody without the primary antibody is shown, with the standard deviation indicated. [Figure 3] Figure 1 shows the ratio 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, and in each, the transduction efficiency was at least 50% relative to the control GFP-containing virus. SMN1 expression was determined at the mRNA and protein levels (see above), and then the ratio of SMN1-GB to SMN1-WT expression was calculated. The figure illustrates the average ratio with the standard deviation. Definitions and General Methods Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0025] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, the classification and methods of cell culture, molecular biology, immunology, bacteriology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and protein and nucleic acid hybridization and chemistry described herein are well known and widely used by those of skill in the art. Enzymatic reactions and purification methods are performed according to manufacturer's guidelines or as described herein, as is common to those of skill in the art.

[0026] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that occurs naturally in an animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a genetically modified cell.

[0027] The terms "naturally occurring," "native," or "wild-type" are used to describe something that can be found in nature, as opposed to something that has been artificially created. For example, a protein or nucleotide sequence that is present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by man in the laboratory is naturally occurring.

[0028] The term "genome" refers to the complete genetic material of an organism.

[0029] As used in this description and the claims that follow, unless the context dictates otherwise, the words "include" and "comprise," or variations thereof such as "having," "includes," "including," "comprises," or "comprising," will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Proteins (peptides) As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no restriction on the maximum number of amino acids that a protein or peptide sequence may contain. A polypeptide includes any peptide or protein containing two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to by those skilled in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to by those skilled in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. nucleic acid molecule The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", used interchangeably in this description, refer to the exact sequence of nucleotides, modified or not, that defines a fragment or region of a nucleic acid, that may or may not contain non-naturally occurring nucleotides, and that is either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA.

[0030] Those skilled in the art have the general understanding that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used in this description, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available to those skilled in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes, using conventional cloning techniques and PCR, etc., as well as synthetic means.

[0031] It should also be noted here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in their natural state. The sequences of the present invention are isolated and / or purified, i.e., sampled directly or indirectly, for example by copying, and their environment is at least partially modified. Thus, isolated nucleic acids obtained by means of recombinant genetics, e.g., host cells, or obtained by chemical synthesis, should also be mentioned here.

[0032] An "isolated" nucleic acid molecule is one that is identified and separated from at least one nucleic acid molecule impurity, with which the former is generally associated in the natural source of the nuclease nucleic acid. An isolated nucleic acid molecule differs from the form or set found under natural conditions. Thus, an isolated nucleic acid molecule differs from the nucleic acid molecule present in cells under natural conditions. However, an isolated nucleic acid molecule includes nucleic acid molecules located in cells where the nuclease is normally expressed, for example, when the nucleic acid molecule has a chromosomal location that differs from its location in cells under natural conditions.

[0033] Unless otherwise specified, the term nucleotide sequence includes its complement. Thus, a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.

[0034] The terms "transformation," "transfection," and "transduction" refer to any method or means by which nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, infection, PEG fusion, etc. Adeno-associated virus (AAV) Viruses in the Parvoviridae family are small DNA-containing animal viruses. The Parvoviridae family can be divided into two subfamilies: the Parvovirinae, whose members infect vertebrates, and the Densovirinae, whose members infect insects. As of 2006, 11 serotypes of adeno-associated viruses have been described [Mori, S., et al., 2004, "Two novel adeno-associated viruses from cynomolgus monkeys: pseudotyping characterization of capsid protein," Virology, Vol. 330(2):375-83]. All known serotypes can infect cells from multiple tissue types. Tissue specificity is determined by the serotype of the capsid protein; therefore, adeno-associated virus-based vectors are constructed by assigning the desired serotype. Further information about parvoviruses and other members of the Parvoviridae family can be found in the literature [Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 of Fields Virology (3rd ed. 1996)].

[0035] The genome organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for replicating nonstructural proteins (Rep) and structural proteins (Cap). The Cap gene encodes the VP proteins (VP1, VP2, and VP3) that form the capsid. The terminal 145 nucleotides are self-complementary and organized to allow the formation of an energetically stable intramolecular duplex that forms a T-shaped hairpin. Such a hairpin structure serves as an origin of replication for viral DNA and as a primer for the cellular DNA polymerase complex. After wild-type AAV (wtAAV) infection in mammalian cells, Rep genes (e.g., Rep78 and Rep52) are expressed using the P5 and P19 promoters, respectively, and both Rep proteins have specific functions in viral genome replication. Splicing events in the Rep open reading frame (Rep ORF) actually result in the expression of four Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that the unspliced ​​mRNAs encoding the Rep78 and Rep52 proteins are sufficient for the production of AAV vectors in mammalian cells. vector As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0036] The terms "infectious unit (iu)," "infectious particle," or "replication unit" as used in reference to viral titer refer to the number of infectious recombinant AAV vector particles as measured by the infectious center assay, also known as the replication center assay, e.g., as described in McLaughlin et al., J. Virol. (1988) 62:1963-1973.

[0037] The term "heterologous" with respect to nucleic acid sequences, such as coding sequences and regulatory sequences, refers to sequences that are not normally linked together and / or not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid that is within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct can include a coding sequence flanked by sequences that are not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from the native gene).

[0038] As used in this description, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter. use "Gene therapy" is the insertion of a gene into a subject's cells and / or tissues to treat a disease, typically a genetic disease, replacing a defective mutant allele with a functional gene.

[0039] "Treat," "treatment," and "therapy" refer to a method of alleviating or eliminating a biological disorder and / or at least one of its attendant symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative, symptomatic, and prophylactic treatment.

[0040] In one aspect, the subject or patient of treatment is a mammal, preferably a human subject. The subject can be either male or female of any age.

[0041] The term "disorder" means any condition that would benefit from treatment according to the present invention, including chronic and acute disorders or diseases, and includes pathological conditions that predispose a mammal to the disorder in question.

[0042] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis, and if the disease is not ameliorated, the animal's health will continue to deteriorate thereafter.

[0043] In this description, the terms "subject," "patient," "individual," etc. are used interchangeably and refer to any animal suitable for the methods described in this description. In certain non-limiting embodiments, the subject, patient, or individual is a human.

[0044] A "therapeutically effective amount" refers to that amount of a therapeutic agent administered during treatment that will relieve to some extent one or more symptoms of the disease being treated. DETAILED DESCRIPTION OF THE INVENTION

[0045] Codon-optimized nucleic acids In one aspect, the present invention relates to an isolated codon-optimized nucleic acid encoding the SMN1 protein (survival motor neuron protein) of SEQ ID NO:1 and comprising the nucleic acid sequence of SEQ ID NO:2.

[0046] The corresponding amino acid sequence of the SMN_HUMAN protein was used as the basis to create a codon-optimized SMN1 gene:

[0047] [ka]

[0048] This amino acid sequence of SEQ ID NO: 1 was translated into a nucleotide sequence by sequentially matching each amino acid starting from the N-terminus of one of the synonymous codons encoding the same amino acid.

[0049] Detailed information regarding the codon-optimized SMN1 gene and final sequence selection is provided in Example 1.

[0050] The final codon-optimized sequence of SMN1 (SMN1-GeneBeam) has the following nucleotide sequence:

[0051] [ka]

[0052] It has.

[0053] This codon-optimized final nucleotide sequence of SMN1 (SMN1-GeneBeam) is identical to the coding sequence of the wild-type SMN gene (SMN1-WT):

[0054] [ka]

[0055] The codon compatibility index (a standard measure for evaluating sequences relative to codon frequency) is increased compared to

[0056] The codon compatibility index of the final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) is 98% relative to the subject sequence and 75% relative to the wild-type sequence.

[0057] The GC content of the wild-type sequence was 45%, i.e., 15% different from the target value, and the GC content of the codon-optimized final nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) for the optimized sequence was 64%, i.e., 4% different from the target value.

[0058] The final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) and the nucleotide sequence of the wild-type SMN1 gene (SEQ ID NO: 3) are 71% identical. Expression cassette, expression vector In one aspect, the present invention relates to an expression cassette comprising the above-described codon-optimized nucleic acid.

[0059] As used herein, the term "expression cassette" specifically refers to a DNA fragment that, under appropriate settings, can induce the expression of the polynucleotide encoding the polypeptide of interest contained in the expression cassette.When introduced into a host cell, the expression cassette is particularly capable of involving the cellular machinery for transcribing the polynucleotide encoding the polypeptide of interest into RNA, which is then generally further processed and ultimately translated into the polypeptide of interest.The expression cassette may be contained in an expression vector.

[0060] The expression cassette of the present invention includes a promoter as an element. As used herein, the term "promoter" specifically refers to a DNA element that promotes transcription of a polynucleotide to which it is operably linked. A promoter may also form part of a promoter / enhancer element. While the physical boundary between a "promoter" and an "enhancer" element 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 transcription is initiated. Enhancers temporally and spatially enhance promoter activity. Many promoters are known to those skilled in the art to be transcriptionally active in a variety of cell types. Promoters can be divided into two classes: those that function constitutively and those that are regulated by induction or derepression. Both classes are suitable for protein expression. Promoters used for high-level production of polypeptides in eukaryotic cells, particularly mammalian cells, should be strong, and preferably active, in a variety of cell types. Strong constitutive promoters capable of driving expression in many cell types are well known to those skilled in the art, and therefore need not be described in detail herein. According to the concept of the present invention, it is preferred to use a cytomegalovirus (CMV) promoter. A promoter or promoter / enhancer obtained from the immediate-early (IE) region of human cytomegalovirus (hCMV) is particularly suitable as a promoter in the expression cassette of the present invention. The immediate-early (IE) region of human cytomegalovirus (hCMV) and functional expression-inducing and / or expression-enhancing fragments obtained therefrom are described, for example, in European Patent Nos. 0173177 and 0323997, and are well known to those skilled in the art. Thus, several fragments of the immediate-early (IE) region of hCMV can be used as a promoter and / or promoter / enhancer. According to one embodiment of the present invention, a human CMV promoter is used in the expression cassette of the present invention.

[0061] In some embodiments, the expression cassette comprises the following elements from the 5' to the 3' end: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; Introns of the hBG1 gene (hemoglobin subunit gamma 1 gene) the above codon-optimized nucleic acid of the SMN1 gene; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Right (second) ITR Includes.

[0062] In some embodiments, the left (first) ITR (inverted terminal repeat) has the following nucleic acid sequence:

[0063] [ka]

[0064] It has.

[0065] In some embodiments, the CMV (cytomegalovirus) enhancer comprises the following nucleic acid sequence:

[0066] [ka]

[0067] It has.

[0068] In some embodiments, the CMV (cytomegalovirus) promoter comprises the following nucleic acid sequence:

[0069] [ka]

[0070] It has.

[0071] In some embodiments, the intron of the hBG1 (hemoglobin subunit gamma 1) gene has the following nucleic acid sequence:

[0072] [ka]

[0073] It has.

[0074] In some embodiments, the hGH1 (human growth hormone 1 gene) polyadenylation signal comprises the following nucleic acid sequence:

[0075] [ka]

[0076] It has.

[0077] In some embodiments, the right (second) ITR has the following nucleic acid sequence:

[0078] [ka]

[0079] It has.

[0080] In some embodiments, the expression cassette comprises the following nucleic acid sequence:

[0081] [ka]

[0082] It has.

[0083] In one aspect, the present invention relates to an expression vector comprising the codon-optimized nucleic acid or the expression cassette.

[0084] In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated.

[0085] In some embodiments, the vector is a viral vector and additional DNA segments may be ligated into the viral genome.

[0086] In some embodiments, vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In further embodiments, the vector (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genes. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0087] Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. DNA molecules can be ligated into vectors such that transcriptional and translational control sequences within the vector perform their intended function of regulating DNA transcription and translation. Expression vectors and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules can be introduced into expression vectors by standard methods (e.g., ligation of complementary restriction sites, or blunt-end ligation if no restriction sites are present).

[0088] The recombinant expression vector can encode a signal peptide that facilitates secretion of the protein of interest from the host cell. The gene for the protein of interest can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the protein of interest. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0089] In addition to the SMN1-GB gene of the present invention, recombinant expression vectors of the present invention can carry regulatory sequences that control 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 choice of host cell to be transformed, the level of expression of the desired protein, and other factors. Preferred regulatory sequences for mammalian expression host cells include viral elements that ensure high-level protein expression in mammalian cells, such as promoters and / or enhancers from retroviral long-term repeats (LTRs), cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the major late promoter adenovirus (AdMLP)), and polyoma virus, as well as strong mammalian promoters such as the native immunoglobulin promoter or actin promoter.

[0090] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0091] The term "promoter" or "transcriptional regulatory sequence" or "regulatory sequence" as used in this description refers to a nucleic acid fragment that controls the transcription of one or more coding sequences, is located upstream in the direction of reading compared to the direction of transcription from the transcription 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 sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, as well as any other nucleotide sequences known to those skilled in the art that directly or indirectly regulate the level of transcription by the promoter. A "constitutive" promoter is a promoter that is active in most tissues under typical 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 only active in specific types of tissues or cells.

[0092] As used herein, the term "enhancers" or "enhancer" can refer to a DNA sequence located adjacent to a DNA sequence that encodes a recombinant product. Enhancer elements are generally located 5' from a promoter element, or can be located downstream of or within a coding DNA sequence (e.g., a DNA sequence that is transcribed or translated into a recombinant product or products). Thus, an enhancer element can be located 100, 200, or 300 or more base pairs upstream of or downstream of a DNA sequence that encodes a recombinant product. An enhancer element can increase the amount of recombinant product expressed from a DNA sequence above the expression level associated with a single promoter element. Multiple enhancer elements are readily available to those of skill in the art.

[0093] In addition to the above-mentioned genes and regulatory sequences, the recombinant expression vector of the present invention may carry additional sequences, such as sequences regulating replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. Selectable marker genes facilitate selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, selectable marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate on the host cells into which the vector has been introduced. Examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells during methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0094] As used herein, the term "expression control sequence" refers to a polynucleotide sequence necessary to achieve the expression and processing of a ligated coding sequence. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include a ribosome binding site, promoter, and transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and transcription termination sequence. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0095] As used herein, the term "operably linked" refers to the linking of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. The term "operably linked" means that the DNA sequences being linked are generally contiguous, and, where necessary to join two protein-coding regions, also contiguous and in reading frame.

[0096] In one embodiment of the present invention, an "expression vector" relates to a vector comprising one or more polynucleotide sequences of interest, a gene of interest, or a "transgene" flanked by parvoviral sequences or inverted terminal repeat (ITR) sequences.

[0097] None of the cassettes or vectors of the present invention contain nucleotide sequences of genes encoding the nonstructural (Rep) and structural (Cap) proteins of adeno-associated virus. AAV9 (adeno-associated virus serotype 9)-based recombinant virus In one aspect, the present invention relates to an AAV9 (adeno-associated virus serotype 9)-based recombinant virus for increasing the expression of the SMN1 gene in target cells, comprising a capsid and the above-described expression cassette.

[0098] As used in this description, the term "AAV-based recombinant virus" (or "AAV-based virus-like particle," or "AAV recombinant virus strain," or "AAV recombinant vector," or "rAAV vector") refers to the expression cassette (or the expression vector) encapsulated in an AAV capsid.

[0099] The Cap gene encodes three capsid proteins (VP1, VP2, and VP3), among other alternative products. VP1, VP2, and VP3 exist in a 1:1:10 ratio to form an icosahedral capsid [Xie Q. et al., The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002;99:10405-10410]. Transcription of these genes is initiated by 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. After transcription, the precursor mRNA can be spliced ​​in two different ways, resulting in the excision of either longer or shorter introns, resulting in the formation of mRNAs of various nucleotide lengths.

[0100] In the generation of recombinant AAV (rAAV)-based viruses, an expression cassette flanked by ITRs is packaged into an AAV capsid. Genes required for AAV replication are not included in the cassette, as described above.

[0101] The expression cassette DNA is packaged into the viral capsid in the form of a single-stranded DNA molecule (ssDNA) approximately 3000 nucleotides in length. When a cell is infected with the virus, the single-stranded DNA is converted into a double-stranded DNA (dsDNA) form. The dsDNA can only be used by cellular proteins to transcribe the present gene or genes into RNA.

[0102] In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP1.

[0103] In some embodiments, the AAV9-based recombinant virus comprises the following amino acid sequence:

[0104] [ka]

[0105] It has a capsid containing the AAV9 protein VP1.

[0106] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 with one or more point mutations.

[0107] In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP2.

[0108] In some embodiments, the AAV9-based recombinant virus comprises the following amino acid sequence:

[0109] [ka]

[0110] The AAV9 protein VP2 has a capsid containing the VP2 protein.

[0111] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP2 having the amino acid sequence of SEQ ID NO:6 containing one or more point mutations.

[0112] In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP3.

[0113] In some embodiments, the AAV9-based recombinant virus comprises the following amino acid sequence:

[0114] [ka]

[0115] It has a capsid containing the AAV9 protein VP3.

[0116] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP3 having the amino acid sequence of SEQ ID NO:7 containing one or more point mutations.

[0117] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 proteins VP1, VP2 and VP3.

[0118] In some embodiments, the AAV9-based recombinant virus has a capsid comprising a VP1 protein having the amino acid sequence of SEQ ID NO:5, a VP2 protein having the amino acid sequence of SEQ ID NO:6, and a VP3 protein having the amino acid sequence of SEQ ID NO:7.

[0119] In some embodiments, the AAV9-based recombinant virus has a capsid comprising a VP1 protein of the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, a VP2 protein of the amino acid sequence of SEQ ID NO: 6 with one or more point mutations, and a VP3 protein of the amino acid sequence of SEQ ID NO: 7 with one or more point mutations.

[0120] The phrase "more point mutations" refers to 2, 3, 4, 5, 6, 7, 8, 9 or 10 point substitutions.

[0121] Particularly preferred embodiments include naturally conservative substitutions (mutations), i.e., substitutions that occur within a family of amino acids connected at the side chain.In particular, amino acids are generally divided 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 reasonably predictable that the single substitution of leucine for isoleucine or valine, aspartic acid for glutamic acid, and threonine for serine, or similar conservative substitutions of amino acids for structurally related amino acids, will not have a significant effect on biological activity. For example, a subject polypeptide can contain up to about 5-10 conservative or non-conservative amino acid substitutions, as long as the desired function of the molecule remains intact.

[0122] The embodiment of point mutation within the sequence of the AAV9 protein VP1, VP2 or VP3 using amino acid substitution is the substitution of at least one amino acid residue within the AAV9 protein VP1, VP2 or VP3 by another amino acid residue.

[0123] Conservative substitutions are shown in Table A under "preferred substitutions."

[0124] [Table 1]

[0125] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:5 containing one or more point mutations, and the expression cassette comprises, from the 5' end to the 3' end, the following elements: CMV enhancer; CMV promoter; introns of the hBG1 gene; the above codon-optimized nucleic acid of the SMN1 gene; hGH1 polyadenylation signal; right ITR Includes.

[0126] In some embodiments, the AAV9-based recombinant virus has a capsid comprising a VP1 protein having the amino acid sequence of SEQ ID NO:5, a VP2 protein having the amino acid sequence of SEQ ID NO:6, and a VP3 protein having the amino acid sequence of SEQ ID NO:7, and the expression cassette comprises, from the 5' end to the 3' end, the following elements: CMV enhancer; CMV promoter; introns of the hBG1 gene; the above codon-optimized nucleic acid of the SMN1 gene; hGH1 polyadenylation signal; right ITR Includes.

[0127] In some embodiments, the AAV9-based recombinant virus has a capsid comprising a VP1 protein of the amino acid sequence of SEQ ID NO:5 with one or more point mutations, a VP2 protein of the amino acid sequence of SEQ ID NO:6 with one or more point mutations, and a VP3 protein of the amino acid sequence of SEQ ID NO:7 with one or more point mutations, and the expression cassette comprises, from the 5' end to the 3' end, the following elements: CMV enhancer; CMV promoter; introns of the hBG1 gene; the above codon-optimized nucleic acid of the SMN1 gene; hGH1 polyadenylation signal; right ITR Includes.

[0128] In some embodiments, the AAV9-based recombinant virus has a capsid comprising the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:5 containing one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO:4.

[0129] In some embodiments, the AAV9-based recombinant virus has a capsid comprising a VP1 protein having the amino acid sequence of SEQ ID NO:5, a VP2 protein having the amino acid sequence of SEQ ID NO:6, and a VP3 protein having the amino acid sequence of SEQ ID NO:7, and the expression cassette comprises the nucleic acid of SEQ ID NO:4.

[0130] In some embodiments, the AAV9-based recombinant virus has a capsid comprising a VP1 protein of the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, a VP2 protein of the amino acid sequence of SEQ ID NO: 6 with one or more point mutations, and a VP3 protein of the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO: 4. Pharmaceutical Composition In one aspect, the present invention relates to a pharmaceutical composition for delivering the SMN1 gene to a target cell, the pharmaceutical composition comprising the AAV9-based recombinant virus described above in combination with one or more pharmaceutically acceptable excipients.

[0131] In certain embodiments, the present invention relates to pharmaceutical compositions comprising the AAV9-based recombinant viruses of the present invention in a pharmaceutically acceptable carrier or other medicinal agent, adjuvant, diluent, etc. For injection, the carrier will generally be a liquid carrier. For other modes of administration, the carrier can be either a solid or a liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline. For inhalation administration, the carrier is respirable and preferably in solid or liquid granular form. As the injection medium, it is preferred to use water containing stabilizers, additives common to injection solutions, such as salts or saline, and / or buffers.

[0132] The term "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 group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, auxiliary agents, dispersing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, and long-term delivery control agents, the selection and proportion of which will depend on the type and route of administration and dosage. The pharmaceutical composition of the present invention and methods for its preparation will be readily apparent to those skilled in the art. Pharmaceutical compositions should preferably be manufactured in accordance with Good Manufacturing Practice (GMP) requirements. The composition may also contain a buffer composition, an isotonicity agent, a stabilizer, and a solubilizer.

[0133] "Pharmaceutically acceptable" means a material that is free of biological or other negative side effects, e.g., the material can be administered to a subject without causing any undesired biological effects. Thus, such pharmaceutical compositions can be used, for example, for transfection of cells ex vivo or for direct administration of the AAV9-based recombinant viruses of the invention to a subject in vivo.

[0134] The term "excipient" is used herein to describe any other component of the present invention. These are inorganic or organic natural substances used in pharmaceutical manufacturing to obtain the necessary physicochemical properties for the formulation.

[0135] "Stabilizer" refers to an excipient or mixture of two or more excipients that provides physical and / or chemical stability to an active agent.

[0136] The terms "buffer," "buffer composition," and "buffering agent" refer to a solution capable of resisting pH changes due to the action of acid-base conjugate components, allowing the rAAV5 vector product to resist pH changes. Generally, pharmaceutical compositions preferably have a pH of 4.0 to 8.0. Examples of buffers that can be used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, and the like.

[0137] A pharmaceutical composition is "stable" if the active agent retains its physical and / or chemical stability and / or biological activity at a storage temperature, e.g., 2-8°C, for a specified shelf life. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The shelf life is adjusted based on the results of stability testing under accelerated or natural degradation conditions.

[0138] The pharmaceutical composition of the present invention can be manufactured, packaged, or widely sold in the form of a ready-to-use preparation in the form of a single unit dose or a plurality of single unit doses.As used herein, the term "single unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dose of active ingredient administered to a subject, or a convenient fraction of such a dose, for example, half or one-third of such a dose. use In one aspect, the present invention relates to the use of the above-described AAV9-based recombinant virus or the above-described composition for delivering the SMN1 gene to target cells.

[0139] Any method of administering an AAV9-based recombinant virus is recognized by those of skill in the art and may be suitably used for the above-described AAV9-based recombinant viruses of the present invention.

[0140] AAV9-based recombinant virus is preferably administered to cells in a biologically effective amount. A "biologically effective" amount of recombinant virus is an amount sufficient to cause infection (or transduction) and expression of a heterologous nucleic acid sequence in cells. When a virus is administered to cells in vivo (for example, when a virus is administered to a subject, as described below), a "biologically effective" amount of a viral vector is an amount sufficient to cause transduction and expression of a heterologous nucleic acid sequence in target cells.

[0141] The cells for administering the AAV9-based recombinant viruses of the present invention can be any type of cell, including, but not limited to, neural 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, cardiac myocytes, 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 for administering the AAV9-based recombinant viruses can be any progenitor cell. Alternatively, the cells can be stem cells (e.g., neural stem cells, hepatic stem cells). Furthermore, the cells can be of any species origin, as defined above.

[0142] The AAV9-based recombinant viruses are not used to modify the genetic integrity of human germline cells. [Example]

[0143] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0144] All publications, patents, and patent applications cited herein are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the accompanying embodiments. Materials and general methods recombinant DNA technology DNA manipulations were performed according to standard techniques described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. Briefly, plasmid DNA was prepared and further manipulated in Escherichia coli (E. coli) cells grown under antibiotic selection pressure to prevent the plasmid from being lost in the cell population. Plasmid DNA was isolated from the cells using a commercially available kit, the concentration was measured, and it was used for cloning by restriction enzyme digestion or PCR amplification. DNA fragments were ligated together using ligase and transformed into bacterial cells, and clones were selected and further manipulated. All of the resulting genetic constructs were confirmed by restriction patterns and complete Sanger sequencing. Gene synthesis The desired gene segments were prepared from chemically synthesized oligonucleotides. Gene segments of 300-1000 bp in length flanked by unique restriction sites were obtained by renaturing the oligonucleotides on top of each other and then PCR-amplified using flanking primers. The resulting mixture of fragments contained the desired fragments. The fragments were cloned into intermediate vectors at the restriction sites, and the DNA sequences of the subcloned fragments were then confirmed by DNA sequencing. DNA sequencing DNA sequences were determined by Sanger sequencing. DNA and protein sequences were analyzed and sequence data were processed with SnapGene Viewer 4.2 or later for sequence generation, mapping, analysis, annotation and illustration. Cell culture incubation Experiments were performed using HEK293 (human embryonic kidney clone 293) and HSMC (human skeletal muscle cell) cell lines. Cells were cultured under standard conditions at 37°C and 5% CO2 in DMEM complete culture medium supplemented with 10% FBS and antibiotics. To culture HSMCs, culture plastic was pre-coated with collagen (Gibco). Cells were passaged when they reached 80-90% confluence. Cell viability was assessed using either trypan blue staining and a hemocytometer or PI staining and flow cytometry. Cell transfection Cell lines were seeded into 6-well plates the day before transfection to reach 70-80% confluence at the time of transfection. Transfection was performed using a commercially available lipofection kit according to the manufacturer's protocol. After 72 hours, cells were treated with trypsin solution or similar, removed from the substrate, washed in phosphate buffer, and harvested for further analysis of target gene and protein expression. For each transfection, a control plasmid expressing GFP was used to control transfection efficiency (percentage of GFP-positive cells). Further analysis was performed only if the transfection efficiency was at least 50%.

[0145] All measurements were performed in three independent experiments. Gene expression analysis SMN1 expression at the mRNA level was assessed by quantitative PCR. Briefly, primers and samples specific for wild-type SMN1 sequence or GeneBeam were used. Primers and samples specific for the GAPDH housekeeping gene were used to control initial RNA levels. Calibration curves were plotted for each set of primers and sample using linearized plasmid DNA of known copy numbers containing the amplified sequence of the corresponding gene. Expression was analyzed by determining the copy numbers of SMN1-GeneBeam, SMN1-WT, and GAPDH in each sample using the calibration curve, and then normalizing the number of SMN1 copies per 10,000 copies of GAPDH. The obtained values ​​were compared for different samples within the same experiment. Determination of SMN1 protein expression by flow cytometry SMN1 protein content in cells was assessed by intracellular staining followed by flow cytometry analysis. Briefly, cells were removed from culture plates using TrypLE, washed in PBS, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, incubated in blocking buffer supplemented with 1-5% BSA, and stained in two steps using a primary antibody against SMN1 and a secondary antibody labeled with Alexa Fluor 488. After staining, cells were washed once in PBS and analyzed by flow cytometry. The average signal intensity was assessed after subtracting the signal from secondary antibody staining without the addition of primary antibody. Assembly and purification of recombinant AAV vector viral particles To assemble AAV particles containing the SMN1 gene or the GFP control gene, HEK293 packaging cells were used and transfected with the following three plasmids: a plasmid containing the AAV genome containing a transgene (SNM1 or GFP) expression cassette; Plasmids for expression of the AAV9 serotype Cap gene and the AAV2 serotype Rep gene, each encoding several protein products using alternative reading frames; A plasmid for the expression of Ad5 (adenovirus serotype 5) genes required for AAV capsid assembly and packaging.

[0146] After 72 hours, cells were lysed, and viral particles were purified and concentrated using filtration and chromatography methods. The titer of viral particles was determined by quantitative PCR using primers specific to the site of recombinant viral genome and sample, and expressed as the number of viral genome copies per mL. Cell culture transduction Cell lines were inoculated as in the transfection experiments, then viral particle-containing products were added and cells were analyzed 72 hours later. Transduction efficiency was estimated by measuring the percentage of GFP+ cells.

[0147] The cultures used were pre-tested for transduction efficiency. Briefly, the AAV9-GFP virus product was transduced into cell lines at different cell-to-viral particle ratios. The ratio of the number of viral particles to the number of cells was referred to as the multiplicity of infection (MOI). The MOI of the AAV9-GFP virus ranged from 50,000 to 1,000,000. Consequently, an MOI range was determined for each line, within which the transduction efficiency varied linearly with the MOI. Further transductions of cell lines were performed within that linear range.

[0148] After transduction, gene and protein expression was analyzed as described above.

[0149] All measurements were performed in three independent experiments.

[0150] Example 1 Methods for creating a codon-optimized SMN1 gene The corresponding amino acid sequence of the SMN_HUMAN protein (SEQ ID NO: 1) was used as the basis for creating a codon-optimized SMN1 gene.

[0151] This 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 the same amino acid, and has the following characteristics: 1) Frequency of codon usage (Yasukazu Nakamura et al., Codon usage tabulated from the international DNA sequence database; its status 1999, Nucleic Acid Research, 1999, Vol. 27, No. 1, doi:10.1093 / nar / 27.1.292); 2) GC content in the terminal regions of the obtained nucleotide sequence (according to the paper 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, the target value for GC content is 60%, and the smaller the difference between the current GC content and that of the target, the more preferred the codon is); 3) The free energy of folding of the terminal regions 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) was taken into consideration, either alone or in combination.

[0152] The construction process further avoided the generation of semantic nucleotide sequences such as restriction sites, internal ribosome entry sites and splicing sites.

[0153] The amino acid sequence of SEQ ID NO: 1 was translated into nucleotide sequences, resulting in an array of codon-optimized nucleotide sequences of the SMN1 gene.

[0154] Some sequences in the above array of codon-optimized nucleotide sequences of SMN1 did not show increased transcription of the SMN1 gene in further studies, i.e., there was no significant increase in mRNA copy number of SMN1-opt compared to SMN1-WT in any of the cell lines used, or this increase was not significant.

[0155] Most of the codon-optimized nucleotide sequences of the SMN1 gene showed a 1.5- to 2-fold increase in SMN1 gene transcription in further studies, i.e., significantly increased the mRNA copy number of SMN1-opt compared to SMN1-WT in all cell lines used.

[0156] In further studies, it was surprisingly found that one sequence from the above array of codon-optimized nucleotide sequences of the SMN1 gene showed a greater than three-fold increase in SMN1 gene transcription, i.e., surprisingly, it increased the mRNA copy number of SMN1-opt compared to SMN1-WT by more than three-fold in all cell lines used (see Examples 3 and 4). This final codon-optimized nucleotide sequence of the SMN1 gene is conventionally referred to as SMN1-GeneBeam (or abbreviated as SMN1-GB).

[0157] The final codon-optimized sequence of SMN1 (SMN1-GeneBeam) has the nucleotide sequence represented by SEQ ID NO:2.

[0158] This codon-optimized final 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 of SEQ ID NO: 3) (Paul M. Sharp et al., The codon adaptation index—a measure of directional synonymous codon usage 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 for evaluating a sequence for codon usage frequencies).

[0159] The codon compatibility index of the final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) is 98%, compared to 75% for the wild-type sequence.

[0160] The GC content of the wild-type sequence was 45%, i.e., 15% different from the target value, and that of the codon-optimized final nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) was 64%, i.e., 4% different from the target value.

[0161] The final codon-optimized nucleotide sequence of the SMN1 gene (SEQ ID NO: 2) and the nucleotide sequence of the wild-type SMN1 gene (SEQ ID NO: 3) are 71% identical.

[0162] Example 2 Assembly of a genetic construct carrying the recombinant AAV genome and encoding the SMN1 gene.

[0163] The wild-type SMN1 gene sequence was generated by amplification using cDNA synthesized from total RNA from HEK293 cells and specific primers. During the amplification process, a Kozak sequence and a ClaI restriction site were added to the 5' end of the gene, and an XbaI restriction site was added to the 3' end. The SMN1 gene sequence was then cloned into the commercially available pAAV-GFP control plasmid (VPK-402) from CellBiolab (USA) at the ClaI and XbaI sites by restriction ligase method, replacing the GFP gene with SMN1, thereby generating the pAAV-SMN1-WT construct.

[0164] The SMN1-GeneBeam sequence was assembled as described above. Given the complex sequence despite its relatively small size, serial subcloning of the gene fragment was performed within the intermediate vector pGEMT, and the sequence was verified for each vector. A full-length version of the gene was then assembled from several intermediate vectors by PCR and cloned into the intermediate vector pGEMT. The construct pAAV-SMN1-WT, in which wild-type SMN1 was replaced with SMN1-GeneBeam at ClaI and XbaI sites added to the ends of the SMN1-GeneBeam sequence by PCR, was used as the final genetic construct.

[0165] The final vector contains the elements necessary for gene expression and assembly as part of the recombinant AAV genome: 1) ITRs at the ends of the sequences encapsidated within the viral capsid; 2) elements for expression of the target gene (promoter, enhancer, intron, Kozak sequence, transgene, polyadenylation site); 3) A bacterial replication origin and antibiotic resistance gene for producing plasmid DNA within bacterial cells Contains everything.

[0166] It is important to note that the genetic constructs containing the SMN1-WT and SMN1-GeneBeam genes differ only in the SMN1 gene sequence and are otherwise completely identical.

[0167] Example 3 Confirmation of SMN1 expression from genetic constructs The genetic constructs pAAV-GFP, pAAV-SMN1-WT, and pAAV-SMN1-GB were transfected into HEK293 cells and HSMCs as described above. 5 μg of DNA was used per well. After 72 hours, cells were harvested and SMN1 expression (normalized to GAPDH) was analyzed as described above.

[0168] We found that codon optimization of the SMN1 gene affected SMN1 transcription, indeed increasing the mRNA copy number of SMN1-GB several-fold compared with SMN1-WT in both cell lines used (Fig. 1). Notably, the normalized expression ratio of SMN1-GB to SMN1-WT was 3.9 in HEK293 cells and 12.8 in HSMCs.

[0169] The data obtained demonstrate that this property of SMN1-GeneBeam is not cell-specific and increases target gene expression several-fold in cells, which may be an important advantage in the development of gene therapy drugs. Furthermore, because this analysis was performed on genetic constructs that were completely identical except for the codon optimization of the SMN1 gene, this property is not due to any differences in the gene expression cassette or the properties of the appropriate viral capsid carrying the SMN1-GeneBeam gene-derived genome.

[0170] HSMCs were selected and SMN1 expression at the protein level was confirmed by flow cytometry as described above. Using 5 μg of DNA per well, the signal of an SMN1-specific antibody in cells transfected with pAAV-SMN1-GB was 12.2-fold higher than in cells transfected with pAAV-SMN1-WT (Figure 2). This observation suggests that although SMN1-GB does not have a translational advantage, increased transcription further increases the final protein level in the cells.

[0171] Example 4 Creation of a viral product expressing SMN1 Plasmids pAAV-SMN1-WT and pAAV-SMN1-GB, as well as other plasmids required for generating recombinant AAV viral particles (see above), were used in the bioprocess for AAV production. The serotypes used were the wild-type AAV9 serotype or those with one or more point mutations.

[0172] In all cases, the properties of wild-type SMN1 and SMN1-GeneBeam were compared only when the serotype and capsid mutation, if any, used were identical. All serotypes based on AAV9, whether wild-type or mutant, are hereafter referred to as AAV9 without specifying the mutation.

[0173] The recombinant viral particles produced in the bioprocess were designated AAV9-SMN1-WT and AAV9-SMN1-GB, as well as the control particle AAV9-GFP. After determining the titer of the viral particles, all three products were used to transduce permissive cells, i.e., primary human muscle cells (HSMCs), at the same MOI (MOI values ​​varied between 50,000 and 200,000 between experiments). Further analysis was performed only if the transduction efficiency was at least 50%.

[0174] After successful transduction, cells were removed from the substrate and washed in phosphate buffer, and SMN1 expression was analyzed at the mRNA and protein levels as described above. The increased transcriptional activity of SMN1-GeneBeam was shown to remain consistent; thus, SMN1-GeneBeam mRNA was detected to be 7.3-fold higher than wild-type SMN1. A similar increase was also observed at the protein level (6.8-fold) (Figure 3), indicating that the advantage of SMN1-GB does not exist at the translational level. However, the detectable increase in transcription efficiency using the AAV9-SMN1-GB product provides higher levels of SMN1 expression in target cells, which could be an important advantage, for example, in the treatment of spinal muscular atrophy, where the level of SMN1 protein expression defines the disease stage from 0 (embryonic lethal) to 4 (no special treatment required). The present invention includes, but is not limited to, the following aspects. [Aspect 1] A codon-optimized nucleic acid encoding the SMN1 protein (survival motor neuron protein) of SEQ ID NO:1 and comprising the nucleic acid sequence of SEQ ID NO:2. [Aspect 2] An expression cassette comprising the codon-optimized nucleic acid of embodiment 1. [Aspect 3] From the 5' to the 3' end, the following elements: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; Introns of the hBG1 gene (hemoglobin subunit gamma 1 gene) a codon-optimized nucleic acid according to embodiment 1; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Right (second) ITR 3. The expression cassette of embodiment 2, comprising: [Aspect 4] 4. The expression cassette of embodiment 3, comprising the nucleic acid of SEQ ID NO:4. [Aspect 5] An expression vector comprising the codon-optimized nucleic acid according to embodiment 1 or the cassette according to embodiments 2 to 4. [Aspect 6] A recombinant virus based on AAV9 (adeno-associated virus serotype 9) for increasing expression of the SMN1 gene in a target cell, comprising a capsid and the expression cassette according to any one of aspects 2 to 4. [Aspect 7] 7. The AAV9-based recombinant virus of embodiment 6, wherein the capsid comprises the AAV9 protein VP1. [Aspect 8] 8. The AAV9-based recombinant virus of embodiment 7, wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5. [Aspect 9] 8. The AAV9-based recombinant virus of embodiment 7, wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5, comprising one or more point mutations. [Aspect 10] the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:5 containing one or more point mutations, and the expression cassette comprises, from the 5' to the 3' end, the following elements: CMV enhancer; CMV promoter; introns of the hBG1 gene; a codon-optimized nucleic acid according to embodiment 1; hGH1 polyadenylation signal; right ITR 10. The AAV9-based recombinant virus according to any one of embodiments 6 to 9, comprising: [Aspect 11] 7. The AAV9-based recombinant virus of embodiment 6, wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:5 containing one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO:4. [Aspect 12] A pharmaceutical composition for delivering the SMN1 gene to a target cell, comprising an AAV9-based recombinant virus according to any one of aspects 6 to 11 in combination with one or more pharmaceutically acceptable excipients. [Aspect 13] Use of an AAV9-based recombinant virus according to any one of embodiments 6 to 11 or a composition according to embodiment 12 for delivering the SMN1 gene to a target cell.

Claims

1. A codon-optimized nucleic acid encoding the SMN1 protein (survival motor neuron protein) of SEQ ID NO:1 and consisting of the nucleic acid sequence of SEQ ID NO:

2.

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

3. From the 5' to the 3' end, the following elements: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; Intron of the hBG1 gene (hemoglobin subunit gamma 1 gene) The codon-optimized nucleic acid of claim 1; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Right (second) ITR 3. The expression cassette of claim 2, comprising:

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

4.

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

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

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

8. 8. The AAV9-based recombinant virus of claim 7, wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:

5.

9. 8. The AAV9-based recombinant virus of claim 7, wherein the capsid comprises AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5 containing one or more point mutations.

10. the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:5 containing one or more point mutations, and the expression cassette comprises, from the 5' to the 3' end, the following elements: CMV enhancer; CMV promoter; introns of the hBG1 gene; The codon-optimized nucleic acid of claim 1; hGH1 polyadenylation signal; Right ITR 10. The AAV9-based recombinant virus according to claims 6 to 9, comprising:

11. 7. The AAV9-based recombinant virus of claim 6, wherein the capsid comprises AAV9 protein VP1 having the amino acid sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:5 containing one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO:

4.

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

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