AAV-based gene therapeutic agent for treating GNE myopathy

Abstract

The present application relates to next-generation biopharmaceutical gene therapy technology, specifically an AAV-based gene therapeutic agent for treating GNE myopathy. The present application provides an isolated nucleic acid having 75% to 99% sequence identity with the nucleotide sequence of SEQ ID NO: 2 and a GC-content ranging from 50% to 60% based on the entire nucleotide sequence, an expression cassette comprising the isolated nucleic acid, a recombinant virus based on adeno-associated virus serotype 9 comprising the expression cassette, and a pharmaceutical composition for preventing or treating GNE myopathy comprising the recombinant virus as an active ingredient.

Description

AAV-based gene therapy for the treatment of GNE myopathy

[0001] This application relates to gene therapy technology, a next-generation biopharmaceutical, and more specifically, to an AAV-based gene therapy for the treatment of GNE muscle disease. This application claims the benefit of Korean Patent Application No. 10-2024-0038584, filed March 20, 2024, and Korean Patent Application No. 10-2024-0163342, filed November 15, 2024, the disclosures of which are incorporated herein by reference.

[0002] GNE myopathy (GNEM) is a rare, intractable, inherited muscle disease caused by mutations in the GNE gene. It causes muscle degeneration, beginning below the knee and extending throughout the body. In patients with GNE myopathy, decreased GNE enzyme activity, leading to decreased sialic acid synthesis, is typically observed, and this mechanism is believed to be a major cause of muscle loss. GNE myopathy typically develops in patients in their 20s and 40s, with the first symptoms manifesting as weakness in the anterior tibialis anterior, making it difficult to lift the ankle, dragging the toes, and an abnormal gait. As the disease progresses, body stability decreases, leading to frequent falls, difficulty running, climbing stairs, and rising from a seated position. With the exception of the quadriceps femoris, which is relatively less affected among the limb muscles, upper limb muscles also deteriorate, causing significant inconvenience in daily life. GNE myopathy progresses over decades, and as the disease progresses, the neck muscles are also affected, causing difficulty in movement and ultimately leading to complete loss of skeletal muscle mass in the upper and lower extremities.

[0003] The GNE protein is an enzyme protein called UDP-N-acetylglucosamine-2-epimerase / N-acetylmannosamine kinase, which is directly involved in the biosynthesis of sialic acid. The GNE protein consists of 753 amino acids and contains two functional domains: the Glucosamine (UDP-N-Acetyl)-2-Epimerase domain (the region from the N-terminus to amino acid position 410) and the N-Acetylmannosamine Kinase domain (the region from amino acid position 411 to the C-terminus). Mutations in the GNE gene can occur anywhere in the DNA sequence encoding the two functional domains mentioned above, and when the GNE enzyme function is reduced or lost due to mutations, it has been reported that the symptoms of GNE myopathy appear due to a decrease in the sialylation level of the glycoprotein expressed on the muscle cell surface.

[0004] Currently, there is no cure for GNE myopathy, and conservative treatment focuses on managing symptoms. While early diagnosis and regular physical therapy and management can slow the progression of GNE myopathy, they do not fundamentally address the muscle wasting caused by continuous muscle use, and their effectiveness in improving patients' quality of life is limited. Therefore, methods such as providing N-acetylmannosamine (ManNAc), an intermediate in the sialic acid biosynthesis pathway, and using gene therapy to induce sustained expression of the normal GNE gene are being attempted, but these are still inadequate.

[0005] Against this backdrop, the present invention was completed based on a novel genetic construct with improved GNE expression efficiency, as a result of extensive efforts to develop an adeno-associated virus vector (AAV)-based gene therapy for treating GNE myopathy.

[0006] As a result, the isolated nucleic acid for treating GNE myopathy, the expression cassette comprising the same, the recombinant virus comprising the same, or the pharmaceutical composition comprising the same, manufactured according to the present invention, can be utilized in the field of next-generation biopharmaceuticals, and can be an innovative new drug as it is a gene therapy agent that has never been used before in relation to GNE myopathy.

[0007] One aspect is to provide an isolated nucleic acid engineered to enhance the expression level of a GNE protein.

[0008] Another aspect is to provide an expression cassette comprising the isolated nucleic acid.

[0009] Another aspect is to provide a recombinant virus based on adeno-associated virus serotype 9 comprising the above expression cassette.

[0010] Another aspect is to provide a pharmaceutical composition for preventing or treating GNE myopathy comprising a vector comprising the isolated nucleic acid.

[0011]

[0012] Other purposes and advantages of this application will be further clarified by the detailed description below, taken in conjunction with the appended claims and drawings. Any details not described herein will be readily apparent and inferred by those skilled in the technical field of this application or similar technical fields, and therefore, their description will be omitted.

[0013] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0014]

[0015] One aspect is to provide an isolated nucleic acid having a sequence identity of 75% to 99% with the nucleotide sequence of SEQ ID NO: 2 and a GC-content in the range of 50 to 60% based on the entire nucleotide sequence.

[0016] As used herein, the term "nucleic acid" refers to a polymeric substance comprising a plurality of nucleotide units, specifically, a polymer in which a plurality of nucleotide units are linked to each other by phosphodiester bonds of a sugar / phosphate backbone. The nucleic acid may be used interchangeably with the terms "polynucleotide" and "nucleic acid molecule." The nucleic acid is a biopolymer essential to living organisms, and may be RNA or DNA, preferably DNA, that encodes genetic information through a unique base sequence. The nucleic acid may be isolated, artificially synthesized, or non-naturally occurring or engineered, wherein "non-naturally occurring or engineered" means a state that is not in its natural state but is produced by artificial modification. Here, the artificial modification is for improving the expression of a GNE protein and may include a coding sequence of a codon-optimized GNE protein.

[0017] As used herein, the term "isolated" nucleic acid (e.g., "isolated DNA") means a polynucleotide that is at least partially separated from other components of a naturally occurring organism or virus, e.g., cellular or viral structural components or other polypeptides or at least some of the nucleic acids with which it is normally found associated with the polynucleotide.

[0018] As used herein, the term "codon optimized" refers to a gene or coding region of a nucleic acid molecule for transformation of various hosts, and refers to altering codons in the gene or coding region of the nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization involves replacing at least one, or more than one, or a significant number of codons with one or more codons that are more frequently used in the genes of that organism.

[0019] As used herein, the term "GNE (glucosamine (UDP-N-acetyl)-2-epimerase / N-acetylmannosamine kinase) protein" is an enzyme involved in the biosynthesis of sialic acid, which is responsible for two enzymatic reactions: from UDP-GlcNAc to ManNAc and from ManNAc to ManNAc6-phosphate. Accordingly, mutations in the GNE gene result in decreased sialic acid production, leading to decreased sialylation of various glycoproteins, including important muscle proteins such as alpha-dystroglycan (α-DG), neural cell adhesion molecule (NCAM) or neprilysin, or to decreased expression of other genes such as ganglioside (e.g., GM3) synthetase, resulting in muscle degenerative changes.

[0020] In one specific example, the GNE protein may be composed of an amino acid sequence of SEQ ID NO: 1, and the wild-type GNE gene encoding the GNE protein may be composed of a nucleotide sequence of SEQ ID NO: 2. The isolated nucleic acid according to one aspect may have a sequence identity of 75% to 99% with the nucleotide sequence of SEQ ID NO: 2 and a GC-content of 50 to 60% based on the entire nucleotide sequence, for enhancing expression of the GNE protein, for example, expression of a target protein under an AAV9-based expression construct.

[0021] As used herein, the term "identity" refers to the overall relatedness between polymer molecules, for example, between nucleic acids (e.g., DNA molecules and / or RNA molecules) and / or between polypeptides. For example, polypeptides are considered to be "substantially identical" to one another if their amino acid sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculating the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). For example, the length of the aligned sequences for comparison purposes is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleic acid or polypeptide sequences at corresponding positions are then compared. Determination of the percent identity between the two sequences and the comparison of the sequences can be accomplished using mathematical algorithms. As is well known to those skilled in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms available in commercial computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSIBLAST for amino acid sequences.

[0022] In one specific embodiment, the isolated nucleic acid may have a sequence identity of 75% to 99% with the nucleotide sequence of SEQ ID NO: 2, for example, the isolated nucleic acid may have a sequence identity of 75 to 99%, 75 to 95%, 75 to 90%, 75 to 85%, 75 to 80%, 80 to 99%, 80 to 95%, 80 to 90%, 80 to 85%, 75 to 80%, 85 to 99%, 85 to 95%, 85 to 90%, or 90 to 95% with the nucleotide sequence of SEQ ID NO: 2.

[0023] In this specification, the term "GC-content" refers to the ratio of guanine (G) to cytosine (C) among the total bases of the target region, and it is known that the higher the GC-content, the higher the DNA density and the less easily it is denatured.

[0024] In one specific embodiment, the isolated nucleic acid may have a GC-content in the range of 50 to 60% based on the entire nucleotide sequence, for example, the GC-content of the isolated nucleic acid may be 50 to 60%, 50 to 58%, 50 to 56%, 50 to 54%, 50 to 52%, 52 to 60%, 52 to 58%, 52 to 56%, 52 to 54%, 54 to 60%, 54 to 58%, 54 to 56%, 56 to 60%, 56 to 58%, or 58 to 60%.

[0025] In one specific example, the isolated nucleic acid may satisfy the following conditions: (a) encoding a GNE protein represented by the amino acid sequence of SEQ ID NO: 1; (b) having a sequence identity of 75 to 80% with the nucleotide sequence of SEQ ID NO: 2; and (c) having a GC content of 55 to 58% based on the entire nucleotide sequence. Specifically, the isolated nucleic acid may be any one selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3, the nucleotide sequence of SEQ ID NO: 5, and the nucleotide sequence of SEQ ID NO: 6.

[0026] In one embodiment, the isolated nucleic acid of the present invention is modified based on the wild-type GNE nucleotide sequence, and when the isolated nucleic acid is applied as a transgene (transgene) in an expression cassette, as illustrated in FIG. 1, the expression level of the GNE protein can be significantly increased.

[0027]

[0028] Another aspect is to provide an expression cassette comprising the isolated nucleic acid.

[0029] Since the above expression cassette contains or utilizes the isolated nucleic acid described above, the common content between them is omitted to avoid excessive complexity of this specification.

[0030] As used herein, the term "expression cassette" refers to a nucleic acid construct containing the elements necessary for expressing a target protein. Specifically, it refers to a nucleic acid construct comprising an expression control sequence including at least a promoter, and a nucleic acid for expressing the target protein operably linked to the expression control sequence.

[0031] As used herein, the term "expression control sequence" refers to a nucleic acid sequence essential for the expression of a coding sequence for a target protein to which it is operably linked in a particular host organism. For example, suitable control sequences for eukaryotic cells include promoters, polyadenylation signals, and enhancers.

[0032] As used herein, the term "promoter" refers to a nucleic acid fragment that regulates the transcription of one or more coding sequences and is located upstream in the reading direction from the transcription start site of the coding sequence, which is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other nucleic acid sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art that directly or indirectly regulate the transcription level of the promoter. Examples of such promoters include any promoter that functions in mammalian cells, and examples thereof include constitutive expression promoters such as the CMV promoter, the SV40 promoter, the EF-1α promoter, the CAG promoter, the PGK promoter, the U3 promoter, the U6 promoter, the H1 promoter, and the like. In addition to these promoters, known inducible promoters, tissue / organ-specific promoters, period-specific promoters, or mutant sequences having functional equivalents thereto may be used in the present invention.

[0033] As used herein, the term "enhancer" may refer to a nucleic acid sequence located adjacent to a sequence encoding a protein of interest. An enhancer element is typically located 5' from a promoter element, or may be located downstream or within the coding nucleic acid sequence (e.g., a DNA sequence that is transcribed or translated into a recombinant product or products). That is, an enhancer element may be located 100 bp (base pairs), 200 bp, or 300 bp upstream or downstream from a nucleic acid sequence encoding a protein of interest, or a greater distance.

[0034] In one specific embodiment, the expression cassette is for enhancing the expression of a target protein under an AAV9-based expression construct, and may comprise or be operably linked to the following elements: a first inverted terminal repeat (ITR); a cytomegalovirus enhancer; a cytomegalovirus promotor; an isolated nucleic acid having at least 75% to 99% sequence identity with the nucleotide sequence of SEQ ID NO: 2 and having a GC-content in the range of 50 to 60% based on the entire nucleotide sequence; a human growth hormone gene 1 polyadenylation signal; and a second inverted terminal repeat (ITR).

[0035] As used herein, the term "operably linked" may refer to nucleotide sequences being linked on a single nucleic acid fragment such that one function is affected by the other.

[0036]

[0037] Another aspect is to provide a recombinant virus based on Adeno-associated virus serotype 9 (AAV9) comprising the above expression cassette.

[0038] Since the recombinant virus based on the above adeno-associated virus serotype 9 contains or utilizes the isolated nucleic acid or expression cassette described above, the common content between them is omitted to avoid excessive complexity of this specification.

[0039] As used herein, the term "adeno-associated virus (AAV)" is a small (26 nm), non-enveloped parvovirus with a single-stranded genome of approximately 4.7 kb, which has been attracting attention as a gene delivery vehicle for disease treatment due to its high transduction efficiency, sustained transgene expression, and low pathogenicity after infection. However, since adeno-associated virus is also a viral vector, it has the problem that systemic administration of high doses can cause serious side effects, as with other viral vectors. The serotype of the adeno-associated virus is mainly determined by the structure of the AAV capsid protein, and since different capsid protein structures recognize different cell surface receptors, the selection of the serotype can affect the infection efficiency, tissue tropism, and expression initiation time of AAV. The serotypes of the above adeno-associated viruses include, for example, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 AAV8, AAV9, AAV10, AAV11, and AAV12.

[0040] As used herein, the term "adeno-associated virus serotype 9-based recombinant virus" refers to the aforementioned expression cassette enclosed within an AAV capsid, and may be used interchangeably with the terms "AAV9-based virus-like particle", "AAV9 recombinant virus strain", "AAV9 recombinant vector", or "rAAV9 vector".

[0041] In one embodiment, the recombinant virus of the present invention comprises an isolated nucleic acid modified based on a wild-type GNE nucleotide sequence, or an expression cassette comprising the nucleic acid. The recombinant virus can be introduced into cells or administered into a subject to enhance the expression efficiency of the GNE protein, thereby enhancing intramuscular sialic acid production. Furthermore, the recombinant virus can significantly enhance the expression level of the GNE protein, thereby reducing the effective dosage for treating the target disease, thereby contributing to alleviating side effects associated with the use of the viral vector.

[0042]

[0043] Another aspect is to provide a pharmaceutical composition for preventing or treating GNE myopathy (GNEM), comprising as an active ingredient a vector comprising any one isolated nucleic acid selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3, the nucleotide sequence of SEQ ID NO: 5, and the nucleotide sequence of SEQ ID NO: 6, and a medicinal use of the pharmaceutical composition for preventing or treating GNE myopathy.

[0044] Since the pharmaceutical composition for preventing or treating the above GNE muscle disease contains or utilizes the isolated nucleic acid or expression cassette described above, the description of common contents between them is omitted to avoid excessive complexity of the present specification.

[0045] As used herein, the term "effective ingredient" means an ingredient in an amount that is effective enough to produce a beneficial or desirable clinical or biochemical result. Specifically, it may refer to an effective amount of a preparation, an active agent, or a recombinant virus. The effective amount may be administered once or more and may be an amount that is effective enough to prevent a disease, or to improve a disease state, including but not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing the disease state (i.e., not worsening), delaying or slowing the progression of the disease, or improving or temporarily alleviating and alleviating (partially or completely) the disease state.

[0046] As used herein, the term "prevention" refers to any action that prevents the onset of a disease, suppresses the disease, or delays its progression. For example, it refers to preventing or interfering with the onset of obesity or its characteristic features, or protecting against or protecting against the onset of GNE myopathy or its characteristic features.

[0047] As used herein, the term "treatment" refers to both therapeutic treatment and preventative or prophylactic measures. It also refers to any action that improves or beneficially alters the symptoms of a disease. For example, it refers to preventing, reducing, or ameliorating the GNE myopathy or its characteristic features, or delaying (attenuating) the progression of the disease or its characteristic features in a subject.

[0048] As used herein, the term "effective amount" refers to its generally accepted meaning in the art. The term may generally refer to the amount of a molecule, compound, or composition that will elicit a desired biological response (e.g., a beneficial response) in a cell, tissue, system, animal, or human, as sought by a researcher, veterinarian, physician, or other clinician. Specifically, a "therapeutically effective amount" may refer to an amount of a molecule, compound, or composition that elicits a desired medical response, such as a therapeutically relevant change in a measurable parameter associated with a disease or disorder, such that a particular clinical treatment can be considered efficacious. A therapeutically effective amount of a drug for treating the disease or disorder may be the amount necessary to effect a therapeutically relevant change in the parameter.

[0049] "GNE myopathy", a disease to be prevented or treated by the above pharmaceutical composition, is an autosomal recessive disease caused by a mutation in the GNE gene that produces an enzyme known as UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase (UDP-GlcNAc 2-Epimerase / ManNAc Kinase: GNE), and is also known by the terms "distal myopathy," "Nonaka myopathy," "vacuolar myopathy sparing the quadriceps femoris," "inclusion body myopathy type 2 (IBM2 or HIBM2)," or "GNE myopathy." Currently, there is no cure for GNE myopathy, and conservative treatment of symptoms is focused on, so a multifaceted approach is needed to treat the disease.

[0050] In one embodiment, the present invention relates to a pharmaceutical composition comprising an AAV9-based recombinant virus comprising the isolated nucleic acid of the present invention in a pharmaceutically acceptable carrier or other pharmaceutical agent, adjuvant, diluent, etc. For injection or systemic administration, the carrier will typically be a liquid carrier. For other administration methods, the carrier can be a solid or liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline solution. As the injection medium, it is preferred to use water containing additives common to injectable solutions, such as stabilizers, salts, or saline and / or buffers.

[0051] The above pharmaceutical composition means a composition comprising the above AAV9-based recombinant virus of the present invention and one or more ingredients selected from the group consisting of pharmaceutically acceptable and pharmacologically appropriate excipients, such as fillers, solvents, diluents, carriers, adjuvants, disintegrants, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, and long-term delivery modifiers, the selection and proportion of which are determined according to the type and route of administration and the dosage. The pharmaceutical composition of the present invention and the method for preparing the same will be without doubt apparent to those skilled in the art. The pharmaceutical composition should preferably be prepared in accordance with Good Manufacturing Practice (GMP) requirements. The composition may also include a buffer composition, a tonicity agent, a stabilizer, and a solubilizer.

[0052] The pharmaceutical composition may be manufactured, packaged, or sold in the form of a ready-to-use formulation, in the form of a single unit dose or multiple single unit doses. As used herein, the term "single unit dose" refers to a discrete quantity of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient typically corresponds to a dosage of the active ingredient to be administered to a subject, or a convenient portion of such a dosage, such as one-half or one-third of the dosage.

[0053]

[0054] One aspect provides a method for preventing or treating GNE myopathy comprising administering the pharmaceutical composition to a subject.

[0055] Since the method for preventing or treating the above GNE muscle disease includes or utilizes the pharmaceutical composition described above, the common content between them is omitted to avoid excessive complexity of this specification.

[0056] As used herein, the term "subject" means a subject in need of treatment for a disease, specifically GNE myopathy, and more specifically, may include any mammal, such as a human or non-human primate, mouse, dog, cat, horse, cow, sheep, pig, goat, camel, or antelope.

[0057] According to an aspect of the invention, an isolated nucleic acid, and an expression cassette or an AAV9-based recombinant virus comprising the isolated nucleic acid, can induce high levels of expression of GNE protein in an administered or introduced subject.

[0058] In addition, the AAV9-based recombinant virus according to the aspect can reduce the effective dosage as an effect resulting from the excellent GNE protein expression efficiency, and thus can reduce the side effects resulting from the application of the viral vector, and thus can contribute to improving the efficacy of gene therapy for treating GNE muscle disease.

[0059] FIG. 1 is a diagram showing the structure of an expression cassette comprising a wild-type GNE nucleotide sequence (GNE wt) according to one embodiment.

[0060] FIG. 2 is a diagram showing the structure of an expression cassette comprising a GNE nucleotide sequence (GNE mod1) according to one embodiment.

[0061] FIG. 3 is a diagram showing the structure of an expression cassette comprising a GNE nucleotide sequence (GNE mod2) according to one embodiment.

[0062] FIG. 4 is a diagram showing the structure of an expression cassette comprising a GNE nucleotide sequence (GNE mod3) according to one embodiment.

[0063] FIG. 5 is a diagram showing the structure of an expression cassette comprising a GNE nucleotide sequence (GNE mod4) according to one embodiment.

[0064] Figure 6 shows the results of confirming the expression level of GNE protein through Western blot after transfecting HEK293T cells with a recombinant plasmid vector according to one embodiment.

[0065] Figure 7 shows the results of confirming the expression level of GNE protein through Western blot after transfecting C2C12 cells with a recombinant plasmid vector according to one embodiment.

[0066] Figure 8 shows the results of Western blot analysis of the expression level of GNE protein after transducing HEK293T cells with a recombinant AAV9 vector according to one embodiment.

[0067] Figure 9 shows the results of Western blot analysis of the expression level of GNE protein after transducing C2C12 cells with a recombinant AAV9 vector according to one embodiment.

[0068] Figure 10 shows the results of Western blot analysis to confirm whether the expression of GNE protein was restored after transducing a recombinant AAV9 vector according to one embodiment into an mGne knock down C2C12 cell line.

[0069] Figure 11 shows whether the sialic acid level was recovered after transducing a recombinant AAV9 vector into an mGne knock down C2C12 cell line according to one embodiment. Figure 11A shows the results of flow cytometry analysis on Neu5AC expressing cells (SNA-FITC), and Figure 11B shows the results of quantitatively comparing the results of the flow cytometry analysis.

[0070] FIG. 12 shows the results of confirming whether the sialic acid level was restored after transducing a recombinant AAV9 vector into an mGne knock down C2C12 cell line according to one embodiment. FIG. 12A shows the results of flow cytometry analysis on galactose expressing cells (PNA-FITC), and FIG. 12B shows the results of quantitatively comparing the results of the flow cytometry analysis.

[0071] Figure 13 shows the results of confirming the expression level of Neu5AC in muscle tissue through SNA staining in a mouse model in which a recombinant AAV9 vector containing a GNE nucleotide sequence was administered at different dosages.

[0072] Figure 14 shows the results of confirming the expression level of Galactose in muscle tissue through PNA staining in a mouse model in which a recombinant AAV9 vector containing a GNE nucleotide sequence was administered at different dosages.

[0073] Figure 15 shows the results of reconfirming the expression level of Neu5AC in muscle tissue through SNA staining in a mouse model administered with different dosages of a recombinant AAV9 vector containing a GNE nucleotide sequence.

[0074] Figure 16 shows the results of reconfirming the expression level of Galactose in muscle tissue through PNA staining in a mouse model administered with different dosages of a recombinant AAV9 vector containing a GNE nucleotide sequence.

[0075] Figure 17 shows the results of confirming the expression level of GNE mRNA in muscle tissue through qRT-PCR in a group (Gne KI Mock) that was administered Mock AAV to a normal mouse (WT) and a disease mouse model according to one embodiment. Figure 17A shows the results comparing the expression levels of the entire experimental group and the control group, and Figure 17B shows the results comparing the expression levels of the control group and the negative control group.

[0076] Figure 18 shows the results of Western blot (detection antibody: #NBP1-81621) to confirm the expression level of GNE protein in muscle tissue in a mouse model administered with different dosages of a recombinant AAV9 vector containing a GNE nucleotide sequence.

[0077] Figure 19 shows the results of Western blot (detection antibody: #25079-1-ap) to confirm the expression level of GNE protein in muscle tissue in a mouse model administered with different dosages of a recombinant AAV9 vector containing a GNE nucleotide sequence.

[0078] Figure 20 shows the results of a treadmill test performed on a mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment.

[0079] Figure 21 shows the results of a treadmill test performed on a mouse model administered a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment, comparing the results before and after administration.

[0080] Figure 22 shows the results of a grip strength test performed on a mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment.

[0081] Figure 23 shows the results of a grip strength test performed on a mouse model administered a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment, comparing the results before and after administration.

[0082] Figure 24 shows the results of confirming the expression level of Neu5AC in muscle tissue through SNA staining in a mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment.

[0083] Figure 25 shows the results of confirming the expression level of Galactose in muscle tissue through PNA staining in a mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment.

[0084] Figure 26 shows the results of confirming the expression level of GNE protein in muscle tissue through Western blot (detection antibody: #25079-1-ap) in a mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment.

[0085] Figure 27 shows the results of comparing the expression levels of GNE protein in muscle tissue in a mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment.

[0086] Figure 28 shows the results of confirming the change in body weight for 8 weeks after administration to a mouse model administered a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment.

[0087] Figure 29 shows the results of a treadmill test performed on a mouse model administered with a recombinant AAV9 vector containing the GNE nucleotide sequence of Example 3.

[0088] Figure 30 shows the results of confirming the change in body weight for 4 weeks after administration to a mouse model administered with a recombinant AAV9 vector containing the GNE nucleotide sequence of Example 3.

[0089] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0090]

[0091] Experimental Example 1. Construction and Comparison of Expression Cassettes for Expression of GNE Proteins

[0092] 1-1. Construction of an expression cassette containing a codon-optimized nucleotide sequence

[0093] In this experimental example, an expression cassette for the expression of the GNE protein was prepared. To this end, as illustrated in FIGS. 1 to 5, each expression cassette was prepared by modifying the nucleotide sequence of the GNE gene in a basic framework comprising, from the 5' end to the 3' end, an AAV2 5' inverted terminal repeat (ITR), a cytomegalovirus (CMV) enhancer, a CMV promoter, a GNE gene (transgene), a human growth hormone gene 1 polyadenylation signal (hGH1 polyadenylation signal), and an AAV2 3' ITR. The GNE gene is a nucleotide encoding a GNE protein consisting of an amino acid sequence of sequence number 1. The wild-type GNE nucleotide sequence (transcript variant 2, NM_005476.7, 2169 bp) and a total of four codon-optimized GNE nucleotide sequences (GNE mod1, GNE mod2, GNE mod3, GNE mod4) were applied. Information on each GNE nucleotide sequence in this experimental example is as shown in Table 1 below.

[0094] [Table 1]

[0095]

[0096]

[0097]

[0098]

[0099] 1-2. Comparison of GNE nucleotide sequences

[0100] In this experimental example, the nucleotide sequences of the GNE gene included in the expression cassette produced in Experimental Example 1-1 were specifically compared, and the results are as shown in Table 2 below.

[0101] [Table 2]

[0102]

[0103]

[0104] As shown in Table 2 above, the codon-optimized nucleotide sequences (GNE mod1, GNE mod2, GNE mod3, GNE mod4) according to one embodiment were confirmed to exhibit sequence similarity / identity of about 75-77% compared to the wild-type GNE, and GC-content (%) in the range of 48.32 to 57.91%.

[0105]

[0106] Experimental Example 2. Evaluation of GNE protein expression levels using recombinant plasmid vectors.

[0107] In this experimental example, the recombinant plasmid vector having the expression cassette of Experimental Example 1 inserted was transfected into HEK293T cells and C2C12 cells, respectively, and the expression level of the GNE protein was evaluated. Specifically, HEK293T cells were seeded at 5.0E+05 cells / 6-well, and C2C12 cells were seeded at 1.25E+05 cells / 6-well, and then 2.5 μg of four types of recombinant plasmid vectors (Example 1 (GNE mod1), Example 2 (GNE mod2), Example 3 (GNE mod3), Example 4 (GNE mod4)) containing the next-day codon-optimized GNE nucleotide sequence were transfected into the cells, respectively. After culturing the transfected cells for 48 hours, proteins were extracted from the cells and GNE protein expression was confirmed through Western blotting. Meanwhile, a group that did not undergo transfection was used as a negative control group, and a group into which a recombinant plasmid vector with a wild-type GNE nucleotide sequence was introduced was used as a comparison group.

[0108] As a result, as shown in Table 3, Table 4, Figures 6 and 7, it was confirmed that the group (Examples 1 to 4) into which the recombinant plasmid vector including the GNE nucleotide sequence according to one embodiment was introduced had a significantly increased expression level of the GNE protein compared to the control group and the comparative group.

[0109] [Table 3]

[0110]

[0111] [Table 4]

[0112]

[0113]

[0114] Experimental Example 3. Evaluation of GNE protein expression levels using recombinant AAV9 vectors.

[0115] In this experimental example, the recombinant AAV9 vector into which the expression cassette of Experimental Example 1 was inserted was transduced into HEK293T cells and C2C12 cells, respectively, and the expression level of the GNE protein was evaluated. Specifically, HEK293T cells were seeded at 5.0E+05 cells / 6-well, and then transduced into the cells at 5.0E+05 VG (vector genome) / cell level with four recombinant AAV9 vectors (Example 1 (GNE mod1), Example 2 (GNE mod2), Example 3 (GNE mod3), Example 4 (GNE mod4)) containing the next-day codon-optimized GNE nucleotide sequence. After 24 hours of transduction, the medium was replaced, and the transduced cells were cultured for an additional 24 hours. In addition, C2C12 cells were seeded at 6.25E+04 cells / 6-well, and then transduced into the cells at 1.0E+03 pfu / cell with four recombinant AAV9 vectors (Example 1 (GNE mod1), Example 2 (GNE mod2), Example 3 (GNE mod3), Example 4 (GNE mod4)) was transduced into cells at a level of 1.0E+06 VG (vector genome) / cell. After 6 hours of transduction, the medium was replaced, and the transduced cells were cultured for an additional 66 hours. Thereafter, proteins were extracted from the HEK293T and C2C12 cells, and GNE protein expression was confirmed through Western blotting. Meanwhile, a group that was not transduced was used as a negative control group, and a group into which a recombinant AAV9 vector with a wild-type GNE nucleotide sequence was introduced was used as a comparison group.

[0116] As a result, as shown in Table 5, Table 6, Figures 8 and 9, it was confirmed that the GNE protein expression level of the group (Examples 1 to 4) introduced with the recombinant AAV9 vector including the GNE nucleotide sequence according to one embodiment was similar to or enhanced compared to the control group. In particular, among the groups introduced with the recombinant AAV9 vector according to one embodiment, the GNE protein expression enhancement effect in Examples 1, 3 and 4, which had a relatively high GC content (%) compared to the wild type, was remarkable.

[0117] [Table 5]

[0118]

[0119] [Table 6]

[0120]

[0121]

[0122] Experimental Example 4. Evaluation of the Restoration Effect of GNE Expression Using a GNE Knockdown Cell Line

[0123] In this experimental example, to evaluate the restoration effect of GNE expression, a C2C12 (mouse, myoblast) cell line in which mGne expression was knocked down was used. The mGne knockdown C2C12 cell line was established using a lentivirus containing mGne shRNA inserted into C2C12 cells. Then, two recombinant AAV9 vectors (Example 3 (GNE mod3), Example 4 (GNE mod4)) containing codon-optimized GNE nucleotide sequences were transduced into the cells at a level of 1E+6 VG (vector genome) / cell, respectively. Meanwhile, as a control group, a C2C12 cell line in which mGne was not knocked down (C2C12 NC), as a negative control group, a group in which no transduction was performed on the mGne knock down C2C12 cell line (C2C12 shGne #1 NC), and as a comparison group, a group in which a recombinant AAV9 vector with a wild-type GNE nucleotide sequence applied to the mGne knock down C2C12 cell line was introduced (C2C12 shGne #1 AAV-GNE (wt)) were used.

[0124]

[0125] 4-1. Evaluation of GNE protein expression levels

[0126] In the same manner as in Experimental Example 3 above, proteins were extracted from the mGne knock down C2C12 cell line and GNE protein expression was confirmed through Western blot.

[0127] As a result, as shown in Table 7 and Fig. 10, the mGne knock down C2C12 cell line in this experimental example showed a significantly reduced GNE expression level compared to the control group, indicating that the Gne knock down C2C12 cell line was stably established. In addition, regarding this reduction in GNE expression level, the control group was not recovered / restored despite the introduction of a recombinant AAV9 vector including a GNE nucleotide sequence, whereas in the group introduced with a recombinant AAV9 vector including a GNE nucleotide sequence according to one embodiment (Examples 3 and 4), it was confirmed that the GNE expression level was not only recovered but also increased above the expression level of the control group.

[0128] [Table 7]

[0129]

[0130]

[0131] 4-2. Sialic acid level assessment

[0132] For each sample, SNA staining was performed for sialic acid (N-Acetylneuraminic acid: Neu5AC) and PNA staining was performed for galactose. Subsequently, flow cytometry (FACS) was performed to quantitatively evaluate the levels of Neu5AC-expressing cells (SNA-FITC) and galactose-expressing cells (PNA-FITC).

[0133] As a result, as shown in Fig. 11, in the mGne knock down C2C12 cell line of this experimental example, the level of Neu5AC expressing cells decreased, whereas in the group introduced with the recombinant AAV9 vector including the GNE nucleotide sequence (Examples 3 and 4) according to one embodiment, the level of Neu5AC expressing cells was confirmed to have recovered to a level similar to that of the control group. In addition, as shown in Fig. 12, in the mGne knock down C2C12 cell line of this experimental example, the level of Galactose expressing cells was significantly increased, whereas in the group introduced with the AAV9 vector according to one embodiment, the level was confirmed to have recovered to a level similar to that of the control group, as above.

[0134]

[0135] Experimental Example 5. Evaluation of the Restoration Effect of GNE Expression by Administration of Recombinant AAV9 Vector Using a Disease Mouse Model

[0136] In this experimental example, a disease mouse model (GNE C44S) to confirm the restoration of GNE expression by administration of recombinant AAV9 vector. For this purpose, 12 or 13-week-old C44S knock-in mice were selected as the disease mouse model, and recombinant AAV9 vectors applied with wild-type GNE nucleotide sequences at different administration doses, i.e., Gne KI LD (low dose) hGNE AAV, Gne KI MD (mid dose) hGNE AAV, and Gne KI HD (high dose) hGNE AAV, were administered to the disease mouse model. After 4 weeks from the date of administration, muscle tissues were obtained from the disease mouse model, and the expression level of GNE or related factors in the tissues was evaluated. Meanwhile, in this experimental example, normal mice (WT) were used as the control group, and the group administered Mock AAV to the disease mouse model (Gne KI Mock AAV) was used as the negative control group. The mouse models and administration conditions in this experimental example are as shown in Table 8 below.

[0137] [Table 8]

[0138]

[0139]

[0140] 5-1. Sialic acid level assessment

[0141] Muscle tissues were obtained from a disease mouse model administered a recombinant AAV9 vector containing the wild-type GNE nucleotide sequence. The muscle tissues were quickly frozen in isopentane, sectioned, and prepared into slides, which were air-dried at room temperature for 20 minutes. Acetone fixation was performed at -20°C for 10 minutes, and after washing with PBS, each sample was stained with SNA for Neu5AC (N-Acetylneuraminic acid) and PNA for galactose. After washing three times with PBS for 3 minutes each time, the expression levels of Neu5AC and galactose were evaluated.

[0142] As a result, as shown in Fig. 13, in the mouse models administered with the recombinant AAV9 vector (Gne KI LD hGNE AAV, Gne KI MD hGNE AAV, Gne KI HD hGNE AAV), it was confirmed that the reduced expression level of Neu5AC caused by the lack of the GNE gene was recovered in the tibialis anterior muscle of the mouse model. In addition, as shown in Fig. 14, in the mouse models administered with the recombinant AAV9 vector (Gne KI LD hGNE AAV, Gne KI MD hGNE AAV, Gne KI HD hGNE AAV), it was confirmed that the increased expression level of Galactose caused by the lack of the GNE gene was recovered to the control level in the tibialis anterior muscle of the mouse model. In particular, this recovery level showed a tendency to be dependent on the administered dose.

[0143] Additionally, 4 weeks after the administration of recombinant AAV9 vectors with wild-type GNE nucleotide sequences at different doses to the disease mouse model, the gastrocnemius muscle of the mouse model was re-evaluated for sialic acid levels by performing the aforementioned SNA staining and PNA staining.

[0144] As a result, as shown in FIGS. 15 and 16, it was confirmed that the reduced expression level of Neu5AC resulting from the lack of the GNE gene was restored by administration of the recombinant AAV9 vector according to one embodiment, and the increased expression level of Galactose was also restored to the control level by administration of the recombinant AAV9 vector according to one embodiment.

[0145] That is, as shown in Figures 13 to 16, recovery of expression levels was confirmed in different muscles, and recovery of sialic acid levels was confirmed throughout the muscles of the mouse model.

[0146]

[0147] 5-2. Evaluation of GNE mRNA levels

[0148] Muscle tissues were obtained from disease mouse models administered with a recombinant AAV9 vector containing a wild-type GNE nucleotide sequence, and the level of GNE mRNA in each sample was measured by q-RT PCR and quantitatively compared through normalization using a T-test.

[0149] As a result, as shown in Fig. 17, in the negative control group (Gne KI Mock) administered with Mock AAV to the disease mouse model, the level of GNE mRNA was reduced compared to the control group, whereas in the disease mouse model (Gne KI LD hGNE AAV, Gne KI MD hGNE AAV, Gne KI HD hGNE AAV) administered with a recombinant AAV9 vector containing the GNE nucleotide sequence, the level of GNE mRNA was increased, and this effect showed a tendency to be dependent on the administered dose.

[0150]

[0151] 5-3. Evaluation of GNE protein expression levels

[0152] Muscle tissues were obtained from disease mouse models administered with a recombinant AAV9 vector containing a wild-type GNE nucleotide sequence, and the expression level of GNE protein was confirmed twice for each sample using Western blot (detection antibodies: #NBP1-81621, #25079-1-ap).

[0153] As a result, as shown in FIGS. 18 and 19, the expression level of GNE protein was increased in the disease mouse models (Gne KI LD hGNE AAV, Gne KI MD hGNE AAV, Gne KI HD hGNE AAV) administered with the recombinant AAV9 vector, and in particular, this effect showed a tendency to be dependent on the administered dose.

[0154]

[0155] Experimental Example 6. Functional Recovery Evaluation Using a Disease Mouse Model

[0156] In this experimental example, a disease mouse model (GNE C44S) to confirm functional recovery by administration of recombinant AAV9 vector through behavioral assessment, etc. For this purpose, C44S knock-in mice over 40 weeks old were selected as a disease mouse model, and a recombinant AAV9 vector (Example 3 (Gne KI GNE mod3 AAV), Example 4 (Gne KI GNE mod4 AAV) containing a GNE nucleotide sequence according to an embodiment) was administered to the disease mouse model. Thereafter, 4 weeks after the administration, a treadmill test and a grip strength test were performed on the individuals of each group. In addition to the behavioral evaluation described above, 8 weeks after the administration, the sialic acid level and the expression level of GNE protein were confirmed in the muscle tissue of each group. Meanwhile, in this experimental example, normal mice (C57BL / 6) were used as a control group, a group administered Mock AAV9 to the disease mouse model (Gne KI Mock AAV) as a negative control group, and a group administered recombinant AAV9 with a wild-type GNE nucleotide sequence applied to the disease mouse model (Gne KI hGNE AAV) as a comparison group. The mouse model and administration conditions in the experimental example are as shown in Table 9 below.

[0157] [Table 9]

[0158]

[0159]

[0160] 6-1. Exercise ability assessment through treadmill test

[0161] A treadmill test was performed on a disease mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment. Specifically, a pre-test was conducted for the first 5 minutes at a 10 degree incline and a speed of 5 m / min. After that, the starting speed was set to 10 m / min and the speed was increased by 1 m / min every minute, up to a maximum speed of 25 m / min. When the mouse reached the limit of its motor ability, it stopped running and was made to run again using an electric shock. After that, if the mouse was unable to run after receiving an electric shock for more than 5 seconds, the test was terminated and the distance traveled (m) was measured.

[0162] As a result, as shown in FIGS. 20 and 21, in the negative control group in which Mock AAV was administered to the disease mouse model and the comparison group in which the wild-type GNE nucleotide sequence was applied, the movement distance of the Treadmill was reduced due to the lack of the GNE gene, whereas in the group in which the recombinant AAV9 vector including the GNE nucleotide sequence according to one embodiment was introduced, the movement distance of the Treadmill was increased.

[0163]

[0164] 6-2. Assessment of motor skills through grip strength testing

[0165] A grip strength test was performed on a disease mouse model administered a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment. Specifically, grip strength was measured by placing the mouse's four paws on a grid and pulling its tail to grasp it. The grip strength / body weight (g) value was then calculated, and the average score was recorded after five repetitions.

[0166] As a result, as shown in FIGS. 22 and 23, it was confirmed that the grip strength was enhanced in the group introduced with the recombinant AAV9 vector including the GNE nucleotide sequence according to one embodiment, similar to the above results.

[0167]

[0168] 6-3. Sialic acid level assessment

[0169] In a disease mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment, at 8 weeks from the date of administration, the expression levels of Neu5AC and Galactose were confirmed, respectively, through SNA staining and PNA staining in the muscle tissue of each group according to the method of Example 5-1.

[0170] As a result, as shown in FIGS. 24 and 25, it was confirmed that the reduced expression level of Neu5AC was restored in the group introduced with the recombinant AAV9 vector including the GNE nucleotide sequence according to one embodiment, and the increased expression level of Galactose was also restored to the control level.

[0171]

[0172] 6-4. Evaluation of GNE protein expression levels

[0173] In a disease mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment, at 8 weeks from the date of administration, the expression level of the GNE protein was confirmed through Western blot (detection antibody: #25079-1-ap) in muscle tissues of each group according to the method of Example 5-3.

[0174] As a result, as shown in FIGS. 26 and 27, in the group introduced with the recombinant AAV9 vector including the GNE nucleotide sequence according to one embodiment, the GNE expression level was restored and showed an expression level similar to or increased compared to the control group.

[0175]

[0176] Experimental Example 7. In vivo safety evaluation using a disease mouse model

[0177] In this experimental example, the survival and body weight changes of the disease mouse model of Experimental Example 6 were measured to confirm the safety of the recombinant AAV9 vector in the body. Specifically, 8 weeks after the recombinant AAV9 vector (Example 3 (Gne KI GNE mod3 AAV), Example 4 (Gne KI GNE mod3 AAV) containing the GNE nucleotide sequence according to one embodiment) was administered to the disease mouse model, the survival of the individual was confirmed, and the body weight was measured a total of 4 times every 2 weeks during the period.

[0178] As a result, all groups introduced with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment survived, and as shown in Fig. 28, it was confirmed that there was no significant change in the body weight of the individuals during the experimental period.

[0179]

[0180] Experimental Example 8. Evaluation of Functional Recovery of Recombinant AAV9 Vector Containing the GNE Nucleotide Sequence of Example 3 Using a Disease Mouse Model

[0181] In this experimental example, we aimed to confirm the functional recovery by administration of a recombinant AAV9 vector containing the GNE nucleotide sequence of Example 3. To this end, C44S knock-in mice aged 40 weeks or older were selected as a disease mouse model, and the recombinant AAV9 vector containing the GNE nucleotide sequence of Example 3, i.e., Gne KI mod3 LD (low dose), Gne KI mod3 MD (Mid dose), and Gne KI mod3 HD (high dose), were administered to the disease mouse model at different doses. Thereafter, 4 weeks after the administration, a treadmill test was performed on the individuals in each group. In addition to the behavioral evaluation described above, body weight changes were measured in the disease mouse model to confirm the in vivo safety of the recombinant AAV9 vector. Meanwhile, in this experimental example, normal mice administered with PBS (WT PBS) served as the control group, and the group administered with the recombinant AAV9 containing the wild-type GNE nucleotide sequence to the disease mouse model (Gne KI hGNE) served as the comparison group. The mouse model and administration conditions in this experimental example are as shown in Table 10 below.

[0182] [Table 10]

[0183]

[0184]

[0185] 8-1. Behavioral Assessment

[0186] A treadmill test was performed according to the method of Experimental Example 6-1 on a disease mouse model administered with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment, to confirm the therapeutic efficacy of the recombinant AAV9 vector containing the GNE nucleotide sequence of Example 3.

[0187] As a result, as shown in Fig. 29, it was confirmed that the movement distance of the treadmill was increased in the group introduced with the recombinant AAV9 vector including the GNE nucleotide sequence according to one embodiment, compared to the comparative group to which the wild-type GNE nucleotide sequence was applied.

[0188]

[0189] 8-2. In vivo safety evaluation

[0190] In order to confirm the safety of the recombinant AAV9 vector comprising the GNE nucleotide sequence of Example 3 in the body, the survival and body weight changes were measured in a disease mouse model administered with a recombinant AAV9 vector comprising the GNE nucleotide sequence according to one embodiment. Specifically, at 4 weeks after the date of administration of the recombinant AAV9 vector comprising the GNE nucleotide sequence according to one embodiment, the survival of the animals was confirmed and their body weights were measured.

[0191] As a result, all groups introduced with a recombinant AAV9 vector containing a GNE nucleotide sequence according to one embodiment survived, and as shown in Fig. 30, it was confirmed that there was no significant change in the body weight of the individuals during the experimental period.

[0192]

[0193] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. An isolated nucleic acid satisfying the following conditions, wherein the isolated nucleic acid is any one selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3, the nucleotide sequence of SEQ ID NO: 5, and the nucleotide sequence of SEQ ID NO: 6: (a) encoding a GNE protein represented by the amino acid sequence number 1; (b) 75 to 80% sequence identity with the nucleotide sequence of SEQ ID NO: 2; and (c) GC content of 55 to 58% based on the entire nucleotide sequence.

2. An expression cassette comprising the isolated nucleic acid of claim 1.

3. In claim 2, the expression cassette comprises the following elements: Inverted terminal repeat (ITR) sequence; cytomegalovirus enhancer; cytomegalovirus promoter; Any one isolated nucleic acid selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3, the nucleotide sequence of SEQ ID NO: 5, and the nucleotide sequence of SEQ ID NO: 6; human growth hormone gene 1 polyadenylation signal (hGH1 polyadenylation signal); and Second inverted terminal repeat (ITR).

4. A recombinant virus based on adeno-associated virus serotype 9 (AAV9) comprising the expression cassette of claim 2 or claim 3.

5. In claim 4, the recombinant virus promotes sialic acid production in muscles.

6. In claim 4, the recombinant virus increases the expression level of the GNE protein.

7. A pharmaceutical composition for preventing or treating GNE myopathy (GNEM), comprising as an active ingredient a vector comprising any one isolated nucleic acid selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3, the nucleotide sequence of SEQ ID NO: 5, and the nucleotide sequence of SEQ ID NO:

6.

8. A pharmaceutical composition for preventing or treating GNE myopathy (GNEM), wherein the vector according to claim 7 is a recombinant virus based on adeno-associated virus serotype 9 (AAV9).

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