Recombinant adeno-associated virus for preventing or treating lafora disease and pharmaceutical composition comprising same

Abstract

The present invention relates to a recombinant adeno-associated virus for preventing or treating Lafora disease and a pharmaceutical composition comprising same. A recombinant adeno-associated virus produced by expressing a vector for producing a recombinant virus, according to an aspect, can effectively deliver a damaged gene without exhibiting pathogenicity to the human body. It was confirmed that, when the recombinant adeno-associated virus is injected into a subject actually showing the symptoms of Lafora disease, a gene was effectively delivered into the body, and accordingly, the effects of alleviating the symptoms of Lafora disease of the subject and improving motor function were confirmed. Therefore, the vector for producing the recombinant virus and the recombinant adeno-associated virus produced by expressing same can be used in various industrial fields for the prevention and treatment of Lafora disease.

Description

Recombinant adeno-associated virus for preventing or treating Lafora disease and pharmaceutical composition comprising the same

[0001] The present invention relates to a recombinant adeno-associated virus for preventing or treating Lafora disease and a pharmaceutical composition comprising the same.

[0002]

[0003] Lafora disease is a hereditary progressive myoclonic epilepsy syndrome, an autosomal recessive disorder that typically begins in adolescence or late childhood. Typical symptoms include myoclonic seizures (irregular, asymmetrical, shock-like muscle contractions) and occipital lobe (the back of the brain) seizures, including visual hallucinations, scotomata, and photoconvulsions. As the disease progresses, it can lead to worsening seizures, severe mental decline, dementia, other neurological deficits, and even death. While some patients may experience delayed onset and a more gradual progression, death typically occurs within 2 to 10 years of the initial onset, with an average of 5 years.

[0004] The exact cause of Lafora disease is not yet known, but previous studies have shown that mutations in the gene encoding the laforin protein located on chromosome 6 (located at 6q24) and mutations in the gene encoding the malin protein are associated with the disease.

[0005] Lafora disease is caused by a loss-of-function mutation in the gene encoding the protein. When either laforin or malin is missing due to the genetic mutation, hyperphosphorylated glycogen is produced, which precipitates, aggregates, and accumulates in Lafora bodies.

[0006] Currently, there is no clear, fundamental treatment for Lafora disease. Initially, seizures and myoclonic epilepsy are treated with anticonvulsants. As symptoms worsen with age, control becomes more difficult, and patients with Lafora disease typically live less than 30 years.

[0007] Accordingly, the present inventors developed a recombinant virus capable of safely carrying a damaged Lafora gene expression gene without infecting the human body, and a vector capable of producing the virus. From this, we confirmed the symptom-alleviating effect and therapeutic potential of Lafora disease, and we aim to provide a pharmaceutical composition containing the virus for the prevention or treatment of Lafora disease.

[0008]

[0009] One aspect provides a vector for producing a recombinant virus comprising a polynucleotide encoding a Laforin protein.

[0010] Another aspect is to provide a recombinant virus comprising a polynucleotide encoding the Laforin protein.

[0011] Another aspect provides a pharmaceutical composition for preventing or treating Lafora disease, comprising a recombinant virus comprising a polynucleotide encoding the Laforin protein.

[0012] Another aspect provides a transformant for producing a recombinant virus, wherein a vector for producing a recombinant virus comprising a polynucleotide encoding the Laforin protein is transfected into a host cell.

[0013] Another aspect provides a method for producing a recombinant virus, comprising the step of transfecting a host cell with a vector for producing a recombinant virus comprising a polynucleotide encoding the Laforin protein.

[0014] Another aspect is to provide a health functional food for preventing or improving Lafora disease, comprising a recombinant virus comprising a polynucleotide encoding the Laforin protein.

[0015] Another aspect provides a method for preventing or treating Lafora disease, comprising administering to a subject in need thereof a recombinant virus comprising a polynucleotide encoding the Laforin protein.

[0016] Another aspect provides the use of a recombinant virus comprising a polynucleotide encoding the Laforin protein for the manufacture of a medicament for the prevention or treatment of Lafora disease.

[0017]

[0018] One aspect provides a vector for producing a recombinant virus comprising a polynucleotide encoding a Laforin protein.

[0019] The above "Laforin" is a protein encoded by the EPM2A (Epilepsy, Progressive Myoclonic, 2A) gene, and is known to play an important role in glycogen metabolism in the body by mainly acting as a phosphatase. The laforin protein is largely composed of the CBM20 (Carbohydrate-Binding Module 20) domain and the DSP (Dual-Specificity Phosphatase) domain. When a mutation or deficiency occurs in the laforin protein, excessive phosphorylation of glycogen occurs, and abnormal glycogen called Lafora bodies accumulate in nerve cells, causing Lafora disease.

[0020] The above “Lafora Disease” is a rare neurological disease that presents with autosomal dominant epilepsy symptoms, known to affect approximately 0.04 out of 10,000 people. Other symptoms of Lafora Disease include gait disturbance, muscle cramps, or dementia. The first symptoms of Lafora Disease typically appear in adolescence, and patients die within about 10 years of the onset of symptoms. Lafora Disease is known to be caused by loss-of-function mutations in the EPM2A gene or NHLRC1 gene, which encode the Laforin and Malin proteins, respectively. When one or more of the Laforin or Malin proteins is missing due to loss-of-function mutations in the above genes, hyperphosphorylated glycogen is produced, which aggregates and accumulates in Lafora bodies. When Lafora bodies accumulate, they can interfere with the function of cells in the brain and cause the death of nerve cells, which can affect various parts of the brain and cause various neurological symptoms such as seizures, muscle stiffness, cognitive impairment, and motor control disorders.

[0021] The term "protein" may be used interchangeably with "polypeptide" and "peptide" and refers to a polymer of amino acids of any length. The protein may include a modified amino acid polymer, for example, a polymer formed by disulfide bond formation, glycosylation, lipidation, or conjugation with a labeling component.

[0022] The term "polynucleotide" above refers to a polymeric substance in which nucleotides are bonded, and refers to a nucleic acid (e.g., DNA or RNA) that encodes genetic information. The polynucleotide may include any nucleic acid composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).

[0023] The term "recombinant virus" refers to a virus whose genome has been artificially manipulated, i.e., by recombinant DNA technology, to include a foreign genetic sequence within its genome. For example, the recombinant virus may be a virus that includes a foreign genetic sequence encoding a polypeptide of interest.

[0024] In one specific example, the recombinant virus may be an adeno-associated virus (AAV), an adenovirus, a retrovirus, a lentivirus, a herpes virus (Herper Simplex Virus (HSV), a bacteriophage, a parvovirus, a baculovirus, or a Sindbis virus, and specifically, may be an adeno-associated virus, a lentivirus, or a herpes virus, and more specifically, may be an adeno-associated virus.

[0025] The term "vector" as used herein refers to a DNA construct comprising a target gene, operably linked to suitable regulatory sequences so as to cause expression of the target gene in a suitable host cell, and may be, specifically, a plasmid. The regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. The vector, after being introduced into a suitable host cell, may replicate or function independently of the host cell's genes, and may be integrated into the genes themselves.

[0026] Specifically, the vector may be a vector derived from a retrovirus such as Sendai Virus, Lentivirus, Human Immunodeficiency Virus (HIV), Murineleukemia Virus (MLV), Avian Sarcoma / Leukosis (ASLV), Spleen Necrosis Virus (SNV), Rous Sarcoma Virus (RSV), Mouse Mammary Tumor Virus (MMTV), Adenovirus, Adeno-associated Virus, or Herpes Simplex Virus, and more specifically, may be a vector derived from Adeno-associated Virus.

[0027] The term "operably" above means that the target gene is linked to regulatory sequences in a manner that allows expression in a host cell. The regulatory sequences are DNA sequences that control the expression of a gene and may include other regulatory elements such as a promoter, enhancer, and polyadenylation.

[0028] The above “promoter” refers to a sequence involved in the binding of RNA polymerase so that transcription can be initiated into a portion of DNA. The promoter is a site where RNA polymerase or a transcription factor, which is a protein that induces RNA polymerase, binds, and is generally located upstream from and adjacent to the target gene. The promoter can induce the enzyme or protein to be located at the correct transcription start site. In other words, the promoter is located at the 5' site of the gene to be transcribed in the sense strand, and plays a role in inducing RNA polymerase to bind to that site directly or through a transcription factor and initiate mRNA synthesis for the target gene.

[0029] In one specific example, the vector for producing the recombinant virus may include a CAG (cytomegalovirus immediate-early enhancer / chicken β-actin) promoter.

[0030] The above “CAG (cytomegalovirus immediate-early enhancer / chicken β-actin) promoter” is a type of modified CMV promoter and is a composite promoter containing the cytomegalovirus immediate-early enhancer, the chicken β-actin promoter, a chimeric intron, exon 1, and exon 2 of the rabbit β-globin gene, and can be used in various modified forms depending on the purpose. The CAG promoter can induce high levels of gene expression in various mammalian cells such as nerve cells, muscle cells, and hepatocytes. In addition, it has high stability in cells, can maintain gene expression continuously for a long period of time, and tends to induce less immune responses, so it is mainly used in gene therapy applications.

[0031] In one specific example, the “CAG (cytomegalovirus immediate-early enhancer / chicken β-actin) promoter” may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 6.

[0032] When the vector for producing the above recombinant virus contains a CAG promoter, the expression rate of the protein encoded by the gene in the vector may increase, and in particular, the expression rate of the protein may increase in mammalian cells.

[0033] In one specific example, the vector for producing the recombinant virus may include a Woodchuck hepatitis B virus post-transcriptional regulatory element (WPRE).

[0034] The above “WPRE (Woodchuck hepatitis B virus post-transcriptional regulatory element)” consists of a DNA sequence approximately 600 bp in length and contributes to mRNA stabilization by promoting the process of transcribed mRNA being processed from the nucleus to the cytoplasm and its transport. In other words, WPRE is a regulatory factor that can increase the level of protein expression by increasing the stability and translation efficiency of mRNA, thereby enabling the mass expression of proteins under the control of the CAG promoter.

[0035] In one specific example, the “WPRE (Woodchuck hepatitis B virus post-transcriptional regulatory element)” may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 7.

[0036] When the vector for producing the above recombinant virus contains WPRE, the expression rate of the protein encoded by the gene in the vector may increase.

[0037] In one specific example, the vector for producing the recombinant virus may further comprise a marker for selecting host cells that have been successfully transfected with the vector. The marker may be a marker that confers an easily selectable phenotype, such as drug resistance, nutrient requirement, resistance to cytotoxic agents, or expression of a surface protein.

[0038] The above "host cell" refers to a cell into which a recombinant vector is transformed, transduced, or introduced. The host cell may be a host cell known in the art. Specifically, the host cell may be NS / O myeloma cells, HEK 293T cells, Chinese hamster ovary cells (CHO cells), HeLa cells, human amniotic fluid-derived cells, COS cells, Kenine D17 cells, Feline PG4 cells, or stem cells, and more specifically, may be HEK 293T cells.

[0039] The above “transformation” may be used interchangeably with “transfection”, and may be the introduction of a gene into a cell according to a biological, chemical, or physical method known in the art. The transfection may be performed by one or more methods selected from the group consisting of lipofectamine treatment, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, polybrene treatment, polyethyleneimine (PEI) treatment, and gene bombardment, and may be specifically performed by polyethyleneimine (PEI) treatment.

[0040] The polyethyleneimine (PEI) treatment described above is one of the transfection methods for delivering genetic material to cells, and is primarily used to introduce foreign genes into cells. Polyethyleneimine is a positively charged polymer that binds to negatively charged DNA or RNA to form nanometer-sized complexes. Because the formed complexes are positively charged by polyethyleneimine, they can easily bind to negatively charged cell membranes. When bound to the cell membrane, the complexes enter the cell via endocytosis. Once inside the cell, the complexes escape the endosomes and are released into the cytoplasm due to the proton sponge effect. The released DNA or RNA can then migrate to the nucleus of the cell and be transfected.

[0041] In one specific example, the polynucleotide encoding the laforin protein may be an EPM2A gene, and specifically, a human EPM2A gene (hEPM2A).

[0042] In one specific example, the hEPM2A gene may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 1.

[0043] In one specific example, the hEPM2A gene may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 1, for example, a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more or 100% homology with the base sequence of SEQ ID NO: 1.

[0044] In one specific example, the polynucleotide encoding the laforin protein may be codon optimized.

[0045] The term "codon optimization" refers to altering codons in a gene or coding region of a nucleic acid molecule to reflect the common codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization involves replacing at least one, more than one, or a significant number of codons with one or more codons that are more frequently used in the genes of the organism. As such codon optimization is performed, the expression rate of the target gene in a particular organism can be increased, and if CpG motifs are removed during the codon optimization process, immunogenicity can be reduced or eliminated at the same time.

[0046] In one specific example, the polynucleotide encoding the laforin protein may have fewer CpG motifs, and specifically, may have no CpG motifs, compared to a polynucleotide consisting of the base sequence of SEQ ID NO: 1. The polynucleotide encoding the laforin protein may be modified to have fewer CpG motifs (i.e., "CpG reduced" or "CpG depleted") through the codon optimization process. For example, a CpG motif located within a codon encoding a specific amino acid may be changed to a codon encoding the same amino acid without the CpG motif.

[0047] The term "CpG motif" above refers to a dinucleotide sequence containing an unmethylated cytosine linked to a guanosine by a phosphate bond. The "CpG motif" may include both methylated and unmethylated CpG dinucleotides. Unmethylated CpG motifs are common in nucleic acids of bacterial and viral origin (e.g., plasmids), but are suppressed and mostly methylated in vertebrate DNA. Therefore, unmethylated CpG motifs can stimulate mammalian hosts to induce a rapid inflammatory response. CpG motifs are known to be associated with the immunogenicity of vectors, and thus, reducing the number of CpG motifs in a vector can reduce or eliminate immunogenicity.

[0048] In one specific example, the nucleotide sequence of human EPM2A comprises 56 CpG motifs, whereas the codon optimized EPM2A gene may comprise less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, less than 10, or less than 5 CpG motifs, specifically less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, less than 10, or less than 5 CpG motifs, and more specifically may comprise 0 CpG motifs, i.e., no CpG motifs are present.

[0049] In one specific example, the laporin protein may have reduced or eliminated immunogenicity for humans, and the reduced or eliminated immunogenicity may be achieved by codon optimization.

[0050] The above term "immunogenicity" dictionary means "the property of acting as an antigen capable of inducing an immune response." Various methods can be used to measure the immunogenicity of a specific antigen, and the methods can be appropriately adopted or designed according to the purpose. That is, as methods for measuring immunogenicity, there are, for example, 1) a method for confirming whether antibodies of the IgG, IgA, and / or IgE type are produced in the body of a subject when the antigen is administered to the body of the subject, 2) a method for confirming the timing of production of antibodies of the IgG, IgA, and / or IgE type depending on the administration cycle of the antigen, 3) a method for confirming the titer of the induced antibody against the antigen, and 4) a method for measuring the effect according to the action mechanism when the action mechanism of the induced antibody is elucidated.

[0051] The polynucleotide encoding the Laforin protein can have its CpG motifs removed during codon optimization. If the CpG motifs are completely removed in a specific manner, the human immunogenicity of the expressed protein can be reduced or eliminated without affecting the protein expression rate. Proteins with reduced or eliminated immunogenicity can persist longer in the human body without causing adverse effects, such as allergic or autoimmune reactions. Therefore, proteins with reduced or eliminated immunogenicity can reach target tissues more effectively, thereby increasing their overall efficacy as therapeutics for specific tissues.

[0052] In one specific example, the polynucleotide encoding the laforin protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 2.

[0053] In one specific example, the polynucleotide expressing the laforin protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 2, for example, a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more or 100% homology with the base sequence of SEQ ID NO: 2.

[0054] In one specific example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 2 may have fewer CpG motifs compared to a polynucleotide comprising a base sequence of SEQ ID NO: 1, for example, may have fewer than 56 CpG motifs, and specifically, may have fewer than 37 CpG motifs.

[0055] In one specific example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 2 may have reduced or eliminated immunogenicity compared to a polynucleotide comprising a base sequence of SEQ ID NO: 1. In addition, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 2 may have a further increased gene expression rate compared to a polynucleotide comprising a base sequence of SEQ ID NO: 1.

[0056] In one specific example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 2 may have a higher gene expression rate and may also have a lower or eliminated immunogenicity compared to a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 3.

[0057] In one specific example, the polynucleotide encoding the laforin protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 3.

[0058] In one specific example, the polynucleotide expressing the laforin protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 3, for example, a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more or 100% homology with the base sequence of SEQ ID NO: 3.

[0059] In one specific example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 3 may have fewer CpG motifs compared to a polynucleotide comprising a base sequence of SEQ ID NO: 1, for example, may have fewer than 56 CpG motifs, and specifically, may have fewer than 49 CpG motifs.

[0060] In one specific example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 3 may have reduced or eliminated immunogenicity compared to a polynucleotide comprising a base sequence of SEQ ID NO: 1. In addition, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 3 may have a further increased gene expression rate compared to a polynucleotide comprising a base sequence of SEQ ID NO: 1.

[0061] In one specific example, the polynucleotide encoding the laforin protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 4.

[0062] In one specific example, the polynucleotide expressing the laforin protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 4, for example, a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more or 100% homology with the base sequence of SEQ ID NO: 4.

[0063] In one specific example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 4 may have fewer CpG motifs compared to a polynucleotide comprising a base sequence of SEQ ID NO: 1, for example, may have less than 56 CpG motifs, and specifically, may have 0 or fewer CpG motifs.

[0064] In one specific example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 4 may have reduced or eliminated immunogenicity compared to a polynucleotide comprising a base sequence of SEQ ID NO: 1. In addition, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 4 may have a further increased gene expression rate compared to a polynucleotide comprising a base sequence of SEQ ID NO: 1.

[0065] In one specific example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 4 may have reduced or eliminated immunogenicity and may also have increased gene expression rate compared to a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 2 and a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 3.

[0066] In one specific example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 4 may have no difference in the number of CpG motifs from a polynucleotide comprising the base sequence of SEQ ID NO: 4. For example, a polynucleotide comprising a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 4 may not have a CpG motif in a base sequence that is not homologous with the base sequence of SEQ ID NO: 4.

[0067] In one embodiment, codon optimization was performed using various methods to increase gene expression and reduce immunogenicity in humans when constructing an AAV vector containing the human EPM2A (hEPM2A) gene. GenScript's GenSmart TMAfter optimization using the codon optimization tool and IDT's codon optimization tool, the expression rate of the genes optimized with each tool was checked, and compared with genes that were not optimized, GenScript's GenSmart TM Using the codon optimization tool, we confirmed that the expression of the optimized gene increased by approximately 70%, and at this time, we confirmed that there were 37 CpG motifs in the optimized gene. When all CpG motifs were removed using the same method to reduce immunogenicity in the gene with the significantly increased expression rate, it was confirmed that the gene expression level was similar to that of the gene that was not optimized (see Example 1). Therefore, GenScript's GenSmart TM The EPM2A gene, in which all CpG motifs were removed using a codon optimization tool, was judged to be most effective because it had reduced immunogenicity in humans without problems such as decreased expression rate.

[0068] In one specific example, the vector for producing the recombinant virus may be a polynucleotide consisting of a base sequence having at least 70% homology with the base sequence of SEQ ID NO: 5.

[0069] In one specific example, the vector for producing the recombinant virus may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 5, for example, a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more or 100% homology with the base sequence of SEQ ID NO: 5.

[0070] In another embodiment, a recombinant AAV9 vector was prepared that contains a CpG-free hEPM2A gene consisting of a CAG promoter, WPRE, and the nucleotide sequence of SEQ ID NO: 4, and that is non-pathogenic to humans and can effectively reach the central nervous system (see Example 1). The entire nucleotide sequence of the ultimately prepared recombinant virus production vector is the same as the nucleotide sequence of SEQ ID NO: 5.

[0071] In one specific example, a vector for producing a recombinant virus comprising nucleotides encoding the Laforin protein may be introduced into a host cell to produce a recombinant virus. The produced recombinant virus comprises nucleotides encoding the Laforin protein and can be effectively delivered to target cells, thereby being used as an active ingredient in a pharmaceutical composition for preventing or treating Lafora disease.

[0072] Another aspect provides a recombinant virus comprising a polynucleotide encoding a Laforin protein.

[0073] The above “Laforin”, “protein”, “polynucleotide” and “recombinant virus” may be within the above-mentioned range.

[0074] In one specific example, the recombinant virus may be at least one selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, herpes simplex virus (HSV), bacteriophage, parvovirus, baculovirus, and sindbis virus, and more specifically, may be an adeno-associated virus.

[0075] In one specific example, the recombinant virus may comprise a polynucleotide encoding the Laforin protein in various forms. Specifically, it may comprise a single-stranded linear DNA, a double-stranded linear DNA, a single-stranded circular DNA, a double-stranded circular DNA, a single-stranded linear RNA, or a double-stranded linear RNA.

[0076] The term "adeno-associated virus" or "AAV" above may include any adeno-associated virus, derivative thereof, or any naturally occurring or recombinant viral subtype used in gene therapy. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeat (TR), Rep protein sequence, and capsid subunit sequences, are known in the art. These sequences can be found in the literature or in public databases such as GenBank. For example, see GenBank Accession No. NC_006152 (AAV-9) and AY530573 (AAV-9).

[0077] The adeno-associated virus may include all known AAV serotypes, and may particularly include AAV serotypes that target the CNS or variants of any AAV serotype that have been genetically engineered to target the CNS. The AAV serotype may specifically be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh43, AAVrh74 or variants thereof, and more specifically may be AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10 or AAV rh43.

[0078] Adeno-associated virus (AAV) is a virus primarily used in gene therapy. It can efficiently deliver genes to various cell types and tissues without causing disease in humans. Furthermore, it has relatively low immunogenicity in the body, making it highly unlikely that the therapeutic effect will be hindered by an immune response. These characteristics make it currently used as a treatment for a variety of diseases.

[0079] The term “variant” above refers to an AAV virus whose genetic sequence or function has been altered due to substitution, insertion, or deletion of one or more amino acids compared to a wild-type AAV virus.

[0080] The term "serotype" above means a subdivision of AAV that can be identified by serological or DNA sequencing methods and distinguished by its antigenic characteristics.

[0081] In one specific example, the polynucleotide encoding the laforin protein may have fewer CpG motifs compared to a polynucleotide consisting of the base sequence of SEQ ID NO: 1.

[0082] In one specific example, the polynucleotide encoding the laforin protein may be composed of a base sequence having 90% or more homology with a polynucleotide composed of the base sequence of SEQ ID NO: 4.

[0083] In one specific example, the laporin protein may have reduced or eliminated immunogenicity for humans.

[0084] In one specific example, the recombinant virus comprising a polynucleotide encoding the Laforin protein can effectively deliver the polynucleotide encoding the Laforin protein without causing side effects in the subject to which it is administered due to reduced or eliminated immunogenicity. When the recombinant virus is administered to a subject, the effects of preventing or treating Lafora disease, such as reducing aggregated Lafora bodies in the subject, alleviating brain inflammation, and restoring motor function, are exhibited. Therefore, the recombinant virus can be used as an active ingredient in a pharmaceutical composition for preventing or treating Lafora disease. The effects can be exhibited in all subjects capable of having Lafora disease, and specifically, the effects can be more excellent in subjects in which the EPM2A or NHLRC 1 gene is damaged, and more specifically, in subjects in which the EPM2A gene is damaged.

[0085] Another aspect provides a pharmaceutical composition for preventing or treating Lafora disease comprising the recombinant virus.

[0086] The above “recombinant virus” may fall within the aforementioned range.

[0087] In one specific example, the recombinant virus may be at least one selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, herpes simplex virus (HSV), bacteriophage, parvovirus, baculovirus, and sindbis virus, and more specifically, may be an adeno-associated virus.

[0088] The above “Lafora Disease” is a rare neurological disease that presents with autosomal dominant epilepsy symptoms, known to affect approximately 0.04 out of 10,000 people. Other symptoms of Lafora Disease include gait disturbance, muscle cramps, or dementia. The first symptoms of Lafora Disease typically appear in adolescence, and patients die within about 10 years of the onset of symptoms. Lafora Disease is known to be caused by loss-of-function mutations in the EPM2A gene or NHLRC1 gene, which encode the Laforin and Malin proteins, respectively. When one or more of the Laforin or Malin proteins is missing due to loss-of-function mutations in the above genes, hyperphosphorylated glycogen is produced, which aggregates and accumulates in Lafora bodies. When Lafora bodies accumulate, they can interfere with the function of cells in the brain and cause the death of nerve cells, which can affect various parts of the brain and cause various neurological symptoms such as seizures, muscle stiffness, cognitive impairment, and motor control disorders.

[0089] In one specific example, the Lafora disease may be caused by damage to the EPM2A gene encoding the Laforin protein or the NHLRC 1 gene encoding the Malin protein due to a loss-of-function mutation, and specifically, the Lafora disease may be caused by damage to the EPM2A gene encoding the Laforin protein due to a loss-of-function mutation, etc.

[0090] The above term "prevention" means any act of suppressing or delaying the onset of Lafora's disease in an individual by administering a pharmaceutical composition according to one aspect.

[0091] The above term "treatment" means any action by which the symptoms of Lafora's disease of an individual are improved or beneficially changed by administration of a pharmaceutical composition according to one aspect.

[0092] The above term “administration” means introducing a given substance into an individual in an appropriate manner.

[0093] The term "subject" above refers to any living organism, including rats, mice, and livestock, including humans, that can harbor Lafora disease. A specific example may be a mammal, including humans.

[0094] In one specific example, the pharmaceutical composition for preventing or treating Lafora's disease may further include one or more active ingredients that exhibit the same or similar function or that exhibit a function that assists the recombinant virus as the active ingredient.

[0095] In one specific example, the pharmaceutical composition may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

[0096] The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories, and may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can be used, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0097] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to prevent or treat Lafora's disease at a reasonable benefit / risk ratio applicable to medical use, and the effective dosage level may be determined based on factors including the type and severity of the individual, age, sex, activity of the drug, weight, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0098] In one specific example, the recombinant virus can be administered at any appropriate dose. The dosage of the recombinant virus can be determined by considering the weight of the subject to achieve a therapeutic effect, and specifically, the dosage is 1 x 10 11 vg / kg to 1 x 10 15 vg / kg, 5 x 10 11 vg / kg to 1 x 10 14 vg / kg, 5 x 10 11 vg / kg to 5 x 10 14 vg / kg, 5 x 10 11 vg / kg to 5 x 10 13 vg / kg, 5 x 10 11 vg / kg to 1 x 10 13 vg / kg or 5 x 10 11 vg / kg to 5 x 10 12 vg / kg, or more specifically about 5 x 10 11 About 1 x 10 15 It can be vg / kg.

[0099] In one specific example, the administration may be to an individual capable of having Lafora's disease, and specifically, the individual may be an individual in which the EPM2A gene encoding the Laforin protein or the NHLRC 1 gene encoding the malin protein is damaged due to a loss-of-function mutation or the like. More specifically, the individual may be an individual in which the EPM2A gene encoding the Laforin protein is damaged. When the recombinant adenovirus according to one embodiment is administered to the individual, an excellent preventive or therapeutic effect for Lafora's disease can be expected, and specifically, when administered to an individual in which the EPM2A gene encoding the Laforin protein is damaged, an even better effect can be expected.

[0100] At this time, the content of the effective ingredient included in the pharmaceutical composition may be 0.0001 wt% to 10 wt%, specifically 0.001 wt% to 1 wt%, based on the total weight of the composition.

[0101] The above pharmaceutical composition comprises, as an active ingredient, a recombinant virus containing a polynucleotide encoding Laforin protein. When the composition is administered orally or parenterally to a subject, it can exhibit excellent preventive or therapeutic effects on Lafora disease, such as reduction of aggregated Lafora bodies in the subject, alleviation of brain inflammation and recovery of motor function in the subject. The above effects can be exhibited in all subjects capable of having Lafora disease, and specifically, it can exhibit excellent effects on subjects in which the EPM2A gene encoding Laforin protein or the NHLRC 1 gene encoding Malin protein is damaged, and more specifically, it can exhibit better effects on subjects in which the EPM2A gene encoding Laforin protein is damaged.

[0102] In one specific example, the pharmaceutical composition may further comprise a pharmaceutical composition for preventing or treating another Lafora disease in addition to the recombinant virus.

[0103] The above pharmaceutical composition may be provided mixed with another composition for preventing or treating Lafora's disease, and the other composition for preventing or treating Lafora's disease may be a conventionally known pharmaceutical composition or a newly developed pharmaceutical composition.

[0104] When the above pharmaceutical composition further comprises a pharmaceutical composition for the prevention or treatment of another Lafora disease, it is important to mix the two in an amount that can achieve the maximum effect with the minimum amount without causing side effects, which can be easily determined by a person skilled in the art.

[0105] When the pharmaceutical composition further comprises another pharmaceutical composition for the prevention or treatment of Lafora disease, a synergistic effect may be exhibited in which the preventive or therapeutic effect of Lafora disease, i.e., the reduction of aggregated Lafora bodies, the alleviation of brain inflammation, and the recovery of motor function, are more pronounced than when the pharmaceutical composition for the prevention or treatment of Lafora disease comprising the recombinant virus is included as an active ingredient alone.

[0106] Furthermore, in one specific embodiment, the pharmaceutical composition may be administered alone or in combination with another pharmaceutical composition for the prevention or treatment of Lafora's disease. Specifically, the pharmaceutical composition may be administered in combination with a known pharmaceutical composition having a preventive or therapeutic effect for Lafora's disease, and may be administered simultaneously, separately, or sequentially, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be readily determined by those skilled in the art.

[0107] When the above pharmaceutical composition is administered in combination with another pharmaceutical composition for the prevention or treatment of Lafora's disease, a synergistic effect may be exhibited in which the effect of preventing or treating Lafora's disease, i.e., reducing aggregated Lafora bodies, alleviating brain inflammation, and restoring motor function, is more pronounced than when the pharmaceutical composition is administered alone.

[0108] The pharmaceutical composition may be administered orally or parenterally, and when administered parenterally, the pharmaceutical composition may be administered by topical application to the skin or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, percutaneous injection, intranasal injection, intraenteric injection, local injection, sublingual injection, rectal injection, ventricular injection, intracerebral microinjection, intrathoracic injection, or intrathecal injection. Specifically, the pharmaceutical composition may be administered by intravenous injection, ventricular injection, or intrathoracic injection.

[0109] In one embodiment, 1 x 10 for 4-month-old mice 10 1 x 10 11 When AAV of vg was administered by intraventricular injection, it was effective in reducing aggregated Laphora bodies in the body, alleviating brain inflammation, and restoring motor function (see Example 3).

[0110] In another example, an experiment was conducted to determine the effect of a recombinant virus containing a polynucleotide encoding the Laforin protein on alleviating Lafora disease symptoms. Mice administered with the recombinant AAV9, produced by expressing the vector, showed a decrease in Lafora bodies accumulated in the body, a reduction in brain inflammation, and a recovery of motor function. As a result, it was confirmed that the degree of reduction in Lafora bodies accumulated in the hippocampus was reduced by approximately 40 to 50% compared to the negative control group. Regarding the degree of alleviation of brain inflammation, the expression level of GFAP, an inflammatory marker, was determined in the hippocampal tissue of the mice, and a similar decrease in expression was confirmed by approximately 40 to 50% compared to the negative control group. Furthermore, to determine the degree of motor function recovery in the mice, a rotarod test, a hanging wire test, and an inverted screen test were performed, and it was confirmed that all mice administered with the recombinant AAV9 recovered motor function to the same level as wild-type mice (see Example 4).

[0111] Another aspect provides a cell for producing a recombinant virus comprising the vector for producing the recombinant virus.

[0112] The above terms “recombinant virus” and “vector” may be within the scope described above.

[0113] In one specific example, the recombinant virus may be at least one selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, herpes simplex virus (HSV), bacteriophage, parvovirus, baculovirus, and sindbis virus, and more specifically, may be an adeno-associated virus.

[0114] In one specific example, the vector for producing the recombinant virus may be transfected into a cell.

[0115] In one specific example, the cell may be at least one selected from the group consisting of NS / O myeloma cells, HEK 293T cells, Chinese hamster ovary cells (CHO cells), HeLa cells, human amniotic fluid-derived cells, COS cells, Kenine D17 cells, Feline PG4 cells, and stem cells, and specifically, may be HEK 293T cells.

[0116] The above transfection may be performed by one or more methods selected from the group consisting of lipofectamine treatment, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, polybrene treatment, polyethyleneimine (PEI) treatment, and gene bombardment, and specifically, may be performed by polyethyleneimine (PEI) treatment.

[0117] Another aspect provides a method for producing a recombinant virus, comprising a step of transfecting the recombinant virus.

[0118] The above terms “recombinant virus”, “transfection” and “vector” may be within the scope described above.

[0119] In one specific example, the recombinant virus may be at least one selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, herpes simplex virus (HSV), bacteriophage, parvovirus, baculovirus, and sindbis virus, and more specifically, may be an adeno-associated virus.

[0120] The above transfection may be performed by one or more methods selected from the group consisting of lipofectamine treatment, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, polybrene treatment, polyethyleneimine (PEI) treatment, and gene bombardment, and specifically, may be performed by polyethyleneimine (PEI) treatment.

[0121] In one specific example, the vector may be transfected simultaneously with the Rep2 / Cap9 plasmid and the helper plasmid.

[0122] In one specific example, the method may further comprise a step of transfecting the Rep2 / Cap9 plasmid and the helper plasmid.

[0123] In one specific example, the step of transfecting the Rep2 / Cap9 plasmid and the helper plasmid may be performed before or after the step of transfecting the vector.

[0124] The above Rep2 / Cap9 plasmid may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 8.

[0125] The above helper plasmid may be a polynucleotide consisting of a base sequence having at least 70% homology with the base sequence of SEQ ID NO: 9.

[0126] The "rep" and "cap" genes of AAV represent polynucleotide sequences encoding replication-associated proteins and encapsidation proteins of AAV, and the Rep2 / Cap9 plasmid provides the Rep protein of AAV2 required for replication of the adeno-associated virus genome and the capsid protein constituting the viral particle of AAV9.

[0127] The above "helper plasmid" comprises adenovirus-derived sequences (E4, E2a, VA, etc.) that facilitate the delivery of genetic material into cells. The helper plasmid may comprise, for example, at least one helper DNA sequence derived from a replication-defective viral genome that encodes in trans all virion proteins necessary for packaging replication-defective AAV and virion proteins capable of packaging replication-defective AAV at high titers without producing replication-defective AAV.

[0128] In one specific example, the vector, the Rep2 / Cap9 plasmid, and the helper plasmid may be transfected at a molar ratio of 1:1 to 2:1 to 3. Specifically, it may be injected at a molar ratio of 1: 1 to 2: 1 to 3, a molar ratio of 1: 1: 1, a molar ratio of 1: 1: 1.5, a molar ratio of 1: 1: 2, a molar ratio of 1: 1: 2.5, a molar ratio of 1: 1: 3, a molar ratio of 1: 1.5: 1.5, a molar ratio of 1: 1.5: 2, a molar ratio of 1: 1.5: 2.5, a molar ratio of 1: 1.5: 3, a molar ratio of 1: 2: 2, a molar ratio of 1: 2: 2.5, a molar ratio of 1: 2: 3, a molar ratio of 1: 2.5: 2.5, or a molar ratio of 1: 2.5: 3, and more specifically, it may be injected at a molar ratio of 1: 1.5: 2.

[0129] In another example, HEK293T cells were used as host cells to produce a recombinant adeno-associated virus by expressing a plasmid containing a polynucleotide encoding the laforin protein. Transfection was performed by treating the plasmid with polyethylenimine to produce a transformant, from which the recombinant adeno-associated virus was produced (see Example 2).

[0130] In another embodiment, an in vivo experiment was performed to confirm the delivery efficiency of the recombinant adeno-associated virus manufactured above. The recombinant adenovirus was administered to mice via intracerebroventricular administration, and the degree of distribution in the body was confirmed by measuring fluorescence. As a result, fluorescence was observed in neurons and glial cells throughout the brain tissue, and it was confirmed that the fluorescence was particularly clearly expressed in the hippocampus and cerebellum (see Example 3).

[0131] Another aspect provides a health functional food for preventing or improving Lafora disease, comprising a recombinant virus comprising a polynucleotide encoding Laforin protein.

[0132] The above “Laforin”, “protein”, “polynucleotide”, “recombinant virus”, “Lafora disease” and “prevention” may be within the scope described above.

[0133] The term "improvement" above may refer to any action that at least reduces a parameter related to the condition being treated, such as the severity of symptoms. In this case, the health functional food may be used before or after the onset of the disease, or concurrently with or separately from a treatment agent, to prevent or improve Lafora's disease.

[0134] The term "health functional food" above encompasses health functional foods, health foods, and health supplements. "Health food" refers to foods that have a more active health maintenance or promotion effect than regular foods, while "health supplement food" refers to foods intended for health supplement purposes.

[0135] The above health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further comprising one or more of a carrier, diluent, excipient, and additive. Foods to which the recombinant virus according to one aspect may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, and health functional foods.

[0136] Specific examples of the carrier, excipient, diluent and additive may include at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

[0137] In the above health functional food, the active ingredient can be added directly to the food or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient mixed can be appropriately determined depending on the intended use (prevention or improvement). Generally, when manufacturing a food or beverage, the health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

[0138] The above health functional food may contain other ingredients as essential ingredients in addition to the effective ingredient. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates may include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as xylitol, sorbitol, erythritol, etc. In addition to the above-mentioned flavoring agents, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by a person skilled in the art.

[0139] In addition to the above, health functional foods according to the aspect may include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0140] In one specific example, the health functional food may further include a health functional food for preventing or improving other Lapora diseases in addition to the recombinant virus.

[0141] The above-mentioned other health functional foods for preventing or improving Lapora disease may be provided in combination with health functional foods for preventing or improving Lapora disease known in the past or health functional foods for preventing or improving Lapora disease that are newly developed.

[0142] If the above health functional food further includes a health functional food for the prevention or improvement of other Lafora diseases, it is important to mix the amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by a person skilled in the art.

[0143] When the above health functional food further includes another health functional food for preventing or improving Lapora disease, a synergistic effect may be produced in which the effect of preventing or improving Lapora disease is more significant than when the health functional food for preventing or improving Lapora disease containing the recombinant virus is included as an active ingredient alone.

[0144] Furthermore, in one specific example, the health functional food may be consumed alone or in combination with other health functional foods for preventing or improving Lapora's disease. That is, the health functional food may be consumed in combination with other known health functional foods or newly developed health functional foods for preventing or improving Lapora's disease, and may be consumed simultaneously, separately, or sequentially, and may be consumed singly or in multiple doses. It is important to take all of the above factors into consideration and consume an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0145] When the above health functional food is taken in combination with other health functional foods for preventing or improving Lapora disease, a synergistic effect may be produced in which the effect of preventing or improving Lapora disease is more pronounced than when the above health functional food is taken alone.

[0146] Another aspect provides a method for preventing or treating Lafora disease, comprising administering to a subject in need thereof a recombinant virus comprising a polynucleotide encoding the Laforin protein.

[0147] The above terms “Laforin”, “protein”, “polynucleotide”, “recombinant virus”, “Lafora disease”, “prevention” and “treatment” may be within the scope described above.

[0148] In one specific example, the recombinant virus may be at least one selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, herpes simplex virus (HSV), bacteriophage, parvovirus, baculovirus, and sindbis virus, and more specifically, may be an adeno-associated virus.

[0149] In one specific example, the subject may be any subject capable of having Lafora disease, and specifically, may be a subject in which the EPM2A gene encoding the Laforin protein or the NHLRC 1 gene encoding the malin protein is damaged due to a loss-of-function mutation or the like, and more specifically, may be a subject in which the EPM2A gene encoding the Laforin protein is damaged due to a loss-of-function mutation or the like.

[0150] In one specific example, the recombinant virus can be administered at any appropriate dose. The dosage of the recombinant virus can be determined by considering the weight of the subject to achieve a therapeutic effect, and specifically, 1 x 10 11 vg / kg to 5 x 10 14 vg / kg, 5 x 10 11 vg / kg to 1 x 10 14 vg / kg, 5 x 10 11 vg / kg to 5 x 10 13 vg / kg, 5 x 1011 vg / kg to 1 x 10 13 vg / kg or 5 x 10 11 vg / kg to 5 x 10 12 vg / kg, or more specifically about 1 x 10 11 About 1 x 10 13 The dose may be vg / kg.

[0151] In one specific example, the administration may be oral or parenteral administration of the recombinant virus, and when parenterally administering the recombinant virus, it may be administered by external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, percutaneous injection, intranasal injection, intraenteric injection, local injection, sublingual injection, rectal injection, ventricular injection, intracerebral microinjection, intrathoracic injection, or intrathecal injection. Specifically, it may be administered by intravenous injection, ventricular injection, or intrathoracic injection.

[0152] Another aspect provides the use of a recombinant virus comprising a polynucleotide encoding the Laforin protein for the manufacture of a medicament for the prevention or treatment of Lafora disease.

[0153] The above terms “Lafora disease”, “prevention”, “treatment”, “Laforin”, “protein”, “polynucleotide” and “recombinant virus” may be within the scope described above.

[0154] In one specific example, the recombinant virus may be at least one selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, herpes simplex virus (HSV), bacteriophage, parvovirus, baculovirus, and sindbis virus, and more specifically, may be an adeno-associated virus.

[0155]

[0156] A recombinant virus produced using a vector for producing a recombinant virus according to one aspect exhibits an excellent preventive or therapeutic effect for Lafora disease along with reduced or eliminated immunogenicity by delivering a polynucleotide encoding a Laforin protein to the administered subject. Specifically, the recombinant virus according to one aspect exhibits preventive or therapeutic effects for Lafora disease, such as reducing aggregated Lafora bodies, alleviating brain inflammation, and restoring motor function, by expressing the Laforin protein in the administered subject. Therefore, the vector for producing a recombinant virus and the recombinant adeno-associated virus produced by expressing the same can be utilized in various industrial fields for the purpose of preventing and treating Lafora disease.

[0157]

[0158] Figure 1a is a schematic diagram showing a manufacturing method for producing an animal model induced by Lafora's disease.

[0159] Figure 1b is a diagram showing the results of PCR performed to confirm whether exon 2 was deleted by extracting DNA from wild-type mice and EPM2A knockout mice, and the results of Western blot performed to compare whether Lafora protein was expressed.

[0160] Figures 2a and 2b are diagrams showing the results of PAS staining to determine whether Lafora bodies were formed in the brain tissue of mice in which the EPM2A gene was knocked out to cause Lafora disease.

[0161] Figure 3 is a diagram showing the results of an experiment to determine whether there is motor function impairment in mice that have Lafora disease induced by knocking out the EPM2A gene.

[0162] Figures 4 and 5 are schematic diagrams showing the important structures and structures of a vector (AAV9-hEPM2A) manufactured to include the human EPM2A gene.

[0163] Figure 6 is a diagram showing the expression level of the hEPM2A gene according to the codon optimization tool of hEPM2A.

[0164] Figure 7 is a schematic diagram illustrating the experimental process for confirming the symptom relief and therapeutic effect by administering recombinant AAV9 produced by expressing AAV9-hEPM2A to mice that have knocked out the EPM2A gene and induced Lafora disease.

[0165] Figure 8 is a schematic diagram showing a method for administering recombinant AAV9 produced by expressing AAV9-hEPM2A into a mouse and the dosage administered.

[0166] Figures 9a and 9b are diagrams showing the results of GFP fluorescence analysis to confirm whether the target gene was effectively delivered to the hippocampus and cerebral tissue of mice administered recombinant AAV9 produced by expressing AAV9-hEPM2A.

[0167] Figure 10a is a diagram showing the degree of reduction in Lafora bodies in the CA1 region of mice administered recombinant AAV9 produced by expressing AAV9-hEPM2A in the negative control group and experimental group.

[0168] Figure 10b is a diagram showing the degree of reduction in Lafora bodies in the CA2 region of the hippocampus in the negative control group and experimental group of mice administered recombinant AAV9 produced by expressing AAV9-hEPM2A.

[0169] Figure 11a is a diagram showing the results of GFAP immunostaining performed in the CA1 and CA2 regions of the experimental group and the negative control group to confirm the degree of alleviation of brain inflammation in mice administered recombinant AAV9 produced by expressing AAV9-hEPM2A.

[0170] Figure 11b is a graph showing the level of GFAP expression in the CA1 and CA2 regions of mice administered recombinant AAV9 produced by expressing AAV9-hEPM2A and the negative control group.

[0171] Figure 12 is a diagram showing the results of a rotarod test performed to confirm the degree of recovery of motor function in mice administered recombinant AAV9 produced by expressing AAV9-hEPM2A.

[0172] Figure 13 is a diagram showing the results of a hanging wire test performed to confirm the degree of recovery of motor function in mice administered recombinant AAV9 produced by expressing AAV9-hEPM2A.

[0173] Figure 14 is a diagram showing the results of an inverted screen test performed to confirm the degree of recovery of motor function in mice using recombinant AAV9 produced by expressing AAV9-hEPM2A.

[0174]

[0175] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0176]

[0177] Reference example

[0178] Reference Example 1. Production of a mouse model with EPM2A knockout

[0179] EPM2A (Epilepsy, Progressive Myoclonus Type 2A) knockout mice were generated by deleting exon 2 of the gene encoding the CBM20 domain of the laforin protein, as shown in Figure 1a. This resulted in suppression of laforin protein expression and glycogen accumulation, enabling the generation of an effective animal model of Lafora disease.

[0180] Specifically, sgRNA and Cas9 complexes designed to delete exon 2 were delivered to mouse embryos via electroporation, and the embryos were then transplanted into surrogate mothers. The gDNA of F0 obtained by the above method was extracted and PCR was performed to confirm that exon 2 was normally deleted (Fig. 1b). F0 offspring with the same genotype were crossed to obtain F1 offspring. Finally, Western blot analysis confirmed that laforin protein was not expressed in the cerebral hippocampal tissue of EPM2A knockout mice.

[0181]

[0182] Reference Example 2. Verification of Lafora disease symptom onset in a mouse model with EPM2A knockout.

[0183] Reference Example 2-1. Confirmation of Lafora body formation in a mouse model with EPM2A knockout.

[0184] Periodic-Acid-Schiff (PAS) staining was performed on brain tissues of 4-month-old and 9-month-old mice in which the EPM2A gene was knocked out, and it was confirmed that Lafora bodies were formed in various regions including the cortex, hippocampus, and cerebellum (Figures 2a and 2b).

[0185]

[0186] Reference Example 2-2. Confirmation of motor function loss in an EPM2A knockout mouse model.

[0187] To confirm whether the EPM2A gene-knocked mice manufactured in the above Reference Example 1 had lost motor function, the rotarod test and hanging wire test were performed.

[0188] As shown in Figure 3, the results showed that mice with the EPM2A gene knockout exhibited significantly reduced motor skills compared to wild-type mice at 9 months of age. In other words, it was confirmed that the symptoms of Lafora disease were severely expressed in the mouse model produced over time.

[0189]

[0190] Example

[0191] Example 1. Preparation of a recombinant vector containing the human EPM2A (hEPM2A) gene and comparison of gene expression levels.

[0192] An Adeno-Associated Virus 9 (AAV9) recombinant vector containing the human EPM2A gene sequence was constructed and verified.

[0193] In order to increase the expression rate and reduce immunogenicity in the production of AAV vectors containing the human EPM2A gene, hEPM2A was optimized using the GenScript codon optimization tool and the IDT codon optimization tool, and CpG was removed in the case of the gene optimized using GenSmart.

[0194] hEPM2A without codon optimization (native hEPM2A, SEQ ID NO: 1), hEPM2A optimized with GenScript tool (Genscript-hEPM2A, SEQ ID NO: 2), hEPM2A optimized with IDT tool (IDT-hEPM2A, SEQ ID NO: 3), and hEPM2A with CpG removed (CpG free-hEPM2A, SEQ ID NO: 4) were cloned into AAV vectors and the expression levels of the hEPM2A gene were analyzed. As a result of comparing the expression rates, the expression rate of EPM2A codon-optimized with GenSmart was the highest, and when even CpG was removed, the expression rate was similar to that of native hEPM2A. However, CpG free-hEPM2A (SEQ ID NO: 4) was selected as the expression vector because of the advantage of reduced immunogenicity due to CpG removal (Fig. 6).

[0195] A recombinant vector (AAV9-hEPM2A) containing the CpG free-hEPM2A sequence (CpG free-hEPM2A) of sequence number 4, a CAG promoter, and WPRE was prepared, which is non-pathogenic to humans and can effectively reach the central nervous system (CNS). After preparation of the vector, it was stored at a temperature of -80°C, and when administered, it was injected into the cerebrospinal fluid (CSF) via the intra-cerebroventricular (ICV) or intra-cisterna magna (ICM) route. The main structure and overall schematic diagram of the prepared recombinant vector are shown in Figures 4 and 5, and the entire base sequence of the recombinant vector is the same as the base sequence of SEQ ID NO: 5.

[0196]

[0197] Example 2. Production of a virus comprising a recombinant vector containing the human EPM2A (hEPM2A) gene

[0198] HEK293T cells were used as host cells to produce a recombinant adeno-associated virus by expressing AAV9-hEPM2A produced in Example 1 above.

[0199] Plasmids were transfected into host cells using polyethylenimine (PEI). 15.49 μg of hEPM2A plasmid, 22.45 μg of Rep2 / Cap9 plasmid, and 46.07 μg of helper plasmid were transfected per 150 mm dish (molar ratio 1:1.5:2). A total of 168 μg of PEI was used.

[0200]

[0201] Example 3. Confirmation of effective in vivo delivery of AAV9-hEPM2A.

[0202] To confirm the in vivo delivery efficiency of AAV9-hEPM2A prepared in Example 1, an in vivo experiment was performed using the mouse model prepared in Reference Example 1. The outline of the experimental process is as shown in Fig. 7.

[0203] In the above Reference Example 2-2, 4-month-old mice were confirmed to have formed Lapora bodies, and 1.3 x 10 of AAV9 containing the AAV9-hEPM2A was injected into the mice. 10 A total of 2 μl of intracerebroventricular administration (ICV) was administered at a concentration of 1 μl per ventricle (vg / μl) (Fig. 8). Afterwards, when the mice administered the AAV9 reached 9 months of age, the distribution of AAV9-hEPM2A was confirmed by measuring the degree of GFP fluorescence expression in the cerebral and cerebellar tissues. As a result, as can be confirmed in Figs. 9a and 9b, GFP expression was observed in neurons and glial cells throughout the brain tissue, and the fluorescence was particularly evident in the hippocampus and cerebellum, confirming that AAV9-hEPM2A was effectively delivered into the mouse body.

[0204]

[0205] Example 4. Confirmation of the effect of alleviating Lafora disease symptoms in mice administered AAV9 containing AAV9-hEPM2A.

[0206] Example 4-1. Confirmation of the effect of reducing Lafora bodies in mice administered AAV9 containing AAV9-hEPM2A.

[0207] To confirm the effect of reducing Lafora bodies in mice administered with recombinant AAV9 produced by expressing AAV9-hEPM2A, PAS (Periodic acid-Schiff) staining of brain tissue was performed. The experimental group used mice that were intracerebroventricularly administered the AAV9 of Example 3 above, and as a control group, 1.3 x 10 AAV9 carrying a vector designed to express only GFP without expressing hEPM2A was administered. 10 A total of 2 μl of the drug was administered intravenously, 1 μl per ventricle at a concentration of vg / μl, for a total of 2.6 x 10 10 Mice administered vg were used.

[0208] To stain Lafora bodies in mouse hippocampal tissue, 10 μm thick hippocampal tissues were stained using a PAS staining kit (Agilent, AR16511-2). The procedures were as follows: periodic acid for 11 minutes at 37°C, Schiff's reagent for 11 minutes at 45°C, hematoxylin for 11 minutes, and Bluing solution for 11 minutes. After each step, all sections were washed in running tap water for 5 minutes. After all steps were completed, dehydration was performed in the following order: 70%, 90%, 100%, 100% EtOH, and neo clear for 1 minute each.

[0209] As a result of the experiment, the mouse model administered with the recombinant AAV9 produced by expressing AAV9-hEPM2A showed a decrease in Lafora bodies by approximately 40% in the CA1 (Cornu Ammonis 1) region and approximately 50% in the CA2 (Cornu Ammonis 2) region compared to the negative control group (Figures 10a and 10b). In other words, it was confirmed that the virus produced by expressing AAV9-hEPM2A successfully induced the expression of the hEPM2A gene in cerebral neurons and glial cells, and consequently alleviated the formation of Lafora bodies, thereby having a therapeutic effect on Lafora disease (p<0.0001, n=18).

[0210]

[0211] Example 4-2. Confirmation of anti-inflammatory effect in mice administered AAV containing AAV9-hEPM2A.

[0212] As it is known that inflammation in the brain is induced as one of the symptoms of Lafora's disease, the degree of inflammation relief was confirmed by staining GFAP (Glial fibrillary acidic protein), which is an inflammatory marker and an indicator of astrocyte activity, in the hippocampal tissue of mice administered recombinant AAV manufactured by expressing AAV9-hEPM2A.

[0213] The hippocampal tissue of the mouse used in Example 4-1 was cut into 15 μm thick sections and treated with 0.2% Triton X-100 for 10 minutes to increase the penetration of the antibody. The sections were treated with 2% normal goat serum (NGS, Abcam, ab7481) solution for 1 hour, GFAP primary antibody (Dako, z0334, dilution 1:500) in 2% NGS for 24 hours, secondary antibody (Jackson, 111-585-144, dilution 1:500) in 2% NGS for 2 hours, and DAPI (Invitrogen, B35451, dilution 1:1,000) in 1x PBS for 5 minutes for nuclear staining. After each process, all sections were washed with 1x PBS for 5 minutes.

[0214] As a result of the verification, as shown in Figures 11a and 11b, it was confirmed that the GFAP staining intensity was reduced in mice administered AAV9 containing AAV9-hEPM2A, and specifically, it was confirmed that it was reduced by about 40% in the CA1 (Cornu Ammonis 1) region and about 50% in the CA2 (Cornu Ammonis 2) region compared to the negative control group. In other words, it was confirmed that when AAV9 manufactured by expressing AAV9-hEPM2A was administered, it had the effect of alleviating brain inflammation, which is one of the symptoms of Lafora disease.

[0215]

[0216] Example 4-3. Confirmation of motor function recovery effect in mice administered AAV containing AAV9-hEPM2A.

[0217] To determine the extent of motor function recovery in mice administered recombinant AAV9 produced by expressing AAV9-hEPM2A, the rotarod test, hanging wire test, and inverted screen test were performed using 9-month-old mice used in Example 3 according to the method of Reference Example 2-2. The negative control group was the same control group used in Example 4-1.

[0218] As can be seen in Fig. 12, the results of the Rotarod analysis showed a significant difference in the motor function of mice administered recombinant AAV9. In the case of mice administered recombinant AAV9, unlike the negative control group, the fall latency significantly increased to approximately 250 seconds from day 3, confirming that the motor function was restored to the same level as that of wild-type mice (WT vs. AAV9-Control; p = 0.0091, AAV9-Control vs. AAV9-hEPM2A; p = 0.0455, n = 18).

[0219] The results of the hanging wire test and inverted screen test analysis also showed that compared to the negative control group, the latency time to fall of mice administered recombinant AAV9 increased by approximately 50 and 120 seconds, respectively, confirming that motor function was restored to the same level as that of wild-type mice (Figures 13 and 14).

Claims

1. A vector for producing a recombinant virus comprising a polynucleotide encoding a Laforin protein.

2. A vector for producing a recombinant virus, wherein the polynucleotide encoding the laforin protein in claim 1 is EPM2A (Epilepsy, Progressive Myoclonic, 2A).

3. A vector for producing a recombinant virus, wherein the polynucleotide encoding the laforin protein according to claim 1 has fewer CpG motifs compared to a polynucleotide consisting of the base sequence of sequence number 1.

4. A vector for producing a recombinant virus, wherein the polynucleotide encoding the laforin protein in claim 1 is composed of a base sequence having 90% or more homology with a polynucleotide composed of the base sequence of SEQ ID NO:

4.

5. A vector for producing a recombinant virus according to claim 1, wherein the laporin protein has reduced or eliminated immunogenicity against humans.

6. A vector for producing a recombinant virus according to claim 1, wherein the vector comprises a CAG (cytomegalovirus immediate-early enhancer / chicken β-actin) promoter.

7. A vector for producing a recombinant virus according to claim 1, wherein the vector comprises WPRE (Woodchuck hepatitis B virus post-transcriptional regulatory element).

8. A vector for producing a recombinant virus according to claim 1, wherein the vector is composed of a base sequence having 90% or more homology with a polynucleotide composed of a base sequence of sequence number 5.

9. A recombinant virus comprising a polynucleotide encoding a Laforin protein.

10. A recombinant virus according to claim 9, wherein the polynucleotide encoding the laforin protein has fewer CpG motifs compared to a polynucleotide consisting of the base sequence of SEQ ID NO:

1.

11. A recombinant virus according to claim 9, wherein the polynucleotide encoding the laforin protein is composed of a base sequence having 90% or more homology with a polynucleotide composed of the base sequence of SEQ ID NO:

4.

12. A recombinant virus according to claim 9, wherein the laporin protein has reduced or eliminated immunogenicity against humans.

13. A recombinant virus according to claim 9, wherein the virus is an adeno-associated virus (AAV).

14. In claim 13, the adeno-associated virus is at least one virus selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh43, AAVrh74, and variants thereof.

15. A pharmaceutical composition for preventing or treating Lafora disease comprising the recombinant virus of claim 9.

16. A pharmaceutical composition according to claim 15, wherein the pharmaceutical composition is administered by intracerebroventricular injection, intravenous injection, or intravenous injection.

17. In claim 15, the dosage of the recombinant virus is 1 x 10 11 vg / kg to 1 x 10 15 A pharmaceutical composition having a dosage of vg / kg.

18. A cell for producing a recombinant virus comprising the vector of claim 1.

19. A cell for producing a recombinant virus according to claim 18, wherein the cell is at least one selected from the group consisting of NS / O myeloma cells, HEK 293T cells, Chinese hamster ovary cells (CHO cells), HeLa cells, human amniotic fluid-derived cells, COS cells, Kenine D17 cells, Feline PG4 cells, and stem cells.

20. A method for producing a recombinant virus, comprising the step of transfecting a cell with the vector of claim 1.

21. A method for producing a recombinant virus according to claim 20, wherein the vector is transfected simultaneously with the Rep2 / Cap9 plasmid and the helper plasmid.

22. A method for producing a recombinant virus according to claim 21, wherein the vector, the Rep2 / Cap9 plasmid, and the helper plasmid are transfected at a molar ratio of 1:1 to 2:1 to 3.

23. A method for producing a recombinant virus according to claim 20, wherein the transfection is performed using a PEI (Polyethyleneimine) treatment method.

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