Gene therapy method for treating muscular dystrophy as genetic defect disease, and gene therapy product using same

Abstract

The present invention relates to a gene therapy method capable of continuously and efficiently correcting genes, and a gene therapy product using same. It is expected that the gene therapy product of the present invention can present clues for gene therapy for muscular diseases including Duchenne muscular dystrophy.

Description

Gene therapy method for treating muscular dystrophy as a genetic defect disease, and gene therapy agent using the same

[0001] The present invention relates to a gene therapy method for treating muscular dystrophy as a genetic defect disease, and a gene therapy agent using the same.

[0002] Genetic disorders are diseases caused by abnormalities in the DNA base sequence, which is the main body of genes. They can be divided into autosomal and sex-linked genetic disorders, and a representative autosomal recessive trait disorder is Duchenne muscular dystrophy. Genetic disorders have been considered incurable because the fundamental treatment is to replace defective genes with normal genes. However, with the recent development of technologies such as CRISPR gene editing that can replace abnormal genes with normal genes, the goal of developing gene therapy for targeted diseases is recognized as a challenging task.

[0003] However, in most cases, the size of the normal gene to be introduced is too large to be delivered in a single viral particle, so only a portion of the normal gene is delivered for therapeutic purposes, resulting in a problem in which the protein capable of treating the target disease is not fully synthesized.

[0004] Accordingly, the inventors of the present invention, after continuous and arduous efforts to resolve the aforementioned problems, have developed a gene therapy method capable of continuously and efficiently correcting genes. Accordingly, the present invention relates to this gene therapy method and a gene therapy product utilizing the same. The gene therapy product of the present invention is expected to provide a clue to the gene therapy of muscle diseases, including Duchenne muscular dystrophy, which is a genetic defect disease.

[0005] The present invention has been devised to solve the above-mentioned problems in conventional technology, and relates to a method for treating muscular dystrophy and a gene therapy using the same.

[0006] Accordingly, the present invention provides a gene therapy method for treating muscular dystrophy, a gene therapy agent using the same, and a method for producing the gene therapy agent.

[0007] More specifically, the present invention provides a novel gene therapy agent for treating Duchenne muscular dystrophy and a method for producing the same.

[0008] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0009] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0010] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0011] In one specific embodiment of the present invention, the term "genetic defect disease" is used synonymously with "hereditary disease," and encompasses diseases caused by abnormalities in the DNA base sequence, which is the main body of genes. These can be categorized into autosomal gene defect diseases and sex-linked gene defect diseases, each of which can be further categorized into recessive trait defect diseases and dominant trait defect diseases.

[0012] Duchenne muscular dystrophy (DMD), the genetic defect of the present invention, is a severe, progressive disease that causes muscle wasting. It occurs in approximately 1 in 5,000 newborn males. The overall number of patients in the United States and Europe is reported to be 8 to 10 per 100,000 people. Symptoms can appear as early as 2 to 3 years of age, leading to difficulty walking and the use of a wheelchair around the age of 12. Even with ongoing medical assistance specialized for the condition, most patients die before their mid-40s from complications involving cardiorespiratory failure. The cause of the disease is a genetic defect that prevents the expression of the dystrophin protein. Dystrophin protein links proteins responsible for muscle rigidity and contractility and helps synchronize the contraction cycle of muscle cells. However, dystrophin gene with 1-2 bases deleted or added due to congenital genetic abnormality or acquired mutation produces small dystrophin protein during the translation process, and these are non-functional or unstable and quickly disappear from muscle cells, causing Duchenne muscular dystrophy. Anti-inflammatory glucocorticoids such as prednisone or deflazacort are prescribed to treat Duchenne muscular dystrophy, but they only have the effect of delaying muscle degeneration by 1-2 years and are not a fundamental treatment, and their mechanism of action is not precisely known. Recently, gene therapy has been attempted to enhance the expression of the dystrophin protein. For example, eteplirsen is a type of antisense molecule that is a small pseudonucleotide that has the effect of inducing the transcription region containing the mutation to be skipped (exon skipping) when the transcript (pre-mRNA) of the dystrophin gene is matured.However, eteplirsen targets only specific mutations and is highly ineffective, applicable to only about 14% of Duchenne muscular dystrophy patients. Furthermore, in patients treated with eteplirsen, dystrophin protein expression, functional recovery, and functional maintenance were not sustained at high levels. Another example of gene therapy, ataluren, also had limitations, failing to significantly induce dystrophin protein synthesis.

[0013] The gene therapy method of the present invention and the gene therapy agent using the same can be used to treat muscular dystrophy, more specifically, Duchenne muscular dystrophy.

[0014] The present invention provides a gene therapy agent for treating the muscular dystrophy disease described above. Gene therapy herein refers to a therapeutic agent for editing a target gene that causes a genetic defect disease, and editing herein includes a series of manipulations for replacing, deleting, or inserting all or part of the gene. The gene therapy agent of the present invention is characterized by using two recombinant adeno-associated virus (AAV) systems. The AAV may optionally include a single-stranded genome (ssAAV) or a self-complementary genome (scAAV), or the AAV may be any one selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and variants modified from any one or more of these. Specifically, the gene therapy agent of the present invention may be a recombinant adeno-associated virus (AAV) system designed as illustrated in FIG. 1 or FIG. 2. The gene therapy agent of the present invention is implemented with two AAV systems, which include a first AAV gene vector that edits a target gene to be treated for a genetic defect, and a second AAV gene vector, wherein the first AAV gene vector includes a nucleic acid encoding an N-terminal region of a target protein from which the target gene is translated, and a nucleic acid encoding a first guide peptide (GP1), and the second AAV gene vector includes a nucleic acid encoding a C-terminal region of the target protein from which the target gene is translated, and a nucleic acid encoding a second guide peptide (GP2). The guide peptide (GP) refers to a peptide that binds to the N-terminal of a target protein and induces overexpression or inhibition of the function of the target protein. Therefore, the target protein is located at the C-terminal of the guide peptide on the side of the fusion protein in which the guide peptide and the target protein are bound.In a preferred embodiment, the guide peptide pair (GP1 and GP2) of the present invention may be a coiled coil peptide pair. Each AAV gene vector constituting the recombinant adeno-associated virus (AAV) system of the present invention comprises a nucleic acid encoding the guide peptide together with all or part of a nucleic acid encoding dystrophin protein as a target gene to be treated for muscular dystrophy, and may further comprise a nucleic acid encoding a promoter, polyadenylation, and / or Kozak, etc. When the gene therapy agent of the present invention is implemented with two AAV systems, it is preferable that the first AAV gene carrier sequentially comprises a promoter, a dystrophin protein (all or part) as a target protein, a guide peptide, and a nucleic acid encoding polyadenylation, and the second AAV gene carrier sequentially comprises a promoter, a guide peptide, a dystrophin protein (all or part) as a target protein, and a nucleic acid encoding polyadenylation. The above promoter may be any one selected from the group consisting of a U6 promoter, a SNR6 promoter, a SNR52 promoter, a SCR1 promoter, a RPR1 promoter, a U3 promoter, a H1 promoter, an EFS promoter, a CMV promoter, a CAG promoter, a PGK1 promoter, a CK8 promoter, a myosin heavy chain kinase (MHCK) promoter, a muscle creatine kinase (MCK) promoter, a synthetic C5-12 (Syn) promoter, and an unc45b promoter, and is preferably, but not limited to, a CAG promoter or a PGK1 promoter. In addition, the recombinant adeno-associated virus (AAV) system of the present invention may further include a nucleic acid motif that functions as a protein translation initiation site, and / or a nucleic acid motif that functions as a termination site.

[0015] Specifically, the present invention provides a gene therapy for treating Duchenne muscular dystrophy. The therapeutic protein thereof is dystrophin, and the nucleic acid encoding the same may be defined as NM_004006.3. In this case, a first AAV gene carrier among the recombinant adeno-associated virus (AAV) systems provided in the present invention may include Rod domain 1 to 3 regions, Rod domain 15 to 17 regions, and Guide peptide 1 among the nucleic acids, and a second AAV gene carrier may include Rod domain 22 to 24 regions, and Guide peptide 2 among the nucleic acids. Specifically, the first AAV gene carrier may include SEQ ID NO: 7, and the second AAV gene carrier may include SEQ ID NO: 8, but is not limited thereto.

[0016] The gene therapeutic agent of the present invention can be replaced with a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to a sequence represented by any one of SEQ ID NOs: 1 to 8.

[0017] In one embodiment of the present invention, a recombinant adeno-associated virus (AAV) system is provided, comprising: a first AAV gene vector comprising a nucleic acid encoding an N-terminal region of a target protein and a nucleic acid encoding a first guide peptide; and a second AAV gene vector comprising a nucleic acid encoding a C-terminal region of the target protein and a nucleic acid encoding a second guide peptide; wherein the target protein is dystrophin.In the recombinant adeno-associated virus (AAV) system of the present invention, the adeno-associated virus (AAV) is any one selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and a variant modified from any one or more of them, and the recombinant adeno-associated virus (AAV) system is provided, wherein the first guide peptide and the second guide peptide are coiled coil peptide pairs, and the first AAV gene vector or the second AAV gene vector further comprises any one or more selected from the group consisting of a promoter and polyadenylation, and the promoter is a U6 promoter, an SNR6 promoter, an SNR52 A recombinant adeno-associated virus (AAV) system is provided, wherein the recombinant adeno-associated virus (AAV) system comprises any one selected from the group consisting of a promoter, an SCR1 promoter, an RPR1 promoter, a U3 promoter, an H1 promoter, an EFS promoter, a CMV promoter, a CAG promoter, a PGK1 promoter, a CK8 promoter, a myosin heavy chain kinase (MHCK) promoter, a muscle creatine kinase (MCK) promoter, a synthetic C5-12 (Syn) promoter, and an unc45b promoter.

[0018] In another specific embodiment of the present invention, a pharmaceutical composition for treating muscular dystrophy disease is provided, comprising the recombinant adeno-associated virus (AAV) system of the present invention as an active ingredient. In the pharmaceutical composition of the present invention, the muscular dystrophy is Duchenne muscular dystrophy, and a pharmaceutical composition for treating muscular dystrophy is provided, wherein the first AAV gene vector comprises Rod domain 1 to 3 regions, Rod domain 15 to 17 regions, and guide peptide 1 of a nucleic acid encoding a dystrophin protein, and the second AAV gene vector comprises Rod domain 22 to 24 regions of a nucleic acid encoding a dystrophin protein, and guide peptide 2, and wherein the first AAV gene vector is represented by SEQ ID NO: 7, and the second AAV gene vector is represented by SEQ ID NO: 8.

[0019] In another specific embodiment of the present invention, a transformant transformed with the recombinant adeno-associated virus (AAV) system of the present invention is provided, wherein the transformant is a bacterium, a plant cell, or an animal cell other than a human.

[0020] In another specific embodiment of the present invention, a method for producing a recombinant adeno-associated virus (AAV) system is provided, comprising the steps of: (a) preparing a first AAV gene vector comprising a nucleic acid encoding an N-terminal region of a target protein and a nucleic acid encoding a first guide peptide; and (b) preparing a second AAV gene vector comprising a nucleic acid encoding a C-terminal region of the target protein and a nucleic acid encoding a second guide peptide, wherein the target protein is dystrophin.In the method for producing a recombinant adeno-associated virus (AAV) system of the present invention, the adeno-associated virus (AAV) is any one selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and a variant modified from any one or more of them, and the method for producing a recombinant adeno-associated virus (AAV) system is provided, wherein the first guide peptide and the second guide peptide are coiled coil peptide pairs, and the first AAV gene vector or the second AAV gene vector further comprises any one or more selected from the group consisting of a promoter and polyadenylation, and the promoter is a U6 promoter, A method for producing a recombinant adeno-associated virus (AAV) system is provided, wherein the recombinant adeno-associated virus (AAV) system comprises any one selected from the group consisting of a SNR6 promoter, a SNR52 promoter, a SCR1 promoter, a RPR1 promoter, a U3 promoter, a H1 promoter, an EFS promoter, a CMV promoter, a CAG promoter, a PGK1 promoter, a CK8 promoter, a myosin heavy chain kinase (MHCK) promoter, a muscle creatine kinase (MCK) promoter, a synthetic C5-12 (Syn) promoter, and an unc45b promoter.

[0021] In another specific embodiment of the present invention, a method for producing a transformant is provided, comprising the step of transfecting a bacterium, a plant cell, or an animal cell other than a human with the recombinant adeno-associated virus (AAV) system of the present invention.

[0022] In another embodiment of the present invention, a method for treating muscular dystrophy is provided, comprising administering to a subject in need of treatment a recombinant adeno-associated virus (AAV) system or pharmaceutical composition of the present invention. In the treatment method of the present invention, the muscular dystrophy may preferably be Duchenne muscular dystrophy.

[0023] In another embodiment of the present invention, the recombinant adeno-associated virus (AAV) system of the present invention, or the pharmaceutical composition thereof, is provided for use in treating muscular dystrophy. In the therapeutic use of the present invention, the muscular dystrophy may preferably be Duchenne muscular dystrophy.

[0024] The present invention relates to a gene therapy method for treating muscular dystrophy, a genetic defect disease, and a gene therapy product using the same. The gene therapy product of the present invention is expected to provide a clue to gene therapy for muscle diseases, including Duchenne muscular dystrophy.

[0025] Figure 1 is a schematic diagram of dual AAV gene therapy according to one embodiment of the present invention. (Figure 1a) Two recombinant AAVs contain a genome consisting of a coding sequence of protein A or protein B, which includes a 5'-ITR, a promoter (P), a polyadenylation signal (A), a 3'-ITR, and a guide peptide (GP1 or GP2, respectively). Upon delivery, protein A-GP1 and GP2-protein B are co-expressed and assembled into heterodimers. (Figure 1b) Heterodimer formation through highly specific and stable interactions of guide peptide pairs is mediated by Cypet / Ypet and dystrophin-derived DNH / Dp116. + It has been proven about.

[0026] Figure 2 illustrates a recombinant vector expressing a protein containing a GP pair according to one embodiment of the present invention. (Figure 2a) pRP-Cypet-GP1 vector map. (Figure 2b) pRP-GP2-Ypet vector map. (Figure 2c) pRP-DNH vector map. (Figure 2d) pRP-Dp116 + Vector map. In the drawing, P represents the CAG promoter, ori represents the pUC replication origin, GP1 and GP2 represent guide peptide 1 and guide peptide 2, and poly(A) represents the BGH polyadenylation signal (Figs. 2a and 2b) or the SV40 late polyadenylation signal (Figs. 2c and 2d).

[0027] Figure 3 shows the results of heterodimerization of Cypet and Ypet through GP pairs according to one embodiment of the present invention. (Figure 3a) rAAV expressing Ypet or GP2-Ypet was added to C2C12 cells cultured in 96-well culture plates at the indicated titers, and the cells were examined under a fluorescence microscope after 2 days. (Figure 3b) C2C12 cells were transduced with pRP vectors expressing the indicated proteins, and the expression of Ypet or GP2-Ypet was examined after 2 days. (Figure 3c) C2C12 cells were transduced with pRP vectors expressing the indicated proteins, and after culturing for 2 days, whole cell lysates were prepared, and a portion (10%) was subjected to Western blot analysis using an anti-GFP antibody. The relative densities of Western signals are indicated at the bottom.

[0028] Figure 4 shows the heterodimerization results between a dystrophin-derived peptide containing a fluorescent protein and GP according to one embodiment of the present invention. (Figure 4a) C2C12 cells were transfected with pRP vectors expressing the designated proteins, and examined under a fluorescence microscope 48 hours later. (Figure 4b) C2C12 cells were transfected with pRP-Cypet-GP1 alone (-) (lanes 2 and 4) or pRP-GP2-Dp116. +Transfected with (lanes 1 and 3). Total cell lysate (TCL) was subjected to IP using M2 affinity gel. Flag-tagged GP2-Dp116 was detected in TCL (4%) and IP pellet (IPP) (10%). + Western blotting was performed to confirm the presence of (left panel) or Cypet-GP1 (right panel). GP2-Dp116 + The slowly moving form is indicated by a triangle, and the slowly moving form is indicated by an asterisk. The relative density of the Western signal is indicated at the bottom. (Fig. 4c) C2C12 cells were treated with pRP-Cypet-GP1 (0.5 μg) and gradually increasing amounts of pRP-GP2-Dp116 + Transformed with. Lane 1, none; lane 2, 0.25 μg; lane 3, 0.5 μg; lane 4, 1.0 μg. After culturing for 2 days, TCL was prepared with RIPA buffer and analyzed by Western blotting using anti-GFP antibody. The relative density of Western signals is indicated at the bottom.

[0029] Figure 5 shows the optimization results of GP pairs for forming heterodimers composed of DNH and Dp116 according to one embodiment of the present invention. DNH and Dp116 with flag tags containing various combinations of GP pairs. + was co-expressed in C2C12 cells. (Fig. 5a) TCL prepared with IP lysis buffer was applied to IP using M2 affinity gel. TCL (4%) and IP pellet (10%) were analyzed by Western blotting for DNH and Dp116. + The presence of DNH was confirmed. The slow-moving form of DNH is indicated by an asterisk. (Fig. 5b) The relative efficiency of heterodimer formation between DNH and Dp116 derivatives is summarized, with '+++' and '-' indicating the highest and undetectable levels, respectively. The footnotes a and b indicate the number of WINZIPB1 and WINZIPA2, respectively.

[0030] Figure 6 is a recombinant vector expressing α1-syntrophin and α-dystrobrevin 2 according to one embodiment of the present invention. (Figure 6a) α1-syntrophin vector map. (Figure 6b) α-dystrobrevin 2 vector map. The full-length coding sequences of hSNTA1 and hDTNA are referenced in NM_003098.3 and NM_032975.4, respectively.

[0031] Figure 7 shows the results of confirming the interaction between Dys203 and α1-syntrophin (α1-Syn) and α-dystrobrevin 2 (α-DB2) according to one embodiment of the present invention. Flag-tagged DNH was co-expressed with designated V5-tagged proteins in C2C12 cells. TLC prepared with IP dissolution buffer was applied to IP using M2 affinity gel, and TLC (2%) and IP pellet (10%) were Western blotted with anti-Flag or anti-V5 antibodies to determine DNH, Dp116. + , the presence of α1-Syn and α-DB2 was examined. Slow-moving forms of DNH and α-DB2 are indicated by asterisks.

[0032] Figure 8 is a schematic diagram of AM203 according to one embodiment of the present invention. (Figure 8a) DNH-GP1 (pDNH) and GP2-Dp116 + (pDp116 + ) is the expression cassette structure. (Fig. 8b) AM203 is rAAV9-DNH and rAAV9-Dp116 + It consists of DNH and Dp116 +is expressed to form a heterodimer Dys203 upon transduction. In the figure, ABD represents the actin-binding domain, H represents the hinge region, SR represents the spectrin repeat, CR represents the cysteine-rich domain, CT represents the C-terminal domain, and ITR represents the AAV2 inverted repeat.

[0033] Figure 9 shows the results of an expression specificity study of promoters used in a gene therapy for DMD according to one embodiment of the present invention. Genes whose expression specificities were compared include DMD (dystrophin; Figure 9a), ACTC1 (actin alpha cardiac muscle 1; Figure 9b), CKM (creatine kinase, M type; Figure 9c), MYH3 (myosin heavy chain 3; Figure 9d), and PGK1 (phosphoglycerate kinase 1; Figure 9e). Tissue expression specificity was referenced from the Human Protein Atlas (www.proteinatlas.org).

[0034] Figure 10 illustrates the structures of dystrophin isoforms and recombinant dystrophin expressed by DMD gene therapy according to one embodiment of the present invention. Full-length dystrophin isoforms such as Dys427, Dys260, Dys140, and Dp116 are depicted. Gene therapy drugs PF06939926 (Pfizer) and SGT-001 (Solid Biosciences) express μDys1 and μDys3, respectively. The former utilizes the MSP promoter, and the latter utilizes the CK8 promoter. SRP9001 (Sarepta Therapeutics) and GNT0004 (Genethon) both express μDys2 and utilize different promoters such as MHCK7 and Spc5.12, respectively. Dys203 is a heterodimeric microdystrophin expressed by AM203 under the control of the mPGK1 promoter.

[0035] Figure 11 shows the results of confirming the therapeutic efficacy of AM203 according to one embodiment of the present invention. (Figure 11a) These are the results of the downhill treadmill running test measured at 4 and 8 weeks after AM203 administration. The mean value of each group data is indicated by a bar. The data were analyzed by one-way ANOVA followed by Tukey post-hoc analysis for multiple comparisons, and **: p<0.01, *: p<0.05. (Figure 11b) These are the results of body weight measured at 0, 4, 8, and 12 weeks after AM203 administration. (Figure 11c) The wire hanging test was performed at 12 weeks after AM203 administration, and the serum CK level was measured 1 day later. G1 is WT (n=6), G2 is mdx treated with saline (n=6), and G3 is AM203 (6x10 12 It represents mdx(n=8) processed with vg capacity.

[0036] Figure 12 shows the results of tracking the biodistribution of AM203 and Dys203 expression in mdx according to one embodiment of the present invention. (Figure 12a) TaqMan qPCR was performed with designated tissue lysates prepared from mdx (n=3) treated with AM203. Data obtained as vg / μg DNA are expressed as mean ± SEM. (Figure 12b) Dys203 expression was confirmed by Western blot analysis using samples of heart extracts. Lane 1 is WT, lane 2 is mdx treated with saline, and lanes 3-5 are mdx treated with AM203. The upper panel shows Dys203, and the lower panel shows GAPDH. (Figures 12c and 12d) Western blot analysis of Dys203 (upper panel) and nNOS (middle panel) expression in designated muscles and organs of mdx treated with AM203. The bottom panel is GAPDH.

[0037] Figure 13 shows the results of confirming the expression and localization of Dys203 in mdx treated with AM203 according to one embodiment of the present invention. Representative immunofluorescence stainings in various muscle tissues are presented. Thin is WT, I is mdx treated with saline, and C and R are mdx treated with AM203. The scale bar represents 100 μm.

[0038] Figure 14 shows the results of confirming the expression of nNOS in mdx treated with AM203 according to one embodiment of the present invention. Representative immunofluorescence staining in GAS sections is presented. a is WT, b is mdx treated with saline, c and d are mdx treated with AM203, and the scale bar represents 100 μm.

[0039] The therapeutic efficacy was explored through preclinical studies using dystrophic animal models. Specifically, 6 × 10 per mouse were administered via tail vein to mdx mice at 5 weeks of age. 12 AM203 was administered at a dose of 2.6× 10 14 Equivalent to vg / kg.

[0040] Downhill running on a treadmill is known to produce eccentric muscle contractions in vivo, but mdx mice have little tolerance for this. In fact, the average running distance of PBS-treated mdx mice (G2) at 4 weeks after treatment was 416±225 m, which was only about 40% of that of wild-type (WT) mice (1027±26 m), and at 8 weeks, the running distance of mdx mice (214±61 m) had decreased to 26% of that of WT mice (829±130 m). In contrast, mdx mice administered AM203 showed significantly improved performance compared to control mdx mice at 4 weeks (967±103 m) and 8 weeks (566±241 m).

[0041] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0042] Example.

[0043] [Research Methods]

[0044] 1. Materials

[0045] The materials used for manufacturing the gene therapy agent of the present invention and evaluating its effectiveness are described below.

[0046] 10x Bolt™ Sample Reducing Agent (Thermo Fisher, B0009), 1kb DNA Marker (Biosesang, GF6055-123-03), 20x Bolt™ MES SDS Running Buffer (Thermo Fisher, B0002), 2-Propanol (SANCHUN, 67-63-0), 4x Bolt™ LDS Sample Buffer (Thermo Fisher, B0007), 50x TAE (LPS Solution, CBT002), Agarose LE (Merck, 11685660001), Antibiotic Antimycotic Solution (Merck, A5955), Antidystrophin Monoclonal Antibody (JF1-022) (Thermo Fisher, MA5-32565), Antidystrophin Antibody, Clone 2C6 (MANDYS106) (Merck, ZMS1051), Anti-FLAG® M2 Affinity Gel (Merck, A2220), anti-gamma tubulin antibody [EPR16793] (abcam, ab179503), anti-GAPDH antibody (Thermo Fisher, PA5-85074), anti-GFP antibody (abcam, ab290), anti-nitric oxide synthase antibody (Merck, N7280), anti-syntrophin alpha 1 antibody [EPR14282] (Abcam, ab188873), Blot™ 4-12% Bis-Tris Plus (Thermo Fisher, NW04125BOX), Blot™ 8% Bis-Tris Plus gel (Thermo Fisher, NW00082BOX), type I collagen solution (Merck, C3867), dimethyl sulfoxide (Merck, D2650), Dulbecco's modified Eagle's medium (DMEM) (Welgene, LM001-05), DPBS (Welgene, LB001-02), DYKDDDDK tag polyclonal antibody (Thermo Fisher, 740001), dystrophin (4C7) (Santa Cruz, sc33697), fetal bovine serum (GW Vitek, US-FBS-500), glycine (LPS solution, GLY05), GFP polyclonal antibody (Thermo Fisher, A-11122),Goat anti-mouse IgG (H+L) secondary antibody, HRP (Thermo Fisher, 62-6520), goat anti-mouse IgG H&L (Alexa Fluor® 488) (Abcam, ab150113), goat anti-rabbit IgG (H+L) secondary antibody, HRP (Thermo Fisher, 62-6120), goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam, ab150077), goat serum donor hub (Merck, G6767), Halt™ protease inhibitor (100x), 0.5 M EDTA solution (Thermo Fisher, 78430), HEPES solution (Merck, H0887), horse serum (Merck, H1270), IGEPAL® CA-630 (Merck, I3021), lipofectamine™ 3000 Transfection reagent (Thermo Fisher, L3000075), methanol (Merck, 1.06009.1011), mouse monoclonal eCFP antibody (Origene, TA180069), M-PER™ buffer (Thermo Fisher, 78503), Opti-MEM™ (Thermo Fisher, 31985062), PageRuler™ and pre-stained protein ladder (Thermo Fisher, 26619), paraformaldehyde (Merck, 8.18715), phenylmethylsulfonyl fluoride (PMSF) (Merck, 11359061001), Pierce™ IP lysis buffer (Thermo Fisher, 87787), poly-D-lysine hydrobromide (Merck, P0899), potassium chloride (Merck, P9333), ProLong™ with DAPI Diamond antifade mountant (Thermo Fisher, P36962), protein analysis staining reagent concentrate (Bio-Rad, 5000006), Amersham™ Hybond P 0.2 PVDF membrane (Cytiva, 10600021), Safe Shine Blue (6x) (Biosesang, GC6050-001-00), SDS solution 20% (w / v) (Bio-Rad, 1610418),Sodium chloride (Merck, 10.6404.5000), SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher, 34094), TaKaRa PCR Mycoplasma Detection Set (TaKaRa, 6601), Tris (LPS solution, TRI05), Triton™ X-100 (Merck, T9284), Trypan Blue Solution (Merck, T8154), Trypsin-EDTA (0.25%) (Thermo Fisher, 25200056), Tween 20 (Bio-Rad, 1706531), V5 Tag polyclonal antibody (Thermo Fisher, PA1-993), Western blotting filter paper (Thermo Fisher, 84783).

[0047] 2. Mammalian expression vectors and recombinant AAV

[0048] pRP-Cypet, pRP-Ypet, pRP-Cypet-GP1, pRP-GP2-Ypet, pRP-DNH, and pRP-Dp116 used in this study + All vectors, including pRP-V5-hSNTA1 and pRP-hDTNA-V5, were produced by VectorBuilder, a CRO company. Their domain structures are shown in Figs. 2 and 6. rAAV9-DNH and rAAV9-Dp116, which constitute AM203 + Vectors were also created in VectorBuilder, and their domain structures are shown in Figures 8 and 10. AM203 (rAAV9-DNH and rAAV9-Dp116 + A 1:1 mixture of AAV stock buffer (1x PBS supplemented with 200 mM NaCl and 0.001% Pluronic F-68) was used to give a final titer of 6x 10 13 It was manufactured to be vg / ㎖.

[0049] 3. Animal models and in vivo gene transfer

[0050] Animal studies were performed by KeyFronBio, a CRO company, in compliance with the Animal Protection Act No. 4379 and its amendment No. 16977, with the approval of the Institutional Animal Care and Use Committee (Approval No. KA24019). Specifically, 4-week-old male dystrophin-deficient C57BL / 10ScSn-Dmdmdx / J and wild-type C57BL / 10 control mice were purchased from Jackson Laboratory, and two mice per cage were housed in Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C under a 12-h light cycle (7:00 AM to 7:00 PM) during the study period. One week after receipt, mice were injected intravenously with AAV storage buffer (n=6) or AM203 (6x 10 12 vg dose) (n=8) 0.1㎖ was injected. Afterwards, the general symptoms of the mice (appearance, behavior, excretion, etc.) were monitored daily, and body weight was measured every two weeks.

[0051] 4. Cell culture and transient DNA transformation

[0052] Mouse myoblast cell line C2C12 obtained from ATCC was cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution in a 5% CO2 incubator at 37°C. 8.0 × 10 5 or 2.0× 10 5 Cells were seeded onto 6-well culture plates coated with poly-D-lysine or type I collagen, cultured overnight, and then transfected the following day with mammalian expression vectors (1.6 μg) premixed with 3.2 μl P3000™ reagent and 7.5 μl lipofectamine™ 3000 in Opti-MEM™ reduced serum medium according to the manufacturer's instructions. When multiple expression vectors were co-transfected, the ratio of total DNA amount (μg) to P3000™ volume (μl) was maintained at 1:2.

[0053] 5. Immunoprecipitation (IP) and Western blot analysis

[0054] Forty-eight hours after transfection, total cell lysates (TCL) were prepared using RIPA, M-PER™, or IP lysis buffer containing Halt™ protease inhibitor (0.5 ml per well). TCL was centrifuged at 13,000–14,000 × g for 10–15 min at 4°C, and the TCL supernatant (400 μl) was transferred to a microcentrifuge tube and mixed with anti-FLAG® M2 affinity gel (20 μl). The mixture was mixed on a nutator at 4°C for 1 h and centrifuged at 8,200 × g for 1 min. Supernatants (SPNT) were transferred to new tubes, and the IP pellet was washed four times with 1x TBS buffer and then added to an equal volume of 2x LDS sample buffer. The mixture was heated at 90°C for 10 minutes, centrifuged at 8,200xg at 4°C for 1 minute, and the supernatant was transferred to a new tube and used for Western blot analysis.

[0055] Total cell lysate (TCL) and IP pellet (IPP) were loaded on Blot™ 8% Bis-Tris plus gels for SDS-PAGE, and immersed in transfer buffer consisting of 48 mM Tris, 38.9 mM glycine, 20% methanol, and 0.05% SDS for 5 minutes. Proteins were then transferred to PVDF membranes using Trans-Blot® Semi-Dry (Bio-Rad 170-3940) at 15 V for 5 minutes, 25 V for 40 minutes, and 15 V for 5 minutes. The PVDF membrane was washed with 1x TBST buffer and treated with blocking buffer (5%, w / v) non-fat dry milk in 1x TBST buffer for 15 minutes. After washing three times with 1x TBST buffer, the PVDF membrane was incubated with the indicated anti-dystrophin, anti-FLAG, or anti-V5 antibodies (1:1000) for 45 minutes. After washing three times with 1x TBST buffer, the PVDF membrane was incubated with goat anti-rabbit IgG-HRP conjugate (1:5000 dilution) or goat anti-mouse IgG-HRP conjugate (1:5000 dilution) for 45 minutes. The membrane was then washed four times with 1x TBST buffer, and the proteins were visualized using enhanced SuperSignal™ west femto maximum sensitivity substrate and a chemiluminescence imaging system (Clinx, ChemiScope 6100) according to the manufacturer's instructions. The density of the Western signal was quantified using the software built into the imaging system.

[0056] 6. Determination of rAAV9 biodistribution

[0057] Tissue samples (1–2 mm) collected from 17-week-old mice were subjected to qPCR analysis using the TaqMan Sample-to-SNP Kit (Thermo Fisher) and TaqMan probes according to the manufacturer's recommended procedures. Briefly, only a portion of genomic DNA (4 μl) prepared using Lysis solution (50 μl) and DNA stabilization solution (50 μl) was added to a PCR reaction (20 μl) provided with two sets of TaqMan pre-made primers (Thermo Fisher Assay ID Hs00124667-cn and Hs00117207-cn) (0.2 μM each) and two MGB probes (0.1 μM each), namely the FAM-MGB probe (493 / 517 nm) for Hs00124667-cn and the VIC-MGB probe (526 / 543 nm) for Hs00117207-cn. Thermo Fisher Assay ID Hs00124667-cn is designed to amplify exon 3 of the dystrophin gene present in rAAV9-DNH. Assay ID Hs00117207-cn is designed to amplify exon 3 of the dystrophin gene present in rAAV9-Dp116. + Amplifies exon 57 encoding SR23 including 40 fg to 40 ng of pRP-Dp116 + Standard qPCR reactions provided were run in parallel to determine the copy number of the rAAV9 vector, which was calculated according to the following equation [Equation 1].

[0058] [Formula 1]

[0059] y = -1.421ln(x) + 40.541 (R² = 0.9992)

[0060] Here, y is the threshold cycle (CT) and x is the copy number.

[0061] The copy number of rAAV9 vector was expressed as vg / μg genomic DNA.

[0062] 7. Immunofluorescence staining

[0063] At 17 weeks of age, the quadriceps femoris, gastrocnemius, tibialis anterior, triceps, biceps, diaphragm, heart, gluteus maximus, testis, kidney, liver, lung, psoas major, spleen, and brain were dissected from all mice and snap-frozen in liquid nitrogen-cooled 2-methylbutane (Sigma Aldrich). 8-mm cross-sections were cut along the entire length of the muscle tissue using a Shandon cryotome (Thermo Fisher) on Superfrost Plus slides (Thermo Fisher) with an inter-slice spacing of 240 mm. Excess tissue between sections was collected into MagNa Lyser Green Beads tubes (Roche) for RT-qPCR and Western blot analysis.

[0064] For immunofluorescence staining of frozen tissue sections, sections were thawed at room temperature and air-dried for 5 minutes. After fixation with cold 4% paraformaldehyde (1% for syntrophin detection) in 1x PBS for 5 minutes, the sections were washed three times in 1x PBS. The sections were permeabilized in 1x PBS containing 0.1% Triton X-100 and 0.1 M glycine for 10 minutes, and then washed three times in 1x PBS. After incubation for 30 min with blocking buffer (5% goat serum, 0.01% Triton X-100 in 1x PBS), sections were reacted overnight at 4°C with the following primary antibodies: anti-dystrophin antibody (Thermo Fisher, MA5-32565, 1:200; Santa Cruz, sc33697, 1:50), anti-nNOS antibody (Merck, N7280, 1:5000), or anti-syntrophin antibody (abcam, ab188873, 1:250). The following day, the sections were washed three times with 1x PBS and treated with secondary antibodies, Alexa 488 goat anti-rabbit IgG (abcam, ab150077, 1:500) or Alexa 488 goat anti-mouse IgG (abcam, ab150113, 1:500), for 1 h. Finally, the sections were washed four times with 1x PBS, treated with DAPI / Permount, and examined with an Axiovert 200 (Carl Zeiss) using appropriate filters.

[0065] 8. Downhill treadmill and wire suspension tests

[0066] After administration of the specified rAAV9 to mice, muscle strength and endurance were assessed every 4 weeks for 12 weeks. Mice were placed on a Simplex II treadmill (Columbus Instruments) and initially run at a speed of 10 m / min for 15 min on a treadmill with a 15° downward angle. The speed was then increased by 5 m / min every 10 min until the mice stopped running. The average running distance and duration (in minutes) of each experimental group were measured.

[0067] Hanging wire time was measured three times with a 1-minute rest between trials, and the longest hanging time was used for further analysis. Although wild-type mice can hang on the wire for much longer periods, the hanging wire test was discontinued at 600 seconds.

[0068] 9. Determine the SCK level

[0069] To measure serum CK levels, mice were anesthetized with isoflurane and laparotomy was performed. Blood was collected from the abdominal vena cava into serum separator tubes. Blood samples were centrifuged at 3,000 rpm for 10 minutes, and CK levels were measured using the JSCC method using a Labospect-006 (Hitachi).

[0070] [Research Results]

[0071] 1. GP-induced heterodimerization

[0072] The GP pair used in the present invention consists of WinZipA2 and WinZipB1. The WinZipA2 / B1 pair is one of the coiled-coil peptides and exhibits very stable thermodynamic properties (Tm=63.2°C, KD=4.5 nM, ΔG=11.2 kcal / mol).

[0073] In the present invention, two fluorescent proteins, Cypet and Ypet, were used as model proteins to explore the optimal GP pair and the related specificity and efficiency for inducing heterodimer formation in mammalian cells. Since the GP pair is expected to be physically close enough to Cypet and Ypet to influence each other's fluorescence properties, shuttle vectors pRP-Cypet-GP1 and pRP-GP2-Ypet (Fig. 2a and Fig. 2b), which can express Cypet and Ypet in mammalian cells, were used directly in transient DNA transduction assays or used as template DNA to produce recombinant AAVs, such as rAAV1-Cypet-GP1 and rAAV1-GP2-Ypet. The apparent maximal expression level determined by fluorescence microscopy was 3 x 10 6 vg / ㎖ of rAAV1-Ypet or 1x 10 7 A relatively low level of fluorescence from rAAV1-GP2-Ypet was observed at vg / ml (Fig. 3a). A relatively low level of fluorescence from rAAV1-GP2-Ypet was also observed in transient DNA transfection assays using the pRP vector (Fig. 3b, left panel). This result may indicate a lower steady-state level or poor dynamic turnover of GP2-Ypet compared to Ypet. Interestingly, however, co-expression with Cypet-GP1 increased the fluorescence of GP2-Ypet, but not Cypet (Fig. 3b, compare middle and right panels).

[0074] As shown in Fig. 3c, Western blot analysis revealed that the steady-state levels of Ypet (lane 1) and Cypet (lane 7) were significantly higher than those of the derivatives containing GPs (lanes 2 and 8). When co-expressed, the bound protein levels of Cypet-GP1 and GP2-Ypet (lane 4) were significantly higher than their simple sum and approached the levels of Cypet and Ypet (compare lanes 4 and 5). In contrast, the levels of Cypet-GP1 appeared unaffected by co-expression with Ypet (compare lanes 3 and 8), and similar results were obtained with GP2-Ypet and Cypet (compare lanes 2 and 6). Taken together, the results in Fig. 3b and Fig. 3c suggest that addition of individual GPs can alter the intracellular levels of the affected proteins, and that co-expression of two proteins containing a GP pair can increase the steady-state levels through heterodimerization.

[0075] 2. Heterodimer formation using Cypet-GP1 and GP2-Dp116+, or DNH-GP1 and GP2-Ypet

[0076] Encouraged by the results in Figure 3, we tested whether GP pairs could induce physical interactions between two heterologous proteins and, furthermore, whether GP pairs could be applied to gene therapy. To this end, Cypet-GP1 or GP2-Ypet were conjugated with GP2-Dp116, respectively. + or co-expressed with DNH-GP1. DNH and Dp116 + All were derived from human dystrophin, and the domain structures (Fig. 2c, Fig. 2d, and Fig. 8) and sequences (SEQ ID NOs. 1 to 8) of the vectors used were described separately.

[0077] As shown in Fig. 4a, the total fluorescence observed in C2C12 cells expressing Cypet-GP1 or GP2-Ypet was 1.5 times higher than that observed in GP2-Dp116 cells, respectively. +or co-expression with DNH-GP1. To determine whether the fluorescence changes were related to protein levels, Cypet-GP1 was expressed alone or in combination with GP2-Dp116. + Total cell lysates (TCL) were prepared from C2C12 cells expressing GP2-Dp116 + After performing immunoprecipitation (IP) on GP2-Dp116, the resulting IP pellet (IPP) was analyzed by Western blotting. + The presence of Cypet-GP1 was confirmed. As a result of the test, GP2-Dp116 was co-expressed with GP2-Ypet (Fig. 2). + Co-expression with GP2-Dp116 + The steady-state level of Cypet-GP1 increased in proportion to the expression level of Cypet-GP1 (Fig. 4c) (Fig. 4b, right panel, compare lanes 1 and 2). Cypet-GP1 and GP2-Dp116 + IP pellets obtained by TCL from cells expressing both GP2-Dp116 + In addition, Cyper-GP1 was also included (Fig. 4b, lane 3). The results of Fig. 4 suggest that Cyper-GP1 and GP2-Dp116 + This supports the idea that steady-state levels increase as stable heterodimers are formed.

[0078] 3. Expression and formation of heterodimer Dys203

[0079] To develop next-generation gene therapy for DMD, the expression cassette of AM203 was designed to consist of the mPGK1 promoter, a GP pair, and selected functional domains of dystrophin, the domain structures of which are shown in Figures 8 and 10.

[0080] The mPGK1 promoter was chosen for the following reasons: Dystrophin isoforms are synthesized using seven independent tissue-specific promoters and two polyadenylation sites, so mutations in DMD can cause complex symptoms in affected patients through changes in the expression profile of dystrophin isoforms. Expression of μDys1-3 is driven by muscle-specific promoters such as CK8, MHCK7, and MCK. In contrast, as shown in Figure 8, rAAV9-DNH and rAAV9-Dp116 + All have the mPGK1 promoter, which drives the expression and formation of Dys203 in a heterogeneous manner in various tissues including muscle, brain, kidney, and other organs (Fig. 9e).

[0081] To optimize the GP pairs and their compositions that support efficient and specific heterodimerization of dystrophin-derived peptides, mammalian expression vectors were constructed containing an epitope-tagged N-terminal fragment (DNH) fused to GP1 at the C-terminus and Dp116 fused to GP2 at the N-terminus. + ) were expressed (Fig. 3). GP1 and GP2 each contain one or two consecutive WinZipA2 and WinZipB1. Flag-DNH with two WinZipA2, DNH* with one WinZipA2, or DNHΔGP lacking WinZipA2 were expressed in C2C12 cells. + , Dp116*, or Dp116 + ΔGP was expressed together, and after 2 days, total cell lysates were prepared and immunoprecipitated (IP) using M2 affinity agarose. The IP pellet was immunoblotted with Flag-tagged proteins such as DNH, DNH*, or DNHΔGP, as well as Dp116. + , Dp116*, Dp116 +The presence of ΔGP was also analyzed by Western blotting. As shown in Figure 5a, Western blot analysis performed with total lysate showed that Dp116 was co-expressed with Flag-DNH. + ΔGP is Dp116 + showed that it was expressed as efficiently as Dp116 (middle panel, lanes 1 and 2). However, co-immunoprecipitation with Flag-DNH showed that Dp116 + was observed only in (lower panel, lanes 1 and 2). In the absence of GP, DNH was Dp116 + failed to interact with DNH and Dp116 (lower panel, compare lanes 2 and 3, Fig. 4a). This result suggests that the presence of GP1 and GP2 induced the formation of Dys203. + suggests that DNH* containing only WinZipA2 in GP1 is essential and sufficient for stable heterodimerization of Dp116 + Although Dp116* containing only WinZipB1 retained the activity of forming heterodimers with DNH (Fig. 5a lower panel, compare lanes 4 and 5), it showed no or low levels of heterodimerization with DNH as summarized in Fig. 5b. Therefore, DNH and Dp116 + It was concluded that each of them formed a stable heterodimer when equipped with at least one WinZipA2 and two consecutive WinZipB1.

[0082] 4. Formation of DAPC by Dys203

[0083] Based on the results in Figure 5, the activity of DNH to form Dys203, which ultimately forms DAPC, was tested. For this purpose, C2C12 cells were co-transfected with a combination of pRP-DNH, pRP-hSNTA1, and pRP-DTNA1, as shown in Figure 6, and pRP-Dp116. +DNH was prepared with or without Dp116. After culturing for 2 days, total cell lysates (TCL) were prepared and immunoprecipitation (IP) was performed using M2 affinity gel to specifically detect DNH. The resulting IP pellet was analyzed using Western blot to detect Dp116. + , the presence of α1-Syn and α-DB2 was examined.

[0084] Under the conditions used in the present invention, relatively similar levels of DNH were detected by M2 affinity gel regardless of the presence or absence of coexpressed proteins, and representative results of Western blot analysis are shown in Figure 7 (middle panel). Densitometry results showed that IP efficiency was estimated to be over 80% across all experimental groups, and among the tested proteins, the expression of α-DB2 was the most prominent. Coexpression of α-DB2 or α1-Syn alone showed no or low levels of association with DNH (Figure 7, lower panel, lanes 2 and 3). The discernible level of α1-Syn was Dp116. + was detected only when co-expressed (Fig. 7, lower panel, lanes 7 and 9), and Dp116 + The Western signal densities of α-DB2 and α1-Syn were estimated to be approximately 2.1 and 0.25, respectively. Taken together, these results suggest that DNH and Dp116 + This means that they stably interact with each other to form a dimer, Dys203, which then forms DAPC together with α-DB2 and α1-Syn. In addition, although α1-Syn positively influences the binding of α-DB2 to DNH, the stable binding of α1-Syn to Dys203 is dependent on Dp116. + This means that the existence of is required.

[0085] 5. Improvement of dystrophic phenotype by AM203

[0086] After confirming efficient expression and heterodimer formation of Dys203, the therapeutic efficacy was explored through preclinical studies using dystrophic animal models. Specifically, 6 × 10 per mouse were injected into mdx mice via tail vein at 5 weeks of age. 12 AM203 was administered at a dose of 2.6×10 14 corresponds to vg / kg. Initial body weight measurements showed that C57BL10 positive control mice were slightly lighter than mdx mice (p < 0.05), and all mice continued to grow, with no significant difference in final body weight between the experimental groups (p < 0.05) (Fig. 11b).

[0087] The dystrophic process affects not only skeletal muscle but also cardiac muscle. The cycle of skeletal muscle necrosis and regeneration begins approximately 3 weeks after birth in mdx mice and continues until approximately 12 weeks of age, whereas characteristic cardiomyopathy is observed only after 21 months of age. Therefore, in the present invention, we primarily evaluated running performance on a treadmill to explore the improvement of dystrophy. In particular, downhill running on a treadmill is known to generate eccentric muscle contractions in vivo, but mdx mice have little tolerance for this. In fact, after 4 weeks of treatment, the average running distance of mdx mice (G2) administered PBS was 416±225 m, only about 40% of that of wild-type (WT) mice (1027±26 m) (Fig. 11a). At 8 weeks, the running distance of mdx mice (214±61 m) decreased to 26% of that of WT mice (829±130 m) (Fig. 11a). In contrast, mdx mice administered AM203 showed significantly improved performance compared to control mdx mice at 4 weeks (967±103 m) and 8 weeks (566±241 m) (Fig. 11a).

[0088] While the downhill running test typically measures muscle function, the wire hanging test assesses subacute muscle function, balance, and coordination. As shown in Figure 11c, all WT mice were willing to hang from the wire, with an average hanging time of 600 seconds, the cutoff point. In contrast, the hanging time in mdx mice treated with PBS (G2) was only 96 ± 16 seconds. However, when treated with AM203 (G3), the hanging time was significantly increased (147 ± 44 seconds).

[0089] Serum CK levels (sCK) in sedentary mdx mice were generally higher than those in WT mice, increasing more than 100-fold 2 h after exercise and then slowly returning to pre-exercise levels within 24 h. Similarly, in this study, sCK levels in mdx mice (4538±4117 U / L) were measured to be significantly higher than those in WT mice (72±29 U / L) (Fig. 11c). However, administration of AM203 significantly reduced sCK levels (2669±1181 U / L) (Fig. 11c). These results, along with the improved performance of AM203-treated mdx in the downhill running and wire hanging tests, support the potential of AM203 as an effective therapeutic option.

[0090] 6. Biological distribution of AM203 and expression of Dys203

[0091] Additionally, the biodistribution of AM203 was determined using real-time qPCR. Varying levels of viral genome copies were detected in tissue samples obtained from mdx mice (Fig. 4a), with an average of approximately 0.2 AM203 copies per cell genome in the examined tissues (Fig. 12a). This is consistent with previously reported AAV9 tropism. Since single muscle fibers from 4- to 5-week-old mice contain an average of 230 nuclei, these results indicate that approximately 45 AM203 copies are present per muscle fiber in mdx mice.

[0092] Consistent with its biodistribution, Dys203 expression was detected in nearly all tissues examined. Because cardiomyopathy is a major cause of death in DMD patients, we examined whether mdx mice express Dys203 in the heart when administered AM203. As shown in Figure 12b, WT mice express dystrophin in the heart, but mdx mice do not (compare lanes 1 and 2). However, AM203 administration increased Dys203 expression in mdx mice to levels comparable to endogenous dystrophin in WT mice (compare lanes 1 and 3–5). These results suggest that AM203, whose expression is induced by mPGK1, may be highly effective in treating cardiomyopathy caused by dystrophin deficiency.

[0093] Although the use of the mPGK1 promoter in AM203 allows for expression of Dys203 in a wide range of tissues, similar to endogenous dystrophin, it should be noted that the relative extent of Dys203 expression is determined not only by AAV9 tropism but also by PGK activity in individual tissues. However, Western blot analysis revealed that Dys203 was expressed in nearly all tissues except the kidney and spleen, albeit at varying levels (Figures 12C and 12D, top panels). Because dystrophin deficiency significantly reduces nNOS expression in both DMD patients and mdx mice, we also investigated whether AM203 administration affected nNOS expression in mdx mice. We detected a marked increase in nNOS expression in AM203-treated mdx mice, with tissue specificity generally consistent with Dys203 expression (Figures 12C and 12D, middle panels).

[0094] Consistent with the results in Figure 12, immunofluorescence staining of muscle tissues also revealed strong Dys203 expression and localization to the muscle cell membrane. Immunostaining for Dys203 was performed in four skeletal muscle tissues: tibialis anterior (TA), triceps brachii, diaphragm, and gastrocnemius (GAS) prepared from WT, mdx, and four mdx mice administered AM203. As shown in Figure 13, dystrophin was detected in the fascicles of all WT tissues (upper panel) and, as expected, was not detected in tissue samples from mdx mice (second lower panel). However, in AM203-treated mdx, Dys203 was detected in approximately 30-50% of the fascicle muscle fibers (third and lower panel). Because the Western blot analysis showed evidence of nNOS expression upon AM203 administration, we additionally investigated nNOS expression and its localization in GAS by immunostaining. As shown in Figure 14, all WT muscle fibers showed nNOS localization to the fascicle (Figure 14a), whereas mdx muscle fibers did not (Figure 14b). Consistent with the results in Figure 12, AM203 administration restored the expression and localization of nNOS to the fascicle (Figures 14c and 14d).

[0095] For reference, information on the constituent sequences for implementing the peptide pair designed in the present invention is shown as sequence numbers 1 to 8.

[0096] Specifically, SEQ ID NO: 1 represents the nucleotide sequence (4701 bp) of pRP-Cypet-GP1. The recombinant vector pRP-Cypet-GP1 consists of the following DNA elements at the indicated positions (pos): 5'-ITR (AAV2) (underlined) (NC_001401.2), 1-141; CAG promoter (italics), 169-752; translation start codon (bold), 783-785; CyPET coding sequence (human optimized), 783-1499; GP1 coding sequence (lowercase), 1500-1691; translation stop codon (ter) (bold), 1692-1694; BGH poly(A) signal, 1749-1956; 3'-ITR(AAV2)(underlined)(NC_001401.2), 1964–2104; Ampicillin resistance gene, 3021–3881; pUC replication origin, 4052–4640. The recombinant vector pRP-Cypet (4509 bp), which expresses Cypet without GP1 (64 amino acid residues), is 192 bp shorter than pRP-Cypet-GP1 due to the absence of GPA, but otherwise has the same nucleotide sequence as pRP-Cypet-GP1.

[0097] Sequence number 2 shows the nucleotide sequence (4704 bp) of pRP-GP2-Ypet. The recombinant vector for expressing Ypet consists of the following DNA elements at the indicated positions: 5'-ITR (AAV2) (underlined), 1-141; CAG promoter (italicized), 169-752; translation start codon (bold), 783-785; GP2 coding sequence (lowercase), 786-977; Ypet coding sequence (human-optimized), 978-1697; ter (bold), 1695-1697; BGH poly(A) signal, 1752-1959; 3'-ITR (AAV2) (underlined), 1967-2107); ampicillin resistance gene, 3024-3884; pUC replication origin, 4055-4643. The recombinant vector pRP-Ypet (4509 bp), which expresses YPET without GP2 (64 amino acid residues), is 192 bp smaller than pRP-GP2-Ypet due to the absence of GP2, and otherwise has the same nucleotide sequence as pRP-GP2-Ypet.

[0098] SEQ ID NO: 3 shows the nucleotide sequence (8332 bp) of pRP-DNH. The recombinant vector pRP-DNH consists of the following DNA elements at the indicated positions (pos): CAG promoter (italics), 211-1943; translation start codon (bold), 1974-1976; 3x Flag epitope (underlined), 1977-2054; DNH, 2055-5207 (Dp427m coding sequence 241-2388, 5860-6861); GP1 (lowercase), 5208-5402; translation termination codon (ter) (bold), 5427-5429; SV40 late poly(A) signal, 5447-5668; pUC replication origin, 5996-6584; Ampicillin resistance gene, 6755-7615; f1 replication origin, 7747-8202. The sequence information of the DNA elements is as follows: Dp427m cDNA, NM_004006.3; SV40 late poly(A) signal, NC_001669.1.

[0099] Sequence number 4 is pRP-Dp116+ The nucleotide sequence (7408 bp) of the recombinant vector pRP-Dp116 + It consists of the following DNA elements at the indicated positions (pos): CAG promoter (italics), 22-1754; translation start codon (bold), 1985-1987; 3x V5 epitope (underlined), 1988-1919; GP2 (lowercase), 1920-2111; Dp116+, 2112-5108 (Dp427m coding sequence 8296-11292); translation stop codon (ter) (bold), 5109-5111; SV40 late poly(A) signal, 5156-5377; pUC replication origin, 5573-6161; ampicillin resistance gene, 6325-7192. The sequence information of the DNA elements is as follows: Dp427m cDNA, NM_004006.3; SV40 late poly(A) signal, NC_001669.1.

[0100] SEQ ID NO: 5 shows the nucleotide sequence (5731 bp) of pRP-V5-hSNTA1. The recombinant vector pRP-V5-hSNTA1 consists of the following DNA elements at the indicated positions (pos): CAG promoter (italics), 22–1754; translation start codon (bold), 1785–1787; 3x V5 epitope (underlined), 1788–1919; hSNTA1 (NM_003098.3), 1920–3431; translation stop codon (ter) (bold), 3432–3434; SV40 late poly(A) signal, 3479–3700; pUC replication origin, 3896–4484; and ampicillin resistance gene, 4655–5515.

[0101] SEQ ID NO: 6 shows the nucleotide sequence (6274 bp) of pRP-hDTNA-V5. The recombinant vector pRP-hDTNA-V5 consists of the following DNA elements at the indicated positions (pos): CAG promoter (italics), 22–1754; translation start codon (bold), 1785–1787; hDTNA1 (NM_032975.4), 1788–3842; 3x V5 epitope (underlined), 3843–3974; translation termination codon (ter) (bold), 3975–3977; SV40 late poly(A) signal, 4022–4243; pUC replication origin, 4439–5027; and ampicillin resistance gene, 5198–6058.

[0102] SEQ ID NO: 7 shows the nucleotide sequence (4636 bp) of rAAV9-DNH. rAAV9-DNH consists of the following DNA elements at the indicated positions: 5'-ITR (underlined), 1-141; PGK1 promoter (italicized), 169-679; Kozak sequence, 704-709; translation start codon (bold), 710-712; DNH, 713-4003 (Dp427m coding sequences 241 / 2388, 5860 / 6861, 7507 / 7647); GP1 (lowercase), 4004-4195 (lowercase); ter, 4196-4201; SV40 late poly(A) signal, 4202-4423; 3'-ITR, 4496-4636 (underlined). The sequence information of the DNA elements is as follows: AAV 5'-ITR and 3'-ITR, NC_001401.2; Dp427m cDNA, NM_004006.3; SV40 late poly(A) signal, NC_001669.1.

[0103] Sequence number 8 represents the nucleotide sequence (4486 bp) of rAAV9-Dp116+. rAAV9-Dp116 +The nucleotide positions of the DNA elements that make up the gene are as follows: 5'-ITR (underlined), 1-141; PGK1 promoter (italicized), 169-679; Kozak sequence, 704-709; translation start codon (bold), 710-712; GP2 (lowercase), 713-904; Dp116 + , 905-4045 (Dp427m coding sequence 7507-7647, 8296-11295); ter, 4046-4051; SV40 late poly(A) signal, 4052-4273; 3'-ITR, 4346-4486 (underlined). The sequence information of the DNA elements is as follows: AAV 5'-ITR and 3'-ITR, NC_001401.2; Dp427m cDNA, NM_004006.3; SV40 late poly(A) signal, NC_001669.1.

[0104] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the technical spirit of the present invention as set forth in the claims.

[0105] It is expected that the gene therapy of the present invention will provide a clue to gene therapy for muscle diseases including Duchenne muscular dystrophy.

Claims

1. A first AAV gene vector comprising a nucleic acid encoding a target protein N-terminal region and a nucleic acid encoding a first guide peptide; and A recombinant adeno-associated virus (AAV) system comprising a second AAV gene vector comprising a nucleic acid encoding a C-terminal region of a target protein and a nucleic acid encoding a second guide peptide; A system wherein the target protein is dystrophin.

2. In paragraph 1, A system wherein the first guide peptide and the second guide peptide are a coiled coil peptide pair.

3. A pharmaceutical composition for treating muscular dystrophy, comprising the recombinant adeno-associated virus (AAV) system of paragraph 1 as an active ingredient.

4. In paragraph 3, A pharmaceutical composition, wherein the above muscular dystrophy is Duchenne muscular dystrophy.

5. In paragraph 3, The first AAV gene vector comprises Rod domain 1 to 3 regions, Rod domain 15 to 17 regions, and Guide peptide 1 of a nucleic acid encoding a dystrophin protein, A pharmaceutical composition, wherein the second AAV gene vector comprises a rod domain 22 to 24 region of a nucleic acid encoding a dystrophin protein, and a guide peptide 2.

6. In paragraph 3, The above first AAV gene vector is represented by sequence number 7, A pharmaceutical composition, wherein the second AAV gene vector is represented by sequence number 8.

7. A transformant transformed with the recombinant adeno-associated virus (AAV) system of paragraph 1.

8. In paragraph 7, A transformant, wherein the transformant is a bacterial cell, a plant cell, or an animal cell other than a human. 9.(a) preparing a first AAV gene vector comprising a nucleic acid encoding a target protein N-terminal region and a nucleic acid encoding a first guide peptide; and, (b) a method for producing a recombinant adeno-associated virus (AAV) system, comprising the step of producing a second AAV gene vector comprising a nucleic acid encoding a C-terminal region of a target protein and a nucleic acid encoding a second guide peptide; A method for producing a transformant, wherein the target protein is dystrophin.

10. A method for producing a transformant, comprising the step of transfecting a bacterium, a plant cell, or an animal cell other than a human with the recombinant adeno-associated virus (AAV) system of paragraph 1.

11. A method for treating muscular dystrophy, comprising administering to a subject in need of treatment the recombinant adeno-associated virus (AAV) system of claim 1 or the pharmaceutical composition of claim 3.

12. Use of the recombinant adeno-associated virus (AAV) system of paragraph 1 or the pharmaceutical composition of paragraph 3 for treating muscular dystrophy.

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