Novel proteins and nucleic acid sequences and their use in the prevention and / or treatment of congenital muscular dystrophies - Patents.com

Abstract

The present invention relates to modified recombinant agrin proteins or fragments thereof and recombinant chimeric laminin-nidogen proteins or fragments thereof, nucleic acid sequences encoding these proteins, vectors containing these nucleic acid sequences, compositions comprising these proteins, and the use of these proteins in the prevention and / or treatment of congenital muscular dystrophies, in particular (laminin-α2) LAMA2-associated muscular dystrophy.

Description

[Technical field]

[0001] The present invention relates to modified recombinant agrin proteins or fragments thereof and recombinant chimeric laminin-nidogen proteins or fragments thereof, nucleic acid sequences encoding these proteins, vectors containing these nucleic acid sequences, compositions comprising these proteins, and the use of these proteins in the prevention and / or treatment of congenital muscular dystrophies, in particular (laminin-α2) LAMA2-associated muscular dystrophies. [Background technology]

[0002] The extracellular matrix (ECM) stabilizes cells and tissues by providing a solid cell adhesion substrate and ultimately linking the ECM to the cellular cytoskeleton. The consequences of ECM binding loss are particularly evident in inherited muscular dystrophies. Mutations in different laminins lead to severe disease caused by disruption of ECM-receptor-cytoskeleton binding. Mutations in the genes encoding these proteins cause muscular dystrophies, supporting the concept that disruption of the transverse connections from the ECM to the cytoskeleton results in impaired sarcolemma stability and muscle function.

[0003] Laminin-211 (α2β1γ1 chain composition; abbreviated as Lm-211) is expressed in adult muscle and peripheral nerves. Mutations in the LAMA2 gene (MIM156225), located on human chromosome 6q2, which encodes the α2 chain of laminin-211 (Helbling-Leclerc A et al. (1995) - Mutations in the laminin-α2 chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy. Nat Genet 11: 216-218), give rise to a specific subtype of congenital muscular dystrophy (CMD) that is characterized by early onset and is often very severe (C. Jimenez-Mallebrera, SC Brown, CA Sewry, F. Muntoni. Congenital muscular dystrophy: molecular and cellular aspects, Cell. Mol. Life Sci. 62 (7-8) (2005) 809-823). The so-called LAMA2 (congenital) muscular dystrophies (or LAMA2 MD, or LAMA2 CMD, previously also called MDC1A) are the largest subgroup of CMD. For example, in a recent study of 249 CMD patients in the UK, 93 patients (37.4%) had LAMA2 mutations (Sgframeli et al., Congenital muscular dystrophies in the UK population: Clinical and molecular spectrum of a large cohort diagnosed over a 12-year period. Neuromuscul Disord 27 (2017) 793-803).Mutations are spread throughout the LAMA2 gene, the majority of which are nonsense mutations that result in premature protein translocation and cause a severe, non-ambulatory dystrophy (Yurchenco PD, McKee KK, Reinhard JR and Ruegg MA. Laminin-deficient muscular dystrophy: Molecular pathogenesis and structural repair strategies. Matrix Biology, 2018; https: / / doi.org / 10.1016 / j.matbio.2017.11.009). Patients with such mutations lack laminin α2 staining in muscle biopsies but express amounts of β1 and γ1 laminin chains similar to those detected in healthy controls. Genetic confirmation is required for diagnosis.

[0004] The clinical pathology of LAMA2 MD congenital muscular dystrophy is already evident a few days or weeks after birth, as summarized by Mercuri E, Bonnemann C, Muntoni F (Mercuri E, Bonnemann CG, Muntoni F. Muscular dystrophies. Lancet 2019;394:2025-38). Affected children present with generalized hypotonia (floppy infant syndrome) and markedly elevated blood creatine kinase levels. Further features include the inability to sit, stand, or walk independently in many cases, as well as failure to thrive, probably due to a weakening of the muscles required for swallowing. Cognitive function is typically normal, but there are characteristic changes in the white matter, and epilepsy occurs in about 20% of patients. Cardiac involvement is also rare. Patients who receive optimal treatment usually survive to early adulthood.

[0005] Recent findings have assessed the effect of a miniaturized engineered version of the ECM molecule agrin in mice lacking laminin α2 with transgenic overexpression. This "mini-agrin" (abbreviated as "mag") was demonstrated to functionally replace the lost link between the cell surface receptor α-dystroglycan and the extracellular laminin network. Expression of this artificial "mini-agrin" significantly extended the life span of laminin α2-deficient mice and improved their motor performance and muscle histology (J. Moll, P. Barzaghi, S. Lin, G. Bezakova, H. Lochmuller, E. Engvall, U. Muller, M. A Ruegg. An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy. Nature. 2001 Sep20;413(6853):302-7). This strategy provides benefits, at least as demonstrated in an animal model of LAMA2 MD, but mag-expressing LAMA2-deficient mice still develop muscular dystrophy and their average life span remains significantly shorter than wild-type controls. Other publications have shown beneficial effects of transgenic expression of mini-agrin (Reinhard JR, Lin S, McKee KK, Meinen S, Stephanie SC, Sury M, Hobbs S, Maier G, Yurchenco PD, Ruegg MA; 2017. Linker proteins restore basement membrane and correct LAMA2-related muscular dystrophy in mice. Sci. Transl. Med. 9, eaal4649). However, in the cited publications, the nucleic acid sequence expressed in the dyW / dyW mouse model of LAMA2 MD resulted in a mini-agrin version that appeared on Western blots as two bands with apparent relative molecular masses of 80 kDa and 110 kDa, respectively (Figure 6). The higher molecular weight band corresponds to the full-length mini-agrin protein, whereas the lower molecular weight band is the result of proteolytic cleavage, indicating proteolysis of the full-length mini-agrin protein.Such proteolysis could severely limit the potential therapeutic use of mini-agrin.

[0006] US2003 / 0224981 teaches a method for treating LAMA2 MD in a mammal, comprising administering a protein comprising at least one binding domain for laminin and at least one binding domain for α-dystroglycan, or a substance that upregulates endogenous agrin. Application of a substance that upregulates endogenous agrin may cause toxicity, since it has been shown that a specific splice variant of agrin expressed only in motor neurons may cause destabilization of the neuromuscular junction (Lin S, Maj M, Bezakova G, Magyar JP, Brenner HR, Ruegg MA (2008) Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice. Proc Natl Acad Sci USA 105:11406-11411).

[0007] Thus, there remains a need to find protein / nucleic acid sequences suitable for achieving therapeutic goals in humans, and more particularly, constructs that are stable and can be efficiently and safely transferred into human patients suffering from LAMA2 MD.

[0008] It is an object of the present invention to provide biomolecules and / or compositions for use in the treatment of congenital muscle diseases, in particular (laminin-α2) LAMA2-associated muscular dystrophy. Summary of the Invention

[0009] The present invention provides modified recombinant agrin proteins or fragments thereof, in particular mini-agrin proteins, and recombinant chimeric laminin-nidogen proteins or fragments thereof, in particular αLNNd proteins, nucleic acid sequences encoding these proteins, vectors containing these nucleic acid sequences, and compositions comprising these proteins, nucleic acid sequences and vectors.

[0010] The present invention also provides said proteins, nucleic acid sequences encoding said proteins, vectors comprising said nucleic acid sequences, and compositions comprising said proteins, nucleic acid sequences and vectors for use in the prevention and / or treatment of congenital muscular dystrophy.

[0011] Surprisingly, the inventors of the present application have found that a recombinant agrin protein or a fragment thereof comprising at least the laminin globular domain 3 (LG3 domain) and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain, in which at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or a fragment thereof has been modified, is resistant to proteolytic cleavage and degradation. [Brief description of the drawings]

[0012] [Figure 1]1 shows a schematic diagram of the modular structure of the agrin protein. The abbreviations used are: SS: signal sequence; NtA: N-terminal agrin domain; FS1: follistatin-like domain 1; FS: follistatin-like domain; LE: laminin epidermal growth factor (EGF) domain; SEA: sperm protein, enterokinase and agrin domain; S / T: serine / threonine rich region; EG1: EGF-like domain 1; EG2: EGF-like domain 2; EG3: EGF-like domain 3; EG4: EGF-like domain 4; LG1: laminin globular domain 1; LG2: laminin globular domain 2; LG3: laminin globular domain 3. The black stretches indicate the amino acid sequence linking the elements designated by the abbreviations. The elements used in the recombinant agrin protein or fragments thereof according to the invention are abbreviated and numbered accordingly. [Diagram 2] 1 shows a schematic diagram of the modular structure of laminin protein. The abbreviations used are: SS: signal sequence; LN: laminin N-terminal domain; LE1: laminin EGF-like domain 1; LE2: laminin EGF-like domain 2; LE3: laminin EGF-like domain 3; LE4: laminin EGF-like domain 4; LE: laminin EGF-like domain; L4: laminin domain IV; LG: laminin globular domain. The black stretches indicate the amino acid sequence connecting the elements designated by the abbreviations. The elements used in the recombinant chimeric laminin-nidogen protein or fragments thereof according to the invention are abbreviated and numbered accordingly. [Diagram 3]1 shows a schematic diagram of the modular structure of nidogen protein. The abbreviations used are: SS: signal sequence; Ty: thyroglobulin type I repeats; G1: globular nidogen domain 1; G2: globular nidogen domain 2; G3: globular nidogen domain 3; EG1: EGF-like domain 1; EG2: EGF-like domain 2; EG3: EGF-like domain 3; EG4: EGF-like domain 4; EG5: EGF-like domain 5. The black stretches indicate the amino acid sequences connecting the elements designated by the abbreviations. The elements used in the recombinant chimeric laminin-nidogen protein or fragments thereof according to the invention are abbreviated and numbered accordingly. [Figure 4] 1 shows a schematic diagram of the modular structure of the agrin protein fragments according to the invention: SS: signal sequence; NtA: N-terminal agrin domain; FS1: follistatin-like domain 1; EG1: EGF-like domain 1; EG2: EGF-like domain 2; EG3: EGF-like domain 3; EG4: EGF-like domain 4; LG1: laminin globular domain 1, LG2: laminin globular domain 2; LG3: laminin globular domain 3; where the indicated stretch between the EG4 and LG3 domains corresponds to the modified amino acid sequence linking the EG4 and LG3 domains. The black stretch indicates the amino acid sequence linking the elements designated by abbreviations. [Diagram 5] 1 shows the single letter amino acid sequence of human mini-agrin hmag974aa protein (SEQ ID NO: 11). The box highlights the single amino acid substitution K187A from the carboxy terminus of the agrin protein or a fragment thereof. [Figure 6]Figure 1 shows a Western blot analysis detecting mini-agrin protein (mag) in triceps lysates from 8-week-old dyW / dyW mice transgenic for expressing mag (compared to lysates from wild-type mice and dyW / dyW mice not expressing mag) according to prior art (J. Moll, P. Barzaghi, S. Lin, G. Bezakova, H. Lochmuller, E. Engvall, U. Muller, M.A. Ruegg. An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy. Nature. 2001 Sep20;413(6853)). GAPDH was used as a loading control. Box: mag runs as two bands on SDS-PAGE, indicating undesired mini-agrin cleavage. [Figure 7] Figure 1 shows a Western blot analysis detecting mini-agrin protein (mag) in triceps lysates from 8-week-old mice transgenic expressing mag (left) or the hmag974aa protein according to the present invention with a single amino acid change at position 187 from the carboxy terminus, i.e. magK187A (right), which represents the final human hmag974aa construct, according to the prior art (J. Moll, P. Barzaghi, S. Lin, G. Bezakova, H. Lochmuller, E. Engvall, U. Muller, M.A. Ruegg. An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy. Nature. 2001 Sep20;413(6853)). GAPDH was used as a loading control. Lysates from wild-type (WT) mice not expressing mag are included for reference. In the case of mag (left), only 51.7±4.2% is present as full-length protein (upper band); in contrast, magK187A (right) is not proteolytically cleaved. [Figure 8]The cloning map of the mag2 925 bp construct containing a promoter sequence (Spc5.12), a bovine growth hormone (BGH) polyadenylation signal (polyA), and 5' and 3' inverted terminal repeat (5'ITR, 3'ITR) sequences for packaging into adeno-associated virus (AAV) is shown. [Figure 9] 1 shows a schematic diagram of the modular structure of recombinant chimeric αLNNd according to the invention. The abbreviations used are: SS: signal sequence; LN: laminin N-terminal domain; LE1: laminin EGF-like domain 1; LE2: laminin EGF-like domain 2; LE3: laminin EGF-like domain 3; LE4: laminin EGF-like domain 4; Ty: thyroglobulin type I repeats; EG1: EGF-like domain 1; EG2: EGF-like domain 2; EG3: EGF-like domain 3; EG4: EGF-like domain 4; EG5: EGF-like domain 5; G2: globular domain 2; G3: globular domain 3. The black stretches indicate the amino acid sequence connecting the elements designated by the abbreviations. [Figure 10] The one-letter amino acid sequence of the hαLNNd1373aa protein (sequence number: 39) is shown. [Figure 11] 1 shows the cloning map of the human αLNNd4 122 bp construct, which contains a promoter sequence (Spc5.12), minimal polyA (pA), and 5' and 3' inverted terminal repeat (5'ITR, 3'ITR) sequences for packaging into adeno-associated virus (AAV). [Figure 12] The cloning map of the human αLNNd 3009 bp construct containing the promoter sequence (CBh), the BGH polyadenylation signal (polyA), and the 5' and 3' inverted terminal repeat (5'ITR, 3'ITR) sequences for packaging into adeno-associated virus (AAV) is shown. [Figure 13] 1 shows Western blot analysis (A) and immunochemistry (B) of an experiment demonstrating AAV-mediated expression of proteins encoding hmag974aa and hαLNNd1373aa according to the present invention in mice. [Figure 14]1 shows a graph depicting the effect on body weight of dyW / dyW mice from postnatal day 1 (P1) to postnatal day 56 (P56), where mice were treated with AAV-mediated delivery of hmag974aa and hαLNNd1373aa (dyW AAV-DL) according to the present invention. [Figure 15] 1 shows a graph depicting the effect of AAV-mediated delivery of hmag974aa and hαLNNd1373aa according to the present invention on muscle mass. [Figure 16] 1 shows histological images (A) and quantification of muscle fiber size (B) demonstrating the effect of AAV-mediated delivery of hmag974aa and hαLNNd1373aa according to the present invention on muscle tissue. [Figure 17] Quantification showing the effect of AAV-mediated delivery of hmag974aa and hαLNNd1373aa according to the present invention on muscle function. [Figure 18] 1 shows Kaplan-Meier survival curves showing the effect of AAV-mediated delivery of hmag974aa and hαLNNd1373aa according to the present invention on survival. [Figure 19] Quantification of the effect on muscle mass of AAV-mediated delivery of hmag974aa and hαLNNd1373aa (both driven by the muscle-specific promoter Spc5.12) or hmag974aa (driven by Spc5.12) in combination with hαLNNd1002aa (driven by the ubiquitous promoter CBh). [Figure 20] Quantification of the effect of AAV-mediated hmag974aa (driven by Spc5.12) in combination with hαLNNd1002aa (driven by the ubiquitous promoter CBh) on walking performance is shown. [Figure 21] The one-letter amino acid sequence of the hαLNNd1002aa protein (sequence number: 68) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention relates to modified recombinant agrin proteins or fragments thereof and recombinant chimeric laminin-nidogen proteins or fragments thereof, nucleic acid sequences encoding these proteins, vectors containing these nucleic acid sequences, compositions comprising these proteins, and the use of these proteins in the prevention and / or treatment of congenital muscular dystrophies, in particular (laminin-α2) LAMA2-associated muscular dystrophy.

[0014] For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa. It shall be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. It shall be understood that features, integers, properties, compounds described in connection with a particular aspect, embodiment or example of the present invention are applicable to any other aspect, embodiment or example described herein, unless inconsistent therewith. All features disclosed in this specification (including the accompanying claims, abstract and drawings), and / or all steps of the disclosed methods or processes, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0015] The term "comprise" and its variants such as "comprises" and "comprising" are generally used in the sense of include, i.e. "including, but not limited to," i.e. permitting the presence of one or more features or components.

[0016] The singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0017] The term "about" refers to values ​​in a range of ±10% of the specified value. For example, the phrase "about 200" includes ±10% of 200, or from 180 to 220.

[0018] As used herein, the terms "polypeptide," "peptide," "protein," "polypeptidic," and "peptidic" are used interchangeably to refer to a series of amino acid residues joined together by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Preferably, a polypeptide has an amino acid sequence containing at least 10 amino acids, more preferably at least 20 amino acids.

[0019] The term "agrin protein" as used herein refers to a large proteoglycan whose main role is in the development of neuromuscular junctions during embryogenesis, including the modular structure shown in FIG. 1. Agrin is named based on its involvement in the aggregation of acetylcholine receptors during synapse formation. In humans, this protein is encoded by the AGRN gene. Preferably, the agrin protein refers to human agrin protein isoform 6, Uniprot (www.uniprot.org) number UniProtKB-O00468-6 (AGRIN_HUMAN). The full-length human agrin sequence is shown in SEQ ID NO: 62. The term "wild-type agrin protein" refers to a naturally occurring agrin protein, preferably human agrin protein isoform 6, Uniprot (www.uniprot.org) number UniProtKB-O00468-6 (AGRIN_HUMAN).

[0020] The term "fragment of the agrin protein" or "fragment thereof" (relating to the agrin protein) as used herein generally refers to a portion of the full-length agrin protein having between 10 and 1500 amino acids, preferably between 50 and 1000 amino acids, more preferably between 100 and 1000 amino acids. A fragment of the agrin protein as defined herein generally has the same functional properties as the agrin protein from which it is derived. The term "the agrin protein from which it is derived" in relation to a fragment refers to the full-length agrin protein from which the fragment is derived. The term "the same functional properties as the agrin protein from which it is derived" refers to a molecular function (or one of the molecular functions) of the full-length protein from which the fragment is derived, which may be, for example, binding to laminin and α-dystroglycan.

[0021] As used herein, the term "at least one amino acid residue is modified" of a given amino acid sequence refers to at least one amino acid substitution, insertion, and / or deletion in a polypeptide sequence. An "amino acid substitution" or "substitution" herein refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, the substitution R94K refers to a variant polypeptide in which the arginine at position 94 is replaced with a lysine. For example, 94K indicates a substitution with a lysine at position 94. For purposes herein, multiple substitutions are typically separated by a slash or a comma. For example, "R94K / L78V" or "R94K,L78V" refers to a double variant containing the substitutions R94K and L78V. An "amino acid insertion" or "insertion" as used herein refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. For example, insertion-94 indicates an insertion at position 94. An "amino acid deletion" or "deletion" as used herein refers to the removal of an amino acid at a particular position in a parent polypeptide sequence. For example, R94- indicates the deletion of arginine at position 94. Preferably, the term "at least one amino acid residue is modified" refers to an amino acid substitution, more preferably one amino acid substitution, even more preferably one amino acid substitution at position 187 from the carboxy terminus of the recombinant agrin protein or a fragment thereof, where the numbering is according to isoform 6 of the agrin protein, and in particular refers to the amino acid substitution K187A from the carboxy terminus of the recombinant agrin protein or a fragment thereof. K187A from the carboxy terminus of the recombinant agrin protein or a fragment thereof means that a lysine to alanine substitution occurs at the 187th amino acid residue from the carboxy terminus of the agrin protein or a fragment thereof.

[0022] The term "recombinant chimeric laminin-nidogen protein" as used herein refers to a fusion protein that contains at least the LN domain of laminin and the G3 domain of nidogen, or at least the LN domain of laminin and the G2 and G3 domains of nidogen, and can be generated by combining portions of two genes, laminin (e.g., laminin-α1) and nidogen (e.g., nidogen-1). Laminin, an essential component of the ECM, is a heterotrimeric protein that contains an α chain, a β chain, and a γ chain. Preferably, the laminin protein refers to the human laminin-α1 protein, Uniprot (www.uniprot.org) number UniProtKB-P25391 (LAMA1_HUMAN). The full-length human laminin-α1 sequence is shown in SEQ ID NO: 63. Nidogens, formerly known as entactins, are a family of glycoproteins located in the ECM. Preferably, the nidogen protein refers to the human nidogen-1 protein with Uniprot (www.uniprot.org) number UniProtKB-P14543 (NID1_HUMAN). The full-length human nidogen-1 sequence is shown in SEQ ID NO: 64. The term "wild-type laminin protein" refers to a naturally occurring laminin protein, preferably the human laminin-α1 protein with Uniprot (www.uniprot.org) number UniProtKB-P25391 (LAMA1_HUMAN). The term "wild-type nidogen protein" refers to a naturally occurring nidogen protein, preferably the human nidogen-1 protein with Uniprot (www.uniprot.org) number UniProtKB-P14543 (NID1_HUMAN).

[0023] The term "fragment of a chimeric laminin-nidogen protein" as used herein generally refers to a portion of a full-length chimeric laminin-nidogen protein having 10 to 1500 amino acids, preferably 50 to 1000 amino acids, more preferably 100 to 1000 amino acids. A fragment of a chimeric laminin-nidogen protein as defined herein generally has the same functional properties as the laminin and nidogen protein from which it is derived. The term "chimeric laminin-nidogen protein from which it is derived" in reference to a fragment refers to the complete chimeric laminin-nidogen protein from which the fragment is derived. The term "same functional properties as the chimeric laminin-nidogen protein from which it is derived" refers to the molecular function (or one of the molecular functions) of the full-length protein from which the fragment is derived, which may be, for example, laminin polymerization and binding to the nidogen binding site of laminin, creating an artificial short arm for laminin polymerization.

[0024] The term "alphaLNNd protein" as used herein refers to a laminin-nidogen protein or fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen, or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, and most particularly an alphaLNNd protein comprising the sequence set forth in SEQ ID NO:39, or an alphaLNNd protein comprising the sequence set forth in SEQ ID NO:68.

[0025] As used herein, a "promoter" refers to a nucleic acid sequence that controls the expression of a transcription unit. A "promoter region" is a control region that is capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Within the promoter region, one will find a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for binding RNA polymerase, such as the putative -35 region and the Pribnow box. The term "operably linked" when describing the relationship between two nucleotides, e.g., DNA regions, simply means that they are functionally related to each other and located on the same nucleic acid fragment. A promoter is operably linked to a structural gene if it controls the transcription of the gene and is located on the same nucleic acid fragment as the gene. Useful promoter sequences are disclosed in the following documents: Synthetic promoter Spc5.12 (Li, X., Eastman, EM, Schwartz, RJ, and Draghia-Akli, R. (1999). Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences. Nature biotechnology 17, 241-245). Muscle creatine kinase (MCK, CK8, CK6) (Himeda, CL, Chen, X., and Hauschka, SD (2011). Design and testing of regulatory cassettes for optimal activity in skeletal and cardiac muscles. Methods Mol Biol 709, 3-19). Truncated MCK (tMCK) (Wang, B., Li, J., Fu, FH, Chen, C., Zhu, X., Zhou, L., Jiang, X., and Xiao, X. (2008). Construction and analysis of compact muscle-specific promoters for AAV vectors. Gene therapy 15, 1489-1499).Myosin heavy chain (MHC) (Pacak, C.A., Sakai, Y., Thattaliyath, B.D., Mah, C.S., and Byrne, B.J. (2008). Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice. Genet Vaccines Ther 6, 13). A hybrid version of MHC and MCK (MHCK7) (Salva, M.Z., Himeda, C.L., Tai, P.W., Nishiuchi, E., Gregorevic, P., Allen, J.M., Finn, E.E., Nguyen, Q.G., Blankinship, M.J., Meuse, L., et al. (2007). Design of tissue-specific regulatory cassettes for high-level rAAV-mediated expression in skeletal and cardiac muscle. Molecular therapy: the journal of the American Society of Gene Therapy 15, 320-329). Desmin (Pacak, C.A., Sakai, Y., Thattaliyath, B.D., Mah, C.S., and Byrne, B.J. (2008). Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice. Genet Vaccines Ther 6, 13).Cytomegalovirus (CMV) (Boshart, M., Weber, F., Jahn, G., Dorsch-Hasler, K., Fleckenstein, B., and Schaffner, W. (1985). A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41, 521-530). Hybrid versions of the cytomegalovirus (CMV) enhancer and chicken beta-actin (CAG, CB, CBA, CBh) (Gray, SJ, Foti, SB, Schwartz, JW, Bachaboina, L., Taylor-Blake, B., Coleman, J., Ehlers, MD, Zylka, MJ, McCown, TJ, and Samulski, RJ (2011). Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Human gene therapy 22, 1143-1153). Elongation factor 1 alpha (EF1alpha, EF1alpha short, EFS) (Sanjana, NE, Shalem, O., and Zhang, F. (2014). Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11, 783-784). Myelin protein zero (Mpz, P0) (Kao SC, Wu H., Xie J., Chang CP, Ranish JA, Graef IA, Crabtree GR Calcineurin / NFAT Signaling Is Required for Neuregulin-Regulated Schwann Cell Differentiation. Science. 2009;323(5914):651-4).

[0026] The term "muscle-specific promoter" refers to a tissue-specific promoter that is only active in muscle fibers, and includes Spc5.12 promoter, muscle creatine kinase (MCK, CK8, CK6) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid version of MHC and MCK (MHCK7) promoter, and desmin promoter. The muscle-specific promoter used in the present invention is preferably selected from the group consisting of Spc5.12 promoter, muscle creatine kinase (MCK, CK8, CK6) promoter, and hybrid version of MHC and MCK (MHCK7) promoter, and is preferably Spc5.12 promoter. The term "ubiquitous promoter" refers to a promoter that is active, preferably strongly active, in the majority of cells, cell states, and tissues, and includes CBh promoter, CBA promoter, CAG promoter, CMV promoter, and EFS promoter. The ubiquitous promoter used in the present invention is preferably selected from the group consisting of CBh promoter, CBA promoter, and EFS promoter, and is more preferably CBh promoter. The term "Schwann cell-specific promoter" refers to a tissue-specific promoter that is active, preferably potently active, in Schwann cells of the peripheral nervous system. A preferred Schwann cell-specific promoter for use in the present invention is the Mpz promoter (Mpz,P0).

[0027] The term "congenital muscular dystrophies" as used herein refers to a group of inherited degenerative muscle diseases (muscular dystrophies) that are evident at birth or within the first few months of life. The most frequent congenital muscular dystrophy in Europe is LAMA2-associated muscular dystrophy (LAMA2 MD), also called congenital type 1A muscular dystrophy (MDC1A or merosin-deficient CMD). It is caused by mutations in the LAMA2 gene, which encodes the α2 subunit of laminin-211 (Lm-211). Most LAMA2 MD patients have a complete lack of laminin-α2, are hypotonic (floppy) at birth, are unable to walk, and die within the first decade of life due to respiratory complications.

[0028] In a first aspect, the present invention provides a recombinant agrin protein or a fragment thereof, the fragment of which comprises at least the laminin globular domain 3 (LG3 domain) and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the recombinant agrin protein, and at least one amino acid residue of the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or a fragment thereof is modified. Usually, the recombinant agrin protein or a fragment thereof is of human origin, i.e., a human agrin protein or a fragment thereof. Preferably, the recombinant agrin protein or a fragment thereof is a recombinant agrin protein or a fragment thereof, the fragment of which comprises at least the EGF-like domain 4 (EG4 domain), the laminin globular domain 3 (LG3 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the recombinant agrin protein, and at least one amino acid residue of the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or a fragment thereof is modified.

[0029] More preferably, the recombinant agrin protein or a fragment thereof is a recombinant agrin protein or a fragment thereof which comprises at least the N-terminal agrin domain (NtA domain), the laminin globular domain 2 (LG2 domain), the laminin globular domain 3 (LG3 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the recombinant agrin protein, and at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or a fragment thereof is modified.

[0030] Even more preferably, the recombinant agrin protein or a fragment thereof is a recombinant agrin protein or a fragment thereof, the fragment of which comprises at least the N-terminal agrin domain (NtA domain), EGF-like domain 4 (EG4 domain), laminin globular domain 2 (LG2 domain), laminin globular domain 3 (LG3 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the recombinant agrin protein, in which at least one amino acid residue of the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or a fragment thereof is modified.

[0031] In one embodiment, the recombinant agrin protein or a fragment thereof is a recombinant wild-type agrin protein or a fragment thereof, the fragment of which comprises at least the laminin globular domain 3 (LG3 domain) and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the wild-type agrin protein, and which comprises an amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant wild-type agrin protein or a fragment thereof, in which at least one amino acid residue of the amino acid sequence linking the EG4 domain and the LG3 domain of the wild-type agrin protein or a fragment thereof has been modified compared to the amino acid sequence linking the EG4 domain and the LG3 domain of the wild-type agrin protein. Preferably, the recombinant agrin protein or a fragment thereof is a recombinant wild-type agrin protein or a fragment thereof, the fragment of which comprises at least EGF-like domain 4 (EG4 domain), laminin globular domain 3 (LG3 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the wild-type agrin protein, and at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant wild-type agrin protein or a fragment thereof is modified compared to the amino acid sequence linking the EG4 domain and the LG3 domain of the wild-type agrin protein.

[0032] More preferably, the recombinant agrin protein or a fragment thereof is a recombinant wild-type agrin protein or a fragment thereof, the fragment of which comprises at least the N-terminal agrin domain (NtA domain), the laminin globular domain 2 (LG2 domain), the laminin globular domain 3 (LG3 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the wild-type agrin protein, and at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant wild-type agrin protein or a fragment thereof is modified compared to the amino acid sequence linking the EG4 domain and the LG3 domain of the wild-type agrin protein.

[0033] Even more preferably, the recombinant agrin protein or a fragment thereof is a recombinant wild-type agrin protein or a fragment thereof, the fragment of which comprises at least the N-terminal agrin domain (NtA domain), EGF-like domain 4 (EG4 domain), laminin globular domain 2 (LG2 domain), laminin globular domain 3 (LG3 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the wild-type agrin protein, and at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant wild-type agrin protein or a fragment thereof is modified compared to the amino acid sequence linking the EG4 domain and the LG3 domain of the wild-type agrin protein.

[0034] In a further embodiment, the recombinant agrin protein is a recombinant human agrin protein or a fragment thereof, the fragment of which comprises at least the laminin globular domain 3 (LG3 domain) and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the recombinant human agrin protein, and at least one amino acid residue of the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant human agrin protein or a fragment thereof is modified compared to the amino acid sequence linking the EG4 domain and the LG3 domain of the human agrin protein. Preferably, the recombinant agrin protein is a recombinant human agrin protein or a fragment thereof, the fragment of which contains at least EGF-like domain 4 (EG4 domain), laminin globular domain 3 (LG3 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the recombinant human agrin protein, and at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant human agrin protein or a fragment thereof has been modified compared to the amino acid sequence linking the EG4 domain and the LG3 domain of the human agrin protein.

[0035] More preferably, the recombinant agrin protein is a recombinant human agrin protein or a fragment thereof, the fragment of which comprises at least the N-terminal agrin domain (NtA domain), the laminin globular domain 2 (LG2 domain), the laminin globular domain 3 (LG3 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the recombinant human agrin protein, and at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant human agrin protein or a fragment thereof has been modified compared to the amino acid sequence linking the EG4 domain and the LG3 domain of the human agrin protein.

[0036] Even more preferably, the recombinant agrin protein is a recombinant human agrin protein or a fragment thereof, the fragment of which comprises at least the N-terminal agrin domain (NtA domain), the laminin globular domain 2 (LG2 domain), the EGF-like domain 4 (EG4 domain), the laminin globular domain 3 (LG3 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain of the recombinant human agrin protein, and at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant human agrin protein or a fragment thereof has been modified compared to the amino acid sequence linking the EG4 domain and the LG3 domain of the human agrin protein.

[0037] In a preferred embodiment, the recombinant agrin protein or a fragment thereof is a recombinant agrin protein or a fragment thereof, wherein the LG2 domain does not contain one or more amino acid insertions at the y site.

[0038] In a preferred embodiment, the recombinant agrin protein or a fragment thereof is a recombinant agrin protein or a fragment thereof, in which the LG3 domain does not contain one or more amino acid insertions at the z site.

[0039] In a more preferred embodiment, the recombinant agrin protein or a fragment thereof is a recombinant agrin protein or a fragment thereof, the LG2 domain of which comprises the amino acid sequence LGESPVPHTV as shown in SEQ ID NO:70.

[0040] In a more preferred embodiment, the recombinant agrin protein or a fragment thereof is a recombinant agrin protein or a fragment thereof, the LG3 domain of which comprises the amino acid sequence AVTESEKALQ as shown in SEQ ID NO:71.

[0041] In a preferred embodiment, at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or a fragment thereof to be modified is a lysine at position 187 from the carboxy terminus of the recombinant agrin protein or a fragment thereof. More preferably, the agrin protein or a fragment thereof is agrin protein isoform 6 or a fragment thereof, and at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or a fragment thereof to be modified is a lysine at position 187 from the carboxy terminus of the recombinant agrin protein or a fragment thereof.

[0042] In a further preferred embodiment, the lysine at position 187 from the carboxy terminus of the recombinant agrin protein or fragment thereof is substituted with an amino acid having a non-polar side chain selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, and methionine.

[0043] In a further preferred embodiment, the lysine at position 187 from the carboxy terminus of the recombinant agrin protein or fragment thereof is substituted with alanine.

[0044] In a further embodiment, the amino acid sequence linking the EG4 and LG3 domains of the recombinant agrin protein or fragment thereof comprises the amino acid sequence KGLVEASAGD as shown in SEQ ID NO:65.

[0045] In a further embodiment, at least one modified amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or fragment thereof is A between E and S of the amino acid sequence KGLVEASAGD shown in SEQ ID NO:65.

[0046] In a further embodiment, the fragment of the recombinant agrin protein further comprises the following polypeptides: a signal sequence (SS), an N-terminal agrin domain (NtA domain), a follistatin-like domain (FS domain), three EGF-like domains (EG domains), and two laminin globular domains (LG domains).

[0047] In a preferred embodiment, the fragment of recombinant agrin protein is a mini-agrin comprising the following polypeptides: signal sequence (SS), N-terminal agrin domain (NtA domain), follistatin-like domain (FS domain), EGF-like domain 1 (EG1 domain), laminin globular domain 1 (LG1 domain), EGF-like domain 2 (EG2 domain), EGF-like domain 3 (EG3 domain), laminin globular domain 2 (LG2 domain), EGF-like domain 4 (EG4 domain), and an amino acid sequence linking EGF-like domain 4 (EG4 domain), the LG3 domain and the laminin globular domain 3 (LG3 domain), wherein at least one amino acid residue of the amino acid sequence linking the EG4 domain and the LG3 domain of the fragment is modified.

[0048] In a further preferred embodiment, the fragment of the recombinant agrin protein is a mini-agrin comprising, from the amino terminus to the carboxy terminus, the signal sequence (SS), the N-terminal agrin domain (NtA domain), the follistatin-like domain (FS domain), the EGF-like domain 1 (EG1 domain), the laminin globular domain 1 (LG1 domain), the EGF-like domain 2 (EG2 domain), the EGF-like domain 3 (EG3 domain), the laminin globular domain 2 (LG2 domain), the EGF-like domain 4 (EG4 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain), the LG3 domain, and the laminin globular domain 3 (LG3 domain), in which at least one amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the fragment is modified.

[0049] In a more preferred embodiment, the recombinant agrin protein or fragment thereof comprises: - a signal sequence (SS) comprising the sequence shown in SEQ ID NO:1, and / or - an N-terminal agrin domain (NtA domain) comprising the sequence shown in SEQ ID NO:2, and / or - a follistatin-like domain (FS domain) comprising the sequence as set forth in SEQ ID NO:3, and / or - an EGF-like domain 1 (EG1 domain) comprising the sequence as set forth in SEQ ID NO: 4, an EGF-like domain 2 (EG2 domain) comprising the sequence as set forth in SEQ ID NO: 6, an EGF-like domain 3 (EG3 domain) comprising the sequence as set forth in SEQ ID NO: 7, and / or an EGF-like domain 4 (EG4 domain) comprising the sequence as set forth in SEQ ID NO: 9, and / or - a laminin globular 1 domain (LG1 domain) comprising the sequence shown in SEQ ID NO: 5, a laminin globular 2 domain (LG2 domain) comprising the sequence shown in SEQ ID NO: 8, an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain shown in SEQ ID NO: 65, and / or a laminin globular 3 domain (LG3 domain) comprising the sequence shown in SEQ ID NO: 10 Includes.

[0050] In an even more preferred embodiment, the recombinant agrin protein or fragment thereof comprises: - a signal sequence comprising the sequence shown in SEQ ID NO:1, - an N-terminal agrin domain (NtA domain) comprising the sequence shown in SEQ ID NO:2, - a follistatin-like domain (FS domain) comprising the sequence shown in SEQ ID NO: 3, - EGF-like domain 1 (EG1 domain) comprising the sequence shown in SEQ ID NO: 4, EGF-like domain 2 (EG2 domain) comprising the sequence shown in SEQ ID NO: 6, EGF-like domain 3 (EG3 domain) comprising the sequence shown in SEQ ID NO: 7, EGF-like domain 4 (EG4 domain) comprising the sequence shown in SEQ ID NO: 9, - a laminin globular 1 domain (LG1 domain) comprising the sequence shown in SEQ ID NO:5, a laminin globular 2 domain (LG2 domain) comprising the sequence shown in SEQ ID NO:8, an amino acid sequence linking the EGF-like domain 4 (EG4 domain) and the LG3 domain shown in SEQ ID NO:65, and a laminin globular 3 domain (LG3 domain) comprising the sequence shown in SEQ ID NO:10 Includes.

[0051] In a particular embodiment, the recombinant agrin protein or fragment thereof comprises a signal sequence, a sperm protein, an enterokinase and an agrin domain, a serine / threonine rich region and / or a further amino acid sequence linking the above-mentioned domains, preferably an amino acid sequence linking the signal sequence, a sperm protein, an enterokinase and an agrin domain, a serine / threonine rich region and / or the above-mentioned domains of the wild-type agrin protein.

[0052] Most preferably, the recombinant agrin protein or fragment thereof comprises the sequence shown in SEQ ID NO: 11. This protein is a fragment of human agrin and is referred to herein as hmag974aa. Figure 5 shows the corresponding amino acid sequence of hmag974aa, with the box indicating the single amino acid substitution K187A from the carboxy terminus of the agrin protein.

[0053] The present invention also relates to a nucleic acid sequence encoding a recombinant agrin protein or fragment. In one embodiment, the nucleic acid sequence comprises: - a signal sequence comprising the sequence shown in SEQ ID NO: 12, and / or - an N-terminal agrin domain comprising the sequence shown in SEQ ID NO: 13, and / or - a follistatin-like domain comprising the sequence as set forth in SEQ ID NO: 14, and / or - an EGF-like domain comprising the sequence as shown in SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18 and / or SEQ ID NO: 20, and / or - a laminin globular domain comprising the sequence shown in SEQ ID NO: 16, SEQ ID NO: 19 and / or SEQ ID NO: 21 Includes.

[0054] In a preferred embodiment, the nucleic acid sequence encoding the recombinant agrin protein or a fragment thereof comprises: - a signal sequence comprising the sequence shown in SEQ ID NO: 12, - an N-terminal agrin domain comprising the sequence shown in SEQ ID NO: 13, - a follistatin-like domain comprising the sequence set forth in SEQ ID NO: 14, - an EGF-like domain comprising the sequence as shown in SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18 and / or SEQ ID NO: 20, - a laminin globular domain comprising the sequence shown in SEQ ID NO: 16, SEQ ID NO: 19 and / or SEQ ID NO: 21 Includes.

[0055] The nucleic acid sequence encoding the recombinant agrin protein or a fragment thereof according to the present invention most preferably comprises SEQ ID NO: 22. This particular nucleic acid sequence is hereinafter referred to as hmag2925bp. hmag2925bp encodes a specially designed mini-agrin protein with optimized codon usage. In a preferred embodiment, the nucleic acid sequence encoding the recombinant agrin protein or a fragment thereof, preferably the nucleic acid sequence encoding the recombinant agrin protein or a fragment thereof comprising SEQ ID NO: 22, further comprises a muscle-specific promoter operably linked to the nucleic acid sequence, preferably the Spc5.12 promoter operably linked to the nucleic acid sequence.

[0056] In addition, to optimize the genetic construct to allow efficient protein expression, the nucleic acid sequence encoding the recombinant agrin protein or a fragment thereof according to the present invention can be codon-optimized to ensure the highest possible expression level upon gene transfer into humans with LAMA2 MD. This can be achieved by a codon optimization process applying a freely available software tool (Puigbo P, Guzma E, Romeu A, Garcia-Vallve S. OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 2007). In addition, a consensus Kozak sequence can be introduced into the genetic construct. Optimizing the codon usage is advantageous to (a) reduce the viral titer required during the manufacturing process (lower titer can reduce manufacturing costs) and (b) address safety concerns related to high concentrations of viral vectors introduced into humans.

[0057] The present invention also relates to a vector comprising the above-mentioned nucleic acid sequence. The vector preferably further comprises a promoter sequence 5' of the nucleic acid sequence, which is a muscle-specific promoter, a ubiquitous promoter or a Schwann cell-specific promoter, where the promoter sequence is more preferably a muscle-specific promoter selected from the group consisting of synthetic promoter Spc5.12, muscle creatine kinase (MCK, CK8, CK6) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, a hybrid version of MHC and MCK (MHCK7) promoter and desmin promoter; a ubiquitous promoter selected from the group consisting of cytomegalovirus (CMV) promoter, a hybrid version of cytomegalovirus (CMV) enhancer and chicken beta-actin promoter (CAG, CB, CBA, CBh) and elongation factor 1 alpha (EF1alpha, EF1alpha short, EFS); or a Schwann cell-specific promoter, preferably the myelin protein zero (Mpz, P0) promoter. The vector more preferably further comprises a promoter sequence of a muscle-specific promoter 5' to the nucleic acid sequence, more preferably selected from the group consisting of synthetic promoter Spc5.12, muscle creatine kinase (MCK, CK8, CK6) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid version of MHC and MCK (MHCK7) promoter, desmin promoter, most preferably the Spc5.12 promoter. The selection of an appropriate promoter depends on the tissue in which protein expression is most desired, taking into account the known tissue specificity of the promoter. The above promoters are suitable for expression in muscle, cardiac and nervous tissues.

[0058] Preferred promoters useful for the expression of the above-mentioned nucleic acid sequences are selected from the group consisting of Spc5.12 (SEQ ID NO:55), EFS (SEQ ID NO:56), CBh (SEQ ID NO:57), CK8 (SEQ ID NO:58), CK8e (SEQ ID NO:59), CMV (SEQ ID NO:60), and MHCK7 (SEQ ID NO:61), Mpz (SEQ ID NO:72), in particular the synthetic promoter Spc5.12 (SEQ ID NO:55).

[0059] In one embodiment, the vector further comprises a polyadenylation (polyA) sequence 3' to the nucleic acid sequence and / or 5' and 3' inverted terminal repeat (5'ITR, 3'ITR) sequences 5' of the promoter and 3' of the polyA sequence, respectively.

[0060] Preferred polyadenylation signal sequences for expression of mag2925bp are the polyA from SV40, 222 base pairs (bp) in length, the synthetic minimal polyA, 49 bp in length, or the polyA from bovine growth hormone (BGH), 208 bp in length, the latter being most preferred.

[0061] The cloning map of the preferred vector mag2925bp is shown in Figure 8, along with the promoter sequence (Spc5.12), the BGH polyadenylation signal (polyA), and the 5' and 3' inverted terminal repeat (5'ITR, 3'ITR) sequences for packaging into adeno-associated virus (AAV).

[0062] The vector is preferably an adeno-associated vector (AAV), more preferably AAV9, AAV8, AAVrh74, AAVMYO, AAVMYO2, AAVMYO3 or MyoAAV, even more preferably AAV9, AAV8, AAVrh74, AAVMYO or MyoAAV.

[0063] In a further aspect, the present invention provides a recombinant chimeric laminin-nidogen protein or fragment thereof, such as an alphaLNNd protein, comprising the sequence set forth in SEQ ID NO: 39 or comprising the sequence set forth in SEQ ID NO: 68. This particular protein is referred to herein as hαLNNd1373aa or hαLNNd1002aa.

[0064] The present invention also relates to a nucleic acid sequence encoding the above-mentioned recombinant chimeric laminin-nidogen protein or a fragment thereof, preferably comprising the sequence shown in SEQ ID NO: 54 or the sequence shown in SEQ ID NO: 69. This particular nucleic acid sequence is referred to below as αLNNd4122bp (SEQ ID NO: 54) or αLNNd3009bp (SEQ ID NO: 69). In one embodiment, the nucleic acid sequence encoding the above-mentioned recombinant chimeric laminin-nidogen protein or a fragment thereof, preferably the nucleic acid sequence encoding the recombinant agrin protein or a fragment thereof comprising SEQ ID NO: 54 or SEQ ID NO: 69, further comprises a muscle-specific or ubiquitous promoter operably linked to the nucleic acid sequence, preferably the Spc5.12 promoter or the CBh promoter operably linked to the nucleic acid sequence, more preferably the CBh promoter.

[0065] To optimize the gene construct to allow efficient protein expression, the nucleic acid sequence of the recombinant chimeric laminin-nidogen protein or fragment thereof, such as the αLNNd of the present invention, can be optimized for codon usage to ensure the highest possible expression level upon transfection in humans with LAMA2 MD. This can be done similarly to the mini-agrin of the present invention. In addition, a consensus Kozak sequence can be introduced into the gene construct.

[0066] The present invention also relates to a vector comprising the above-mentioned nucleic acid sequence encoding a recombinant chimeric laminin-nidogen protein or a fragment thereof, such as alpha LNNd protein. The vector preferably further comprises a promoter sequence 5' of the nucleic acid sequence, which is a muscle-specific promoter, a ubiquitous promoter or a Schwann cell-specific promoter, more preferably a muscle-specific promoter selected from the group consisting of synthetic promoter Spc5.12, muscle creatine kinase (MCK, CK8, CK6) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, a hybrid version of MHC and MCK (MHCK7) promoter and desmin promoter; a ubiquitous promoter selected from the group consisting of cytomegalovirus (CMV) promoter, a hybrid version of cytomegalovirus (CMV) enhancer and chicken beta-actin promoter (CAG, CB, CBA, CBh) and elongation factor 1 alpha (EF1alpha, EF1alpha short, EFS); or a Schwann cell-specific promoter, preferably the myelin protein zero (Mpz, P0) promoter. More preferably, the vector further comprises a promoter sequence 5' of the nucleic acid sequence, which is a muscle-specific promoter or a ubiquitous promoter, more preferably the Spc5.12 promoter or the CBh promoter, most preferably the CBh promoter. The selection of a suitable promoter depends on the tissue in which protein expression is most desired, taking into account the known tissue specificity of the promoter. The above promoters are suitable for expression in muscle, cardiac and nervous tissue. References and sequences for the above mentioned promoters have already been disclosed in the section on agrin according to the present invention.

[0067] The vector further preferably comprises a polyadenylation (polyA) sequence 3' of the nucleic acid sequence and / or 5' and 3' inverted terminal repeat (5'ITR, 3'ITR) sequences 5' of the promoter and 3' of the polyA sequence, respectively. The preferred polyadenylation signal sequence for the expression of αLNNd4122bp is a synthetic minimal polyA of 49bp in length or a synthetic polyA of a length different from 49bp, the first being the most preferred, as long as the selected polyA tail allows the complete gene construct to be packaged into a suitable viral gene transfer vector. The preferred polyadenylation signal sequence for the expression of hαLNNd3009bp is a polyA from SV40 of 222 base pairs (bp) in length, a synthetic minimal polyA of 49bp in length, or a polyA from bovine growth hormone (BGH) of 208bp in length, the latter being the most preferred.

[0068] The cloning map of the preferred vector αLNNd4122bp is shown in FIG. 11 along with the promoter sequence (Spc5.12), minimal polyA (pA), 5′ and 3′ inverted terminal repeat (5′ITR, 3′ITR) sequences for packaging into adeno-associated virus (AAV).

[0069] The cloning map of the preferred vector αLNNd3009bp is shown in Figure 12 along with the promoter sequence (CBh), the BGH polyadenylation signal (polyA), and the 5' and 3' inverted terminal repeat (5'ITR, 3'ITR) sequences for packaging into adeno-associated virus (AAV).

[0070] The vector is preferably an adeno-associated vector (AAV), more preferably AAV9, AAV8, AAVrh74, AAVMYO, AAVMYO2, AAVMYO3 or MyoAAV, even more preferably AAV9, AAV8, AAVrh74, AAVMYO, or MyoAAV.

[0071] In a further aspect, the present invention provides a composition comprising the above-mentioned agrin protein or a fragment thereof and a recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen.

[0072] In a preferred embodiment, the recombinant chimeric laminin-nidogen protein or fragment thereof comprises the laminin (LN) binding domain (LN domain) of laminin and the globular domain 3 (G3 domain) of nidogen, and further comprises a signal sequence, and / or a laminin EGF-like domain (LE domain), and / or an EGF-like domain (EG domain), and / or thyroglobulin type I repeats (Ty).

[0073] In a further preferred embodiment, the recombinant chimeric laminin-nidogen protein or fragment thereof comprises the laminin (LN) binding domain of laminin (LN domain), the globular domain 2 of nidogen (G2 domain), and the globular domain 3 of nidogen (G3 domain), and further comprises a signal sequence, and / or a laminin EGF-like domain (LE domain), and / or an EGF-like domain (EG domain), and / or thyroglobulin type I repeats (Ty).

[0074] In a more preferred embodiment, the recombinant chimeric laminin-nidogen protein or fragment thereof comprises, from the amino terminus to the carboxy terminus, a signal sequence, the laminin (LN) binding domain of laminin (LN domain), four laminin EGF-like domains (LE domains), two EGF-like domains (EG domains), thyroglobulin type I repeats (Ty), globular domain 3 of nidogen (G3 domain) and the EGF-like domain (EG domain).

[0075] In an even more preferred embodiment, the recombinant chimeric laminin-nidogen protein or fragment thereof comprises, from the amino terminus to the carboxy terminus, a signal sequence, a laminin (LN) binding domain of laminin (LN domain), four laminin EGF-like domains (LE domains), globular domain 2 of nidogen (G2 domain), four EGF-like domains (EG domains), thyroglobulin type I repeats (Ty), globular domain 3 of nidogen (G3 domain) and an EGF-like domain (EG domain).

[0076] In a particular embodiment, the recombinant chimeric laminin-nidogen protein or fragment thereof comprises a laminin (LN) binding domain (LN domain) comprising the sequence set forth in SEQ ID NO:26, and a nidogen globular domain 3 (G3 domain) comprising the sequence set forth in SEQ ID NO:37, and further comprises a signal sequence (SS) comprising SEQ ID NO:25, and / or a laminin EGF-like domain (LE domain) comprising the sequence set forth in SEQ ID NO:27 or SEQ ID NO:28 or SEQ ID NO:29 or SEQ ID NO:30, and / or an EGF-like domain (EG domain) comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35, and / or a thyroglobulin type I repeat (Ty) comprising the sequence set forth in SEQ ID NO:36, and / or an EGF-like domain (EG domain) comprising the sequence set forth in SEQ ID NO:38.

[0077] In a further particular embodiment, the recombinant chimeric laminin-nidogen protein or fragment thereof comprises a laminin (LN) binding domain (LN domain) comprising the sequence set forth in SEQ ID NO:26, and a globular domain 3 (G3 domain) of nidogen comprising the sequence set forth in SEQ ID NO:37, and further comprises a signal sequence (SS) comprising SEQ ID NO:25, and / or a laminin EGF-like domain (LE domain) comprising the sequence set forth in SEQ ID NO:27 or SEQ ID NO:28 or SEQ ID NO:29 or SEQ ID NO:30, and / or a globular domain 2 (G2 domain) of nidogen comprising the sequence set forth in SEQ ID NO:31, and / or an EGF-like domain (EG domain) comprising the sequence set forth in SEQ ID NO:32 or SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35, and / or a thyroglobulin type I repeat (Ty) comprising the sequence set forth in SEQ ID NO:36, and / or an EGF-like domain (EG domain) comprising the sequence set forth in SEQ ID NO:38.

[0078] In a more specific embodiment, the recombinant chimeric laminin-nidogen protein or fragment thereof comprises, from the amino terminus to the carboxy terminus: A signal sequence comprising the sequence shown in SEQ ID NO: 25, A laminin (LN) binding domain (LN domain) comprising the sequence set forth in SEQ ID NO: 26; Laminin EGF-like domain (LE domain) including the sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; An EGF-like domain (EG domain) containing the sequences shown in SEQ ID NO: 34 and SEQ ID NO: 35; Thyroglobulin type I repeat comprising the sequence shown in SEQ ID NO: 36; The globular domain 3 (G3 domain) of Nidogen comprising the sequence shown in SEQ ID NO: 37, and EGF-like domain (EG domain) containing SEQ ID NO: 38 Includes.

[0079] In an even more specific embodiment, the recombinant chimeric laminin-nidogen protein or fragment thereof comprises, from the amino terminus to the carboxy terminus: A signal sequence comprising the sequence shown in SEQ ID NO: 25, A laminin (LN) binding domain (LN domain) comprising the sequence set forth in SEQ ID NO: 26; Laminin EGF-like domain (LE domain) including the sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; Nidogen globular domain 2 (G2 domain) comprising the sequence shown in SEQ ID NO: 31; An EGF-like domain (EG domain) containing the sequence shown in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35; Thyroglobulin type I repeat comprising the sequence shown in SEQ ID NO: 36; The globular domain 3 (G3 domain) of Nidogen comprising the sequence shown in SEQ ID NO: 37, and EGF-like domain (EG domain) containing SEQ ID NO: 38 Includes.

[0080] In a particular embodiment, the recombinant chimeric laminin-nidogen protein or a fragment thereof comprises a signal sequence and / or a further amino acid sequence linking the above-mentioned domains, preferably comprising a signal sequence and / or an amino acid sequence linking the above-mentioned domains for each of the wild-type laminin protein and the wild-type nidogen protein.

[0081] In an even more specific embodiment, the recombinant chimeric laminin-nidogen protein or fragment thereof comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen is an alpha LNNd protein, most particularly an alpha LNNd protein comprising the sequence shown in SEQ ID NO: 39 or an alpha LNNd protein comprising the sequence shown in SEQ ID NO: 68.

[0082] The present invention also relates to a nucleic acid sequence encoding a recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, said protein being an alpha LNNd protein. A signal sequence comprising the sequence shown in SEQ ID NO: 40; A laminin (LN) binding domain (LN domain) comprising the sequence set forth in SEQ ID NO: 41; Laminin EGF-like domain (LE domain) including the sequence shown in SEQ ID NO: 43, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45; Nidogen globular domain 2 (G2 domain) comprising the sequence shown in SEQ ID NO: 46; An EGF-like domain (EG domain) containing the sequence shown in SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50; Thyroglobulin type I repeat comprising the sequence shown in SEQ ID NO: 51; The globular domain 3 (G3 domain) of Nidogen comprising the sequence set forth in SEQ ID NO: 52, and EGF-like domain (EG domain) containing SEQ ID NO: 53 Includes.

[0083] In a preferred embodiment, the composition comprises the above-mentioned agrin protein or a fragment thereof and a recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the above-mentioned LN domain of laminin and the G3 domain of nidogen or comprising at least the above-mentioned LN domain of laminin and the G2 and G3 domains of nidogen, preferably the above-mentioned agrin protein or a fragment thereof and the above-mentioned alphaLNNd protein, more preferably the above-mentioned agrin protein or a fragment thereof and the alphaLNNd protein comprising the sequence shown in SEQ ID NO: 39 or SEQ ID NO: 68. Most preferred is a composition comprising the agrin protein or a fragment thereof comprising the sequence shown in SEQ ID NO: 11 and the alphaLNNd protein comprising the sequence shown in SEQ ID NO: 39 or SEQ ID NO: 68.

[0084] The present invention also provides a composition comprising a nucleic acid sequence encoding the above-mentioned agrin protein or a fragment thereof and a nucleic acid encoding the above-mentioned recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, or a vector comprising a nucleic acid sequence encoding the above-mentioned agrin protein or a fragment thereof and a nucleic acid encoding the above-mentioned recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, or a vector comprising a nucleic acid sequence encoding the above-mentioned agrin protein or a fragment thereof and a nucleic acid encoding the above-mentioned recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, preferably an AAV vector comprising these nucleic acid sequences. Most preferred is an AAV vector comprising the nucleic acid sequence shown in SEQ ID NO:22, SEQ ID NO:54 or SEQ ID NO:69, in particular an AAV vector comprising the nucleic acid sequence shown in SEQ ID NO:22, SEQ ID NO:54 or SEQ ID NO:69, and further comprising the above-mentioned promoter sequence 5' of each nucleic acid sequence. The above disclosed proteins, nucleic acid sequences, vectors and / or compositions are effectively used for the prevention and / or treatment of congenital muscular dystrophy.

[0085] Thus, in a further aspect, the present invention provides a composition comprising the above-mentioned agrin protein or a fragment thereof, and / or the above-mentioned recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising the LN domain of laminin and the G2 and G3 domains of nidogen, or the above-mentioned agrin protein or a fragment thereof and the above-mentioned recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising the LN domain of laminin and the G2 and G3 domains of nidogen, for use in the prevention and / or treatment of congenital muscular dystrophy, as well as the respective nucleic acid sequences and vectors as described above. Similarly, the present invention provides a method for preventing and / or treating congenital muscular dystrophy in a subject, comprising administering to the subject the above-mentioned agrin protein or a fragment thereof, and / or the above-mentioned recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, or the above-mentioned composition, wherein the protein is administered in an amount sufficient to treat the subject.

[0086] Similarly, the present invention provides the use of the above-mentioned agrin protein or a fragment thereof, and / or a recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, or the above-mentioned composition, in the manufacture of a medicament for the prevention and / or treatment of congenital muscular dystrophy in a subject.Similarly, the present invention provides the use of the above-mentioned agrin protein or a fragment thereof, and / or a recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, or the above-mentioned composition, for the prevention and / or treatment of congenital muscular dystrophy in a subject.

[0087] The congenital muscular dystrophy is preferably (laminin-α2) LAMA2-associated muscular dystrophy. Preferably, (laminin-α2) LAMA2-associated muscular dystrophy patients express a version of Lm-211 in which the α2 chain is truncated at its N-terminal region encoding the endogenous LN domain. In one embodiment, an AAV vector for the expression of the above-mentioned agrin protein or a fragment thereof and an AAV vector for the expression of a recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, or one vector for the expression of both proteins, is used in combination with an immunosuppressant or omigapil (N-(dibenz(b,f)oxepin-10-ylmethyl)-N-methyl-N-prop-2-ynylamine).

[0088] The dosing regimen in human subjects is preferably an AAV vector for expression of a recombinant chimeric laminin-nidogen protein or fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, at 1E13 to 3E14 vector genomes per kilogram (vg / kg) of body weight, and an AAV vector for expression of a recombinant agrin protein or fragment thereof, at 1E13 to 3E14 vector genomes per kilogram (vg / kg) of body weight. EXAMPLES

[0089] Example 1: Generation of protein constructs encoding hmag974aa, hαLNNd1373aa, and hαLNNd1002aa: The Spc512 promoter and minimal polyA signal were generated by de novo DNA synthesis. The hmag974aa (SEQ ID NO:11), hαLNNd1373aa (SEQ ID NO:39), and hαLNNd1002aa proteins (SEQ ID NO:68) were codon-optimized for human use, generated by de novo DNA synthesis, and inserted into an AAV expression vector containing ITRs by standard restriction enzyme-based cloning. A single amino acid substitution, K187A, from the carboxy-terminus of the agrin protein fragment was generated by PCR-based site-directed mutagenesis using the oligo 5'-CCT GGT GGA GGC CAG CGC CGG CG-3' (SEQ ID NO:23) and the oligo 5'-CCC TTC TCG CAG TGG GGG-3' (SEQ ID NO:24).

[0090] Example 2: Packaging of protein constructs into AAV viruses AAV9 particles were generated by packaging a cell line (HEK293T) by triple transfection using recombinant AAV plasmids for protein expression described in Example 1, together with a plasmid containing the essential rep and cap genes and a third adenovirus-derived helper plasmid. AAV particles were purified using IDX gradient ultracentrifugation to remove impurities and empty capsids. Viral titers were quantified using SYBR Green-based qPCR with primers targeting the ITR sequences (5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 66) and 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO: 67). AAV particle purity was assessed by 4-12% SDS-acrylamide gel electrophoresis and Coomassie blue staining.

[0091] Example 3: AAV-mediated expression of proteins encoding hmag974aa and hαLNNd1373aa in mice Purified AAV9 particles were diluted in PBS to a titer of 1E13 vector copies (vg) / ml. Neonatal mice were co-injected with AAV9-hαLNNd1373aa (AAV-αLNNd) and AAV9-hmag974aa (AAV-mag) at a dose of 1.5E14 vg / kg (each vector) via the temporal vein on postnatal day 1 (P1). Muscles were harvested 8 weeks after injection and processed for Western blot analysis and immunohistochemistry. Figure 13A shows that Western blot analysis confirmed the presence of hmag974aa (mag) and hαLNNd1373aa in various muscles, namely, triceps brachii (TRC), quadriceps (Quad), tibialis anterior (TA) and gastrocnemius (Gastro). GAPDH was used as a loading control. Figure 13B shows that immunohistochemistry of triceps brachii muscle cross sections using antibodies against human agrin and human laminin-α1 confirmed the presence of hmag974aa and hαLNNd1373aa proteins in the extracellular matrix of muscle fibers. Scale bar, 100 μm.

[0092] Example 4: Effect of treatment with AAV-mediated delivery of hmag974aa and hαLNNd1373aa according to the present invention on body weight The therapeutic effect of AAV9-mediated delivery of hmag974aa and hαLNNd1373aa as described in Examples 1-3 above was evaluated in a mouse model of LAMA2-associated muscular dystrophy (dyW mice). After genotyping on the day of birth, mice were randomly assigned to treatment or vehicle groups. On postnatal day 1, mice were co-injected via the temporal vein with AAV9-hαLNNd1373aa and AAV9-hmag974aa (dyW AAV-DL) or vehicle only (dyW) at a dose of 1.5E14vg / kg (each vector). Body weight was assessed weekly. Starting at 4 weeks of age, dyW mice treated with hmag974aa and hαLNNd1373aa gained significantly more body weight compared to growth-impaired vehicle controls. Results are shown in FIG. 14. Data are mean ± SEM. *** P<0.001, Student's t test. n=4-5 mice per group.

[0093] Example 5: Effect of treatment with AAV-mediated delivery of hmag974aa and hαLNNd1373aa according to the present invention on muscle mass The therapeutic effect of AAV9-mediated delivery of hmag974aa and hαLNNd1373aa described in Examples 1-3 above was evaluated in a mouse model of LAMA2-associated muscular dystrophy (dyW mice). After genotyping on the day of birth, mice were randomly assigned to treatment or vehicle groups. On postnatal day 1, mice were co-injected via the temporal vein with AAV9-hαLNNd1373aa and AAV9-hmag974aa (dyW AAV-DL) or vehicle only (dyW) at a dose of 1.5E14vg / kg (each vector). Mice were euthanized at 8 weeks of age and muscle tissue was harvested. Injection of AAV9-hαLNNd1373aa and AAV9-hmag974aa significantly increased muscle weight in all muscles analyzed. Results are shown in Figure 15. Data are mean ± SEM. *** P<0.001. ** P<0.01. Student's t test. n=4-5 mice per group. TA: tibialis anterior muscle.

[0094] Example 6: Effect of treatment with AAV-mediated delivery of hmag974aa and hαLNNd1373aa according to the present invention on muscle histology The therapeutic efficacy of AAV9-mediated delivery of hmag974aa and human hαLNNd1373aa as described in Examples 1-3 above was evaluated in a mouse model of LAMA2-associated muscular dystrophy (dyW mice). After genotyping on the day of birth, mice were randomly assigned to treatment or vehicle groups. On postnatal day 1, mice were co-injected via the temporal vein with AAV9-hαLNNd1373aa and AAV9-hmag974aa (dyW AAV-DL) or vehicle only (dyW) at a dose of 1.5E14vg / kg (each vector). Mice were euthanized at 8 weeks of age. Figure 16A shows histology of the diaphragm muscle, assessed by staining cross sections with hematoxylin and eosin (H&E). Note that diaphragm thickness and histological signs of muscular dystrophy were strongly improved by injection of AAV9-hαLNNd1373aa and AAV9-hmag974aa. Scale bar, 50 μm. FIG. 16B is a graph showing the distribution of muscle fiber diameters in the triceps muscle. Treatment with AAV9-hαLNNd1373aa and AAV9-hmag974aa significantly shifted fiber size toward larger fibers. Data are mean ± SEM. * P < 0.05. n = 3–4 mice per group.

[0095] Example 7: Effect of treatment with AAV-mediated delivery of hmag974aa and hαLNNd1373aa according to the present invention on muscle strength The therapeutic effect of AAV9-mediated delivery of hmag974aa and human hαLNNd1373aa as described in Examples 1-3 above was evaluated in a mouse model of LAMA2-associated muscular dystrophy (dyW mice). After genotyping on the day of birth, mice were randomly assigned to treatment or vehicle groups. On postnatal day 1, mice were co-injected via the temporal vein with AAV9-hαLNNd1373aa and AAV9-hmag974aa (dyW AAV-DL) or vehicle only (dyW) at a dose of 1.5E14vg / kg (each vector). Muscle function was assessed at 8 weeks of age. Treatment with AAV9-hαLNNd1373aa and AAV9-hmag974aa significantly increased total forelimb grip strength (A) and specific grip strength (B). C) Locomotor activity was increased in treated dyW mice. The results are shown in Figure 17. Data are means ± SEM. *** P<0.001, Student's t test. n=5-8 mice per group.

[0096] Example 8: Effect of treatment with AAV-mediated delivery of hmag974aa and hαLNNd1373aa according to the present invention on survival rate The therapeutic effect of AAV9-mediated delivery of hmag974aa and hαLNNd1373aa described in Examples 1-3 above was evaluated in a mouse model of LAMA2-associated muscular dystrophy (dyW mice). After genotyping on the day of birth, mice were randomly assigned to treatment or vehicle groups. On postnatal day 1, mice were co-injected via the temporal vein with AAV9-hαLNNd1373aa and AAV9-hmag974aa (dyW AAV-DL) or vehicle only (dyW) at a dose of 1.5E14vg / kg (each vector). The life span of the mice is shown as Kaplan-Meier cumulative survival curves. Marks indicate mice that were still alive. Animals treated with AAV9-hαLNNd1373aa and AAV9-hmag974aa had a higher probability of survival. The results are shown in Figure 18.

[0097] Example 9: Effect of treatment with AAV9-mediated delivery of Spc5.12-hmag974aa and Spc5.12-hαLNNd1373aa or Spc5.12-hmag974aa and CBh-hαLNNd1002daa according to the present invention on muscle mass The therapeutic efficacy of AAV9-mediated delivery of hmag974aa and hαLNNd1373aa, both driven by a muscle-specific promoter (Spc5.12), or hmag974aa (driven by the Spc5.12 promoter) and hαLNNd1002aa (driven by the ubiquitous promoter CBh), was evaluated in a mouse model of LAMA2-associated muscular dystrophy (dyW mice). After genotyping on the day of birth, mice were randomly assigned to treatment or vehicle groups. On postnatal day 1, mice were co-injected via the temporal vein with AAV9-Spc5.12-hαLNNd1373aa and AAV9-Spc5.12-hmag974aa (dyW AAV-Spc5.12-DL), or AAV9-CBh-hαLNNd1002aa and AAV9-Spc5.12-hmag974aa (dyW AAV-Spc5.12 / CBh-DL), or vehicle only (dyW) at a dose of 1.5E14vg / kg (each vector). Mice were euthanized at 8 weeks of age and muscle tissue was harvested. The specific combination of the Spc5.12 promoter driving hmag974aa and CBh of hαLNNd1002aa significantly increased muscle weight compared to muscle-specific expression. Results are shown in Figure 19. Data are mean ± SEM. ** P<0.01. * P<0.05. One-way ANOVA with Bonferroni post-hoc test. n = 4–5 mice per group. TA: tibialis anterior muscle.

[0098] Example 10: Effect of treatment with AAV9-mediated delivery of Spc5.12-hmag974aa and CBh-hαLNNd1002daa according to the present invention on locomotion The therapeutic effect of AAV9-mediated delivery of hmag974aa (driven by Spc5.12) and hαLNNd1002 (driven by the ubiquitous promoter CBh) was evaluated in a mouse model of LAMA2-associated muscular dystrophy (dyW mice). After genotyping on the day of birth, mice were randomly assigned to treatment or vehicle groups. On postnatal day 1, mice were co-injected with AAV9-CBh-hαLNNd1002aa at a dose of 1.5E14vg / kg (each vector) and AAV9-Spc5.12-hmag974aa (dyW AAV-Spc5.12 / CBh-DL) or vehicle only (dyW) via the temporal vein. Ambulation ability was assessed at 8 weeks of age. The specific combination of Spc5.12 promoter driving hmag974aa and CBh of hαLNNd1002aa significantly increased the walking ability of dyW mice. The results are shown in Figure 20. Data are mean ± SEM. ** P<0.01. * P<0.05, Student's t test, n=3-4 mice per group.

[0099] [advantage] The recombinant agrin protein or a fragment thereof according to the present invention, alone or in combination with a recombinant chimeric laminin / nidogen protein according to the present invention comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, effectively enables the polymerization of laminin and the stabilization of muscle fibers by binding laminin to cell membranes.

[0100] The recombinant agrin protein or fragments thereof according to the invention are significantly more stable than the mini-agrin proteins according to the prior art.

[0101] The recombinant agrin protein or fragment thereof construct of the present invention, alone or in combination with a recombinant chimeric laminin / nidogen protein of the present invention comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, is suitable to be fitted into an AAV vector and efficiently expressed.

[0102] The recombinant agrin protein or fragments thereof according to the present invention, alone or in combination with the recombinant chimeric laminin / nidogen protein according to the present invention comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, induce a low immune response in vivo, since they are composed of protein domains present in LAMA2 MD patients.

[0103] The recombinant agrin protein or a fragment thereof according to the present invention, alone or in combination with a recombinant chimeric laminin / nidogen protein according to the present invention containing at least the LN domain of laminin and the G3 domain of nidogen or containing at least the LN domain of laminin and the G2 and G3 domains of nidogen, is efficiently secreted and acts outside the muscle fiber.

[0104] As shown by the in vivo experiments disclosed above, the recombinant agrin protein or a fragment thereof according to the present invention, alone or in combination with a recombinant chimeric laminin / nidogen protein according to the present invention comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, delivered via AAV significantly ameliorates the disease phenotype in a mouse model of LAMA2 MD by increasing body and muscle weight, improving muscle histology, fiber number, fiber size, and reducing inflammatory responses in muscle.

Claims

1. A recombinant agrin protein or a fragment thereof, the fragment comprising at least an N-terminal agrin domain (NtA domain), a laminin globular domain 2 (LG2 domain), a laminin globular domain 3 (LG3 domain), and an amino acid sequence linking EGF-like domain 4 (EG4 domain) and the LG3 domain of the recombinant agrin protein, in which at least one amino acid residue of the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or a fragment thereof is modified, and the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein comprises the amino acid sequence KGLVEASAGD shown in SEQ ID NO:

65.

2. The recombinant agrin protein or a fragment thereof according to claim 1, wherein at least one modified amino acid residue in the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein or a fragment thereof is A between E and S of the amino acid sequence KGLVEASAGD shown in SEQ ID NO:

65.

3. 3. The recombinant agrin protein or a fragment thereof according to any one of claims 1 to 2, wherein the fragment of the recombinant agrin protein further comprises the following polypeptides: a signal sequence (SS), a follistatin-like domain (FS domain), three EGF-like domains (EG domain), and one laminin globular domain (LG domain).

4. 3. The recombinant agrin protein or a fragment thereof according to claim 1, wherein the recombinant agrin protein fragment comprises the following polypeptides: a signal sequence (SS), an N-terminal agrin domain (NtA domain), a follistatin-like domain (FS domain), an EGF-like domain 1 (EG1 domain), a laminin globular domain 1 (LG1 domain), an EGF-like domain 2 (EG2 domain), an EGF-like domain 3 (EG3 domain), a laminin globular domain 2 (LG2 domain), an EGF-like domain 4 (EG4 domain), and an amino acid sequence linking the EGF-like domain 4 (EG4 domain), the LG3 domain, and the laminin globular domain 3 (LG3 domain), wherein at least one amino acid residue of the amino acid sequence linking the EG4 domain and the LG3 domain of the fragment is modified, and the amino acid sequence linking the EG4 domain and the LG3 domain of the recombinant agrin protein comprises the amino acid sequence KGLVEASAGD shown in SEQ ID NO:

65.

5. 5. The agrin protein or a fragment thereof according to any one of claims 1 to 4, comprising the sequence shown in SEQ ID NO:

11.

6. A nucleic acid sequence encoding the agrin protein or a fragment thereof according to any one of claims 1 to 5.

7. 7. The nucleic acid sequence of claim 6, further comprising a muscle-specific promoter operably linked to the nucleic acid sequence encoding the agrin protein or a fragment thereof.

8. A vector comprising the nucleic acid sequence of claim 6 or claim 7.

9. A recombinant chimeric laminin-nidogen protein or a fragment thereof comprising the sequence shown in SEQ ID NO:39 or the sequence shown in SEQ ID NO:

68.

10. A nucleic acid sequence encoding the protein of claim 9.

11. 11. The nucleic acid sequence of claim 10, further comprising a muscle-specific or ubiquitous promoter operably linked to the nucleic acid sequence encoding the protein of claim 9.

12. A vector comprising the nucleic acid sequence of claim 10 or claim 11.

13. A composition comprising an agrin protein or a fragment thereof according to any one of claims 1 to 5 and a recombinant chimeric laminin-nidogen protein or a fragment thereof comprising at least the LN domain of laminin and the G3 domain of nidogen or comprising at least the LN domain of laminin and the G2 and G3 domains of nidogen, preferably alpha LNNd protein, more preferably a recombinant chimeric laminin-nidogen protein or a fragment thereof according to claim 9, or a nucleic acid sequence according to claim 6 or 7 and a nucleic acid sequence according to claim 10 or 11, or a vector according to claim 8 and a vector according to claim 12, or a vector comprising a nucleic acid sequence according to claim 6 or 7 and a nucleic acid sequence according to claim 10 or 11, preferably an AAV vector comprising a nucleic acid sequence according to claim 6 or 7 and a nucleic acid sequence according to claim 10 or 11.

14. An agrin protein or a fragment thereof according to any one of claims 1 to 5 and / or a recombinant chimeric laminin-nidogen protein or a fragment thereof according to claim 9, or a nucleic acid sequence according to claim 6 or 7 and / or a nucleic acid sequence according to claim 10 or 11, or a vector according to claim 8 and / or a vector according to claim 12, or a vector comprising the nucleic acid sequence according to claim 6 or 7 and the nucleic acid sequence according to claim 10 or 11, and / or a composition according to claim 13, for use in the prevention and / or treatment of congenital muscular dystrophy.

15. 15. The protein, nucleic acid sequence, vector or composition for use according to claim 14, wherein the congenital muscular dystrophy is (laminin-α2) LAMA2-associated muscular dystrophy.

16. 16. The protein, nucleic acid sequence, vector or composition for use according to claim 14 or 15, wherein the AAV vector for the expression of the recombinant laminin-nidogen protein and the AAV vector for the expression of the agrin protein or a fragment thereof are used in combination with an immunosuppressant or omigapil (N-(dibenz(b,f)oxepin-10-ylmethyl)-N-methyl-N-prop-2-ynylamine).

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