Recombinant adeno-associated virus products and methods for treating dystroglycanopathy and laminin-deficient muscular dystrophy

Abstract

To provide products and methods for treating dystroglycanopathies and laminin-deficient muscular dystrophies.SOLUTION: In methods, a protein including a linker domain, such as the heparin-binding domain of Heparin-Binding Epidermal Growth Factor-Like Growth Factor (HBEGF), is delivered to patients. Provided herein are methods and products for treatment of CMDs such as dystroglycanopathies and laminin-deficient muscular dystrophies. The products include therapeutic proteins and rAAV encoding disclosed therapeutic proteins.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 686,522, filed on 18 June 2018, which is incorporated herein by reference in its entirety. This invention was made with government support under AR070604 granted by the National Institutes of Health, USA. The government has certain rights in this invention.

[0002] Integration by referencing electronically submitted documents This application includes, as a separate part of the present disclosure, a computer-readable sequence listing in its entirety incorporated herein by reference and recognized as filename: 53147A_Seqlisting.txt, size: 125,439 bytes, created: June 18, 2019.

[0003] Products and methods for treating dystroglycanopathy and laminin-deficient muscular dystrophy are provided. In this method, a protein containing a linker domain, such as the heparin-binding domain of heparin-binding epidermal growth factor-like growth factor (HBEGF), is delivered to the patient. This linker protein helps target the transgene to the extracellular matrix (ECM) of muscle cells. [Background technology]

[0004] Muscular dystrophy (MD) is a group of genetic disorders characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while others may not appear until middle age or later. The disorder varies in terms of the distribution and extent of muscle weakness (some forms of MD also affect the myocardium), age of onset, rate of progression, and mode of inheritance.

[0005] Congenital muscular dystrophy (CMD) represents a group of muscular disorders (MDs) in which the loss of structural components of muscle leads to neonatal hypotonia and progressive skeletal muscle weakness. These disorders are often associated with significant extramuscular complications, including brain and eye developmental disorders, cognitive impairment, seizures, and respiratory and cardiac abnormalities, and require regular medical management by an interdisciplinary team. The estimated incidence of CMD is 1 in 21,500 births worldwide. Despite the severity of these disorders, there are currently no approved effective therapies. Dystroglycanopathy and merosin-deficient CMD1A (MDC1A) are two of the most common forms of CMD [Sframeli et al., Neuromuscul Disord., 27(9):793-803 (2017)].

[0006] Dystroglycanopathy is caused by mutations in any of the 18 or more genes required for the glycosylation of α-dystroglycan. Proper glycosylation allows α-dystroglycan to bind to elements of the extracellular matrix (ECM). α-dystroglycan is then fixed to the muscular sheath by binding to β-dystroglycan, a transmembrane protein. Due to the number of susceptibility genes, the development of a single gene replacement therapy for dystroglycanopathy is not feasible. Examples of dystroglycanopathy include: Walker-Warburg syndrome (WWS), which involves gene mutations in B3GLNT2, B4GAT1, DAG1, FKRP, FKTN, GMPPB, ISPD, or LARGE; Myoophthalmoencephalopathy (MEB), which involves gene mutations in B3GLNT2, B4GAT1, DAG1, FKRP, FKTN, GMPPB, ISPD, or LARGE; and Fukuyama-type CMD, which involves mutations in the FKTN gene. The group of congenital muscular dystrophy with cognitive impairment is due to mutations in FKRP, LARGE, POMT1, POMT2, or POMGNT1. The group of CMD without cognitive impairment is a result of gene mutations in FKRP or FKTN. Limb-girdle muscular dystrophy LGMD2I, 2K, 2M, 2N, and 2O are associated with glycosylation abnormalities due to gene mutations in FKRP, FKTN, POMGNT1, POMT1, or POMT2. Limb-girdle muscular dystrophy LGMD2T and 2U are a result of gene mutations in GMPPB and ISPD, respectively. Other mutated genes in dystroglycanopathy include DOLK, DPM1, DPM2, DPM3, GTDC2 / AG061, TMEM5, and SK196.

[0007] MDC1A is caused by a mutation in the LAMA2 gene, which encodes laminin-α2, a key ECM protein that binds to glycosylated α-dystroglycans in the sarcoplasmic sheath. The complete LAMA2 gene is over 9,000 base pairs long.

[0008] A study by Reinhard et al., Sci Transl Med., 9(396), (2017) showed that germline expression of a fusion domain from laminin-α4 and miniagrin resulted in incomplete remission of disease symptoms in a dyW / dyW mouse model of MDC1A.

[0009] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains two 145-nucleotide reverse-ended repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the AAV serotype genomes are known. For example, the complete genome of AAV-1 is available under GenBank acceptance number NC_002077, the complete genome of AAV-2 is available under GenBank acceptance number NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is available under GenBank acceptance number NC_1829, the complete genome of AAV-4 is available under GenBank acceptance number NC_001829, the genome of AAV-5 is available under GenBank acceptance number AF085716, the complete genome of AAV-6 is available under GenBank acceptance number NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are available under GenBank acceptance numbers AX753246 and AX753249, respectively, and the genome of AAV-9 is available under Gao et al. The AAV-10 genome is available in al., J. Virol., 78:6381-6388 (2004), the AAV-11 genome is available in Mol. Ther., 13(1):67-76 (2006), the AAV-11 genome is available in Virology, 330(2):375-383 (2004), a portion of the AAV-12 genome is available under GenBank access number DQ813647, and a portion of the AAV-13 genome is available under GenBank access number EU285562. The sequence of the AAVrh.74 genome is available under U.S. Patent No. 9,434,928, which is incorporated herein by reference. The Cis action sequence that directs viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration is contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 relative to their relative map locations) promote the expression of two AAV internal open reading frames that encode rep and cap genes.Coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), two rep promoters (p5 and p19) generate four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production of the three related capsid proteins. A single-consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are described in Muzyczka, Current Topics in. This was reviewed in Microbiology and Immunology, 158:97-129 (1992).

[0010] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cellular, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, enabling the potential to target many different tissues in vivo. Additionally, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA within a plasmid, enabling the construction of a recombinant genome. Furthermore, since signals directing AAV replication and genomic capsid formation are contained within the ITR of the AAV genome, some or all of the internal approximately 4.3 kb of genome (rep-cap, encoding replication and structural capsid proteins) may be replaced with foreign DNA. To generate an AAV vector, the rep and cap proteins can be supplied trans. Another important characteristic of AAV is that it is an extremely stable and robust virus. This makes it easy to withstand the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), reducing the importance of chilling AAV. AAV can be freeze-dried. Finally, AAV-infected cells do not show resistance to co-infection. The need for this technology for the treatment of CMDs such as dystroglycanopathy and MDC1A remains. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Sframeli et al., Neuromuscul Disord.,27(9):793-803(2017) [Non-Patent Document 2] Reinhard et al.,Sci Transl Med.,9(396),(2017) [Non-Patent Document 3] Srivastava et al., J.Virol., 45:555-564(1983)

Summary of the Invention

Means for Solving the Problems

[0012] Methods and products for the treatment of CMDs such as dystroglycanopathies and laminin-deficient muscular dystrophy are provided herein. The products include therapeutic proteins and rAAVs encoding the disclosed therapeutic proteins.

[0013] a) A first domain comprising the heparin-binding domain of heparin-binding epidermal growth factor-like growth factor (HBEGF), and a second domain comprising the G1-G5 domains of the human laminin alpha 2 (LAMA2) gene, b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G1-G5 domains of the human LAMA2 gene, c) A first domain comprising the heparin-binding domain of HBEGF, and a second domain comprising the G3-G5 domains of the human LAMA2 gene, d) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G3-G5 domains of the human LAMA2 gene, e) A first domain comprising the heparin-binding domain of HBEGF, and a second domain comprising DAG1 alpha, or f) A polynucleotide encoding a protein comprising a first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising DAG1 alpha is provided.

[0014] In one embodiment, the provided polynucleotide encodes a protein, the first domain of the protein being encoded by the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and the second domain of the protein being encoded by the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0015] For example, the provided polynucleotide may include the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 15, or SEQ ID NO: 13 and SEQ ID NO: 16, or SEQ ID NO: 14 and SEQ ID NO: 15, or SEQ ID NO: 14 and SEQ ID NO: 16, or SEQ ID NO: 13 and SEQ ID NO: 17, or SEQ ID NO: 14 and SEQ ID NO: 17.

[0016] In one embodiment, the provided polynucleotide comprises one of the following: i) the nucleotide sequence shown in Figure 3, ii) the nucleotide sequence containing nucleotides 14-3235 shown in Figure 3, iii) the nucleotide sequence of SEQ ID NO: 1, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 19.

[0017] In another embodiment, the provided polynucleotide includes one of the following: i) the nucleotide sequence shown in Figure 4, ii) the nucleotide sequence containing nucleotides 14-3361 shown in Figure 4, iii) the nucleotide sequence of SEQ ID NO: 3, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 20.

[0018] In further embodiments, the provided polynucleotide includes one of the following: i) the nucleotide sequence shown in Figure 5, ii) the nucleotide sequence containing nucleotides 14-1930 shown in Figure 5, iii) the nucleotide sequence of SEQ ID NO: 5, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 21.

[0019] In another embodiment, the provided polynucleotide comprises one of the following: i) the nucleotide sequence shown in Figure 6, ii) the nucleotide sequence containing nucleotides 14-2056 shown in Figure 6, iii) the nucleotide sequence of SEQ ID NO: 7, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22.

[0020] In one embodiment, the provided polynucleotide includes one of the following: i) the nucleotide sequence shown in Figure 7, ii) the nucleotide sequence containing nucleotides 14-1360 shown in Figure 7, iii) the nucleotide sequence of SEQ ID NO: 9, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23.

[0021] In another embodiment, the provided polynucleotide includes one of the following: i) the nucleotide sequence shown in Figure 8, ii) the nucleotide sequence containing nucleotides 14-1486 shown in Figure 8, iii) the nucleotide sequence of SEQ ID NO: 11, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24.

[0022] A therapeutic protein encoded by any of the provided polynucleotides is also provided. For example, the provided protein contains the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0023] In addition, this disclosure provides recombinant host cells comprising any of the polynucleotides described herein. In exemplary embodiments, the host cells and polynucleotides are operably linked to transcriptional regulators, and these host cells express any of the polynucleotides disclosed herein. For example, the host cells are Chinese hamster ovary (CHO) cells or human HEK293 cells.

[0024] Recombinant adeno-associated viruses (rAAVs) are further provided, the genome of which the rAAV comprises one of the polynucleotides described herein. For example, an rAAV is provided, the genome of which the rAAV comprises the polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11. In exemplary embodiments, the rAAV genome further comprises muscle-specific transcriptional regulators such as the CMV promoter (SEQ ID NO: 18), MCK, NHCK, LAMA2, or tMCK. Any of the rAAVs described herein comprises an AAV9, AAV10, AAVrh74, AAV8, or AAV6 capsid.

[0025] rAAV is also provided, and the rAAV genome includes nucleotides 3590-8215 of SEQ ID NO: 2, nucleotides 3590-8341 of SEQ ID NO: 4, nucleotides 3609-6929 of SEQ ID NO: 6, nucleotides 3590-7036 of SEQ ID NO: 8, nucleotides 3590-6340 of SEQ ID NO: 10, nucleotides 3590-6049 of SEQ ID NO: 12, the nucleotide sequence shown in Figure 13, or the nucleotide sequence shown in Figure 14.

[0026] rAAV particles comprising any of the rAAVs described herein are also provided. The disclosure also provides compositions comprising any of the polynucleotides disclosed herein, any of the rAAVs disclosed herein, any of the rAAV particles disclosed herein, or any of the proteins disclosed herein.

[0027] A method for treating laminin-deficient muscular dystrophy is provided, which involves administering rAAV to patients in need of treatment, and the genome of rAAV is a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G1-G5 domains of the human LAMA2 gene, b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G1-G5 domains of the human LAMA2 gene, c) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene, or d) A polynucleotide encoding a protein comprising a first domain containing the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene.

[0028] For example, a method for treating laminin-deficient muscular dystrophy includes administering to a patient in need of treatment either one of the polynucleotides disclosed herein that encode a protein comprising LAMA2(G1-G5) or LAMA2(G3-G5) as a second domain, or one of the rAAVs or rAAV particles disclosed herein that encode a polynucleotide that encodes a protein comprising LAMA2(G1-G5) or LAMA2(G3-G5) as a second domain.

[0029] Further methods have been provided for treating laminin-deficient muscular dystrophy, including, for patients who require treatment, a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G1-G5 domains of the human LAMA2 gene, b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G1-G5 domains of the human LAMA2 gene, c) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene, or d) The procedure involves administering a protein comprising a first domain containing the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene.

[0030] For example, a method for treating laminin-deficient muscular dystrophy involves administering a protein to a patient in need of treatment, the protein comprising the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0031] Compositions for treating laminin-deficient muscular dystrophy, including rAAV, are also provided, and the rAAV genome is, a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G1-G5 domains of the human LAMA2 gene, b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G1-G5 domains of the human LAMA2 gene, c) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene, or d) A polynucleotide encoding a protein comprising a first domain containing the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene.

[0032] For example, a composition for treating laminin-deficient muscular dystrophy includes any of the following: any of the polynucleotides disclosed herein that encode a protein comprising LAMA2(G1-G5) or LAMA2(G3-G5) as a second domain; or any of the rAAVs or rAAV particles disclosed herein that include a polynucleotide encoding a protein comprising LAMA2(G1-G5) or LAMA2(G3-G5) as a second domain.

[0033] Further provided are compositions for treating laminin-deficient muscular dystrophy, the protein being: a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G1-G5 domains of the human LAMA2 gene, b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G1-G5 domains of the human LAMA2 gene, c) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene, or d) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G3-G5 domain of the human LAMA2 gene.

[0034] For example, a composition for treating laminin-deficient muscular dystrophy comprises a protein, the protein comprising the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0035] This disclosure also provides the use of rAAV for the preparation of therapeutic agents in patients requiring treatment for laminin-deficient muscular dystrophy, and the rAAV genome is a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G1-G5 domains of the human LAMA2 gene, b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G1-G5 domains of the human LAMA2 gene, c) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene, or d) A polynucleotide encoding a protein comprising a first domain containing the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene.

[0036] For example, the Disclosure also provides the use of any of the rAAVs or rAAV particles disclosed herein, comprising any of the polynucleotides disclosed herein that encode a protein comprising LAMA2(G1-G5) or LAMA2(G3-G5) as a second domain, for the preparation of a drug for therapeutic purposes in patients requiring treatment for laminin-deficient muscular dystrophy.

[0037] This disclosure further provides the use of a protein for the preparation of a therapeutic agent in patients requiring treatment for laminin-deficient muscular dystrophy, and the protein is a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G1-G5 domains of the human LAMA2 gene, b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G1-G5 domains of the human LAMA2 gene, c) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the G3-G5 domain of the human LAMA2 gene, or d) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G3-G5 domain of the human LAMA2 gene.

[0038] For example, the disclosure also provides the use of a protein for the preparation of a drug to perform treatment in patients requiring treatment for laminin-deficient muscular dystrophy, the protein comprising the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0039] Methods for treating dystroglycanopathy are also provided, which include administering rAAV to patients in need of treatment, and the rAAV genome is a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing DAG1 alpha, or b) A polynucleotide encoding a protein comprising a first domain containing the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain containing DAG1 alpha.

[0040] For example, a method for treating dystroglycanopathy includes administering to a patient in need of treatment any of the following: any of the polynucleotides disclosed herein that encode a protein comprising DAG1 alpha as a second domain, or any of the rAAVs or rAAV particles disclosed herein that comprise a polynucleotide encoding a protein comprising DAG1 alpha as a second domain.

[0041] Further methods for treating dystroglycanopathy have been provided, including for patients who need treatment. a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing DAG1 alpha, or b) The procedure involves administering a protein comprising a first domain containing the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain containing DAG1 alpha.

[0042] For example, a method for treating dystroglycanopathy, comprising administering a protein to a patient in need of treatment, wherein the protein comprises the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24.

[0043] Compositions for treating dystroglycanopathy containing rAAV are also provided, and the rAAV genome is a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing DAG1 alpha, or b) A polynucleotide encoding a protein comprising a first domain containing the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain containing DAG1 alpha.

[0044] For example, the present disclosure provides a composition for treating dystroglycanopathy comprising any of the following: any of the polynucleotides disclosed herein encoding a protein containing DAG1 alpha as a second domain, or any of the rAAVs or rAAV particles disclosed herein comprising a polynucleotide encoding a protein containing DAG1 alpha as a second domain.

[0045] A composition for treating dystroglycanopathy containing a protein is also provided, the protein being: a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing DAG1 alpha, or b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising DAG1 alpha.

[0046] For example, a composition for treating dystroglycanopathy comprises a protein, the protein comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24.

[0047] The use of rAAV for the preparation of therapeutic drugs in patients requiring treatment for dystroglycanopathy is also provided, and the rAAV genome is, a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing DAG1 alpha, or b) A polynucleotide encoding a protein comprising a first domain containing the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain containing DAG1 alpha.

[0048] For example, this disclosure provides the use of any of the polynucleotides disclosed herein that encode a protein containing DAG1 alpha as a second domain, or any of the rAAVs or rAAV particles disclosed herein that encode a polynucleotide that encodes a protein containing DAG1 alpha as a second domain, for the preparation of a drug for therapeutic purposes in patients requiring treatment for dystroglycanopathy.

[0049] The use of proteins for the preparation of therapeutic drugs in patients requiring treatment for dystroglycanopathy is also provided, and the proteins are, a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing DAG1 alpha, or b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising DAG1 alpha.

[0050] For example, this disclosure provides the use of a protein for the preparation of a drug to perform treatment in patients requiring treatment for dystroglycanopathy, wherein the protein comprises the amino acid sequence of SEQ ID NO: SEQ ID NO: 23 or SEQ ID NO: 24.

[0051] In the methods, uses, or compositions provided for treating laminin-deficient muscular dystrophy, laminin-deficient muscular dystrophy may be, for example, MDC1A.

[0052] In any method, use, or composition for treating dystroglycanopathy, dystroglycanopathy is, for example, Walker-Warburg syndrome, myophthalmoencephalopathy, Fukuyama congenital muscular dystrophy, MDC1C, MDC1D, LGMD2I, LGMD2K, LGMD2M, LGMD2N, LGMD2O, LGMD2P, LGMD2T, or LGMD2U.

[0053] Examples of the proteins provided are shown in Table 1. [Table 1]

[0054] The patent or application file must include at least one drawing made in color. A copy of the publication of this patent or patent application, including the color drawings and color photographs, will be provided by the Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0055] [Figure 1-1] [Figure 1A] Shows the dystrophin-associated glycoprotein (DAG) complex. [Figure 1B] Shows that α-dystroglycan is not only abnormally glycosylated (losing its normal laminin-binding function) in dystroglycanopathy, but also that the α-dystroglycan protein is reduced in affected muscle. [Figure 1C] Shows that the therapeutic proteins described herein enable α-dystroglycan to bind to the fascia by binding to β-dystroglycan, which is present in normal amounts, and to bind to the ECM even without its appropriate ECM-binding glycan, via the binding of HBEGF to heparin sulfate proteoglycan in the ECM. This reconstitutes the lost binding of α-dystroglycan to the ECM and fascia. The use of the methods described herein that provide these therapeutic proteins is applicable to the treatment of all 18 or more genotypes of dystroglycanopathy, and the methods represent a powerful alternative to gene replacement strategies, which require the development of different gene therapies for each dystroglycanopathy. [Figure 1-2] Same as above. [Figure 2-1][Figure 2A] This shows that MDC1A is caused by a loss-of-function mutation in the LAMA2 gene, which encodes laminin α2, an extracellular matrix (ECM) protein that surrounds each muscle cell in the body. LAMA2 is required for muscle cell adhesion to the ECM and for fascial stability. [Figure 2B] This shows that the therapeutic proteins described herein can fix the LAMA2 G1-G5 domain to the ECM where the LAMA2 G1-G5 domain normally exists, so that the LAMA2 G1-G5 domain can function similarly to natural laminin α2. Therefore, the use of the methods described herein that provide these therapeutic proteins is applicable to MDC1A. [Figure 2-2] Same as above. [Figure 3-1] This shows the polynucleotide sequence encoding the therapeutic protein HB-EGF (terminating with a heparin-binding domain)-LAMA2 G1-G5. The therapeutic protein is encoded by the nucleotides of the present invention and includes nucleotides 14-3235, which also correspond to Sequence ID No. 1. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 4-1] This shows the polynucleotide sequence encoding the therapeutic protein HB-EGF (fully soluble)-LAMA2 G1-G5. The therapeutic protein is encoded by the nucleotides of the present invention and contains nucleotides 14-3361, which also correspond to Sequence ID No. 3. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. [Figure 4-6] Same as above. [Figure 4-7] Same as above. [Figure 5-1]This shows the polynucleotide sequence encoding the therapeutic protein HB-EGF (terminating with a heparin-binding domain)-LAMA2 G3-G5. The therapeutic protein is encoded by the nucleotides of the present invention and contains nucleotides 14-1930, which also correspond to Sequence ID No. 5. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 6-1] This shows the polynucleotide sequence encoding the therapeutic protein HB-EGF (fully soluble)-LAMA2 G3-G5. The therapeutic protein is encoded by the nucleotides of the present invention and contains nucleotides 14-2056, which also correspond to Sequence ID No. 7. [Figure 6-2] Same as above. [Figure 7-1] This shows a polynucleotide sequence encoding the therapeutic protein HB-EGF (terminated with a heparin-binding domain)-DAG1 (a naturally processed alpha-DG gene). The therapeutic protein is encoded by the nucleotides of the present invention and contains nucleotides 14-1360, which also correspond to Sequence ID No. 9. [Figure 7-2] Same as above. [Figure 8-1] This shows the polynucleotide sequence encoding the therapeutic protein HB-EGF (fully soluble)-DAG1 (naturally processed alpha-DG gene). The therapeutic protein is encoded by the nucleotides of the present invention and contains nucleotides 14-1486, which also correspond to Sequence ID No. 11. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 9-1] This shows the expression of the therapeutic proteins described herein by recombinant mammalian host cells. [Figure 9-2] Same as above. [Figure 10] This shows that sHB-EGF can be secreted from muscle and adhere to the extracellular matrix. [Figure 11]This study demonstrates that sHB-EGF induces the expression of therapeutic surrogate muscular dystrophy genes. Full-length HBEGF does not induce therapeutic gene expression. [Figure 12] This study demonstrates that sHB-EGF can induce the Akt tyrosine kinase cascade in skeletal muscle, stimulating muscle growth and regeneration. [Figure 13-1] This shows an exemplary rAAV genome encoding the therapeutic protein HB-EGF (fully soluble)-LAMA2 G1-G5. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above. [Figure 14-1] This shows an exemplary rAAV genome encoding the therapeutic protein HB-EGF (fully soluble)-DAG1 (a naturally processed alpha-DG gene). [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 15] This document provides immunohistochemical staining of HB-EGF and LG5 (represented as 4H8-2 in the figure) after intramuscular injection of rAAV9.CMV vectors containing HBEGF, HBEGF.LAMA2(G1-G5), HBEGF.LAMA2(G3-G5), HB.LAMA2(G1-G5), HB.LAMA2(G3-G5), or LAMA2(G1-G5) in wild-type mice. Mice injected with sham injections (buffer only) are shown as negative controls. 4H8-2 is an anti-laminin antibody representing muscle cells in tissue sections. [Figure 16] This provides immunohistochemical staining of HB-EGF and laminin globular domains (LG5) in muscles injected with rAAV9.HBEGF-LAMA2(G1-G5), HB-LAMA2(G1-G5), or LAMA2(G1-G5) via immunoimmunochemical injection. The lower panel below shows staining with secondary antibodies only. [Figure 17]This graph shows that rAAV9.CMV.HB.LAMA(G1-G5) prevented muscle weakness in dy / dy mice. Mice were intravenously injected with 1 × 10¹² vg of rAAV9.CMV vector containing HBEGF.LAMA2(G1-G5), HB.LAMA2(G1-G5), or HB.LAMA2(G3-G5). Mice were compared to sham-injected dy / dy disease controls and wild-type normal controls 2 and 3 months after injection. Mixed (50:50) female:male sex was used in all groups. Error was calculated using SEM of n=12 animals per group (wild-type and sham-dy / dy), 6 animals (sHB-EGF.LAMA2G1-G5 and HB.LAMA2G1-G5), or 5 animals (HB.LAMA2G3-G5), with 5 measurements averaged per data point. *p<0.05, **p<0.01, ***p<0.001 [Figure 18] To demonstrate the expression of HB.LAMA2(G1-G5) in 4-month-old dy / dy muscle (triceps) after IV injection in P1, we provide immunohistochemical staining of HB-EGF and LG5. Muscle sections from the triceps of 4-month-old wild-type and dy / dy mice, either sham-injected or injected with 1 × 10¹² vg of rAAV9.CMV.HB-LAMA2(G1-G5), were stained with antibodies specific to HBEGF (green) to recognize the transgenic protein and to collagen IV (Col(IV), red) to recognize all muscle cells. DAPI was added in blue to stain the nuclei. A combined tricolor image is shown. [Figure 19-1] This provides the plasmid sequences (SEQ ID NO: 2) for pAAV.CMV.HB.LAMA1(G1-G5), with the rAAV genome corresponding to nucleotides 3590-8215. [Figure 19-2] Same as above. [Figure 19-3] Same as above. [Figure 19-4] Same as above. [Figure 20-1] This provides the plasmid sequences (SEQ ID NO: 4) for pAAV.CMV.HBEGF LAMA2 (G1-G5), with the rAAV genome corresponding to nucleotides 3590-8341. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 20-4] Same as above. [Figure 21-1] This provides the plasmid sequence (SEQ ID NO: 6) for pAAV.CMV.HB LAMA2(G3-G5), with the rAAV genome corresponding to nucleotides 36909-6929. [Figure 21-2] Same as above. [Figure 21-3] Same as above. [Figure 21-4] Same as above. [Figure 22-1] This provides the plasmid sequences (SEQ ID NO: 8) for pAAV.CMV.HBEGF.LAMA2(G3-G5), with the rAAV genome corresponding to nucleotides 3590-7036. [Figure 22-2] Same as above. [Figure 22-3] Same as above. [Figure 23-1] The plasmid sequence (SEQ ID NO: 10) of pAAV.CMV.HB.DAG1 (alpha) is provided, and the rAAV genome corresponds to nucleotides, with the rAAV genome corresponding to nucleotides 3590-6340. [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 24-1] This provides the plasmid sequence (SEQ ID NO: 12) for pAAV.CMV.HB.DAG1 (alpha), with the rAAV genome corresponding to nucleotides 3590-6049. [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Modes for carrying out the invention]

[0056] This specification provides methods and products for the treatment of dystroglycanopathy (including, but not limited to, Walker-Warburg syndrome, myophthalmoencephalopathy, Fukuyama congenital muscular dystrophy, MDC1C, MDC1D, LGMD2I, LGMD2K, LGMD2M, LGMD2N, LGMD2O, LGMD2P, LGMD2T, and LGMD2U) and laminin-deficient muscular dystrophy (including, but not limited to, MDC1A), which utilize the lysine-rich heparin-binding domain of HBEGF. Heparin sulfate proteoglycans are abundant in the extracellular matrix (ECM), and as shown herein, overexpression of HBEGF in muscle leads to localization of HBEGF in the muscle ECM. In the methods described herein, membrane fixation defects in dystroglycanopathy and laminin-deficient muscular dystrophy are treated using the heparin-binding domain of HBEGF as the "linker" domain of the therapeutic protein.

[0057] Here, the term "HBEGF" refers to the entire HBEGF sequence up to the bioactive EGF domain, but lacks the transmembrane domain, thus enabling HBEGF secretion (Figure 4). The HBEGF fragment contains four domains from the HBEGF gene: a signal peptide that enables entry into the secretory pathway, a prepropeptide that enables protein folding and stabilization, a heparin-binding domain that enables increased interaction with the extracellular matrix, and a bioactive EGF domain that enables HBEGF signaling. In the proteins disclosed herein, the coding sequences of these domains are then ligated to a laminin alpha-2 or dystroglycan coding sequence. A second "HB" fragment is also used (Figure 3). The HB fragment contains only three of the four domains found in HBEGF: the signal peptide, the prepropeptide, and the heparin-binding domain (hence HB lacks the bioactive EGF domain). When ligated to a laminin alpha-2 or dystroglycan protein fragment, the HB domain can increase association with the ECM, but does not increase EGF or HBEGF signaling.

[0058] The HBEGF or HB linker domain targets the protein against the extracellular matrix of a cell, acting to fix this protein to the extracellular domain of a cell, such as a muscle cell. A polynucleotide encoding the therapeutic protein is delivered to the patient (e.g., by recombinant AAV encoding the therapeutic protein), or the therapeutic protein itself is delivered to the patient.

[0059] For example, in all cases of dystroglycanopathy, the coding sequence of the HBEGF heparin-binding domain is fused to the coding sequence of α-dystroglycan to create a polynucleotide encoding the therapeutic protein HBEGF-DAG1(α). In addition to hypoglycosylation of α-dystroglycan, α-dystroglycan protein levels are reduced in dystroglycanopathy. In the method described herein, the HBEGF domain of HBEGF-DAG1(α) binds to ECM heparin sulfate proteoglycan, while the α-dystroglycan domain binds to β-dystroglycan, linking the muscular sheath to the ECM despite hypoglycosylation in dystroglycanopathy. Four examples of such HBEGF-DAG1(α) therapeutic proteins are: HB-EGF (terminates with heparin-binding domain)-LAMA2 G1-G5 (encoded by polynucleotides in Figure 3), HB-EGF (fully soluble)-LAMA2 G1-G5 (encoded by polynucleotides in Figure 4), HB-EGF (terminating with a heparin-binding domain)-LAMA2 G3-G5 (encoded by the polynucleotides in Figure 5), and HB-EGF (fully soluble)-LAMA2 G3-G5 (encoded by the polynucleotides in Figure 6).

[0060] In this specification, the term “fully soluble” indicates that the therapeutic protein contains the HBEGF heparin-binding domain and the EGF-like domain, but does not contain the transmembrane portion of HBEGF. The combination of the HBEGF heparin-binding domain and the EGF-like domain of HBEGF corresponds to a soluble isoform of the cleaved activity of HBEGF. This term is referred to herein as “HBEGF.”

[0061] In this specification, the term "terminated with a heparin-binding domain" indicates that the therapeutic protein contains only the HBEGF heparin-binding domain and does not contain the EGF-like domain or transmembrane portion of HBEGF. This term is abbreviated as "HB" in this specification.

[0062] For example, in the case of laminin-deficient muscular dystrophy such as MDC1A, the coding sequence of the HBEGF heparin-binding domain is fused to the coding sequences of the globular (G) domains 1-5 of laminin-α2 to create a polynucleotide encoding the therapeutic protein HBEGF-LAMA2(G1-5). The laminin-α2G domain also binds to glycosylated α-dystroglycans in the sarcosium and to integrins, and is encoded by a portion of the LAMA2 gene. In the method described herein, the HBEGF domain of HBEGF-LAMA2(G1-5) binds to ECM heparin sulfate proteoglycans, and the G domain binds to α-dystroglycans, linking the sarcosium to the ECM even though full-length laminin-α2 is not present in MDC1A. Two examples of such HBEGF-LAMA2(G1-5) therapeutic proteins are: HB-EGF (terminates with heparin-binding domain)-DAG1 (naturally processed alpha-DG gene) (encoded by polynucleotides in Figure 7) and HB-EGF (fully soluble)-DAG1 (naturally processed alpha-DG gene) (encoded by polynucleotides in Figure 8).

[0063] Furthermore, both dystroglycanopathy and laminin-deficient muscular dystrophy (such as MDC1A) are associated with reduced muscle regeneration, and in embodiments of the method described herein, in which the therapeutic protein comprises an HBEGF heparin-binding domain and an HBEGF EGF-like domain, the patient also benefits from the nutritional signaling of the HBEGF EGF-like domain of the therapeutic protein, which alters the expression of genes including Pax7, MyoD, myogenin, and Myh3, resulting in increased myogenesis and muscle regeneration.

[0064] Therefore, polynucleotides encoding therapeutic proteins are provided. Embodiments include polynucleotides comprising the polynucleotide sequence shown in Figures 3, 4, 5, 6, 7, or 8. Other embodiments include polynucleotides encoding the same amino acid sequence as the polynucleotide sequence shown in Figures 3, 4, 5, 6, 7, or 8. Yet another embodiment includes polynucleotides comprising the polynucleotide sequence shown in Figures 3, 4, 5, 6, 7, or 8.

[0065] In one embodiment, the provided polynucleotide comprises one of the following: i) the nucleotide sequence shown in Figure 3, ii) the nucleotide sequence containing nucleotides 14-3235 shown in Figure 3, iii) the nucleotide sequence of SEQ ID NO: 1, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 19.

[0066] In another embodiment, the provided polynucleotide includes one of the following: i) the nucleotide sequence shown in Figure 4, ii) the nucleotide sequence containing nucleotides 14-3361 shown in Figure 4, iii) the nucleotide sequence of SEQ ID NO: 3, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 20.

[0067] In further embodiments, the provided polynucleotide includes one of the following: i) the nucleotide sequence shown in Figure 5, ii) the nucleotide sequence containing nucleotides 14-1930 shown in Figure 5, iii) the nucleotide sequence of SEQ ID NO: 5, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 21.

[0068] In another embodiment, the provided polynucleotide comprises one of the following: i) the nucleotide sequence shown in Figure 6, ii) the nucleotide sequence containing nucleotides 14-2056 shown in Figure 6, iii) the nucleotide sequence of SEQ ID NO: 7, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22.

[0069] In one embodiment, the provided polynucleotide includes one of the following: i) the nucleotide sequence shown in Figure 7, ii) the nucleotide sequence containing nucleotides 14-1360 shown in Figure 7, iii) the nucleotide sequence of SEQ ID NO: 9, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23.

[0070] In another embodiment, the provided polynucleotide includes one of the following: i) the nucleotide sequence shown in Figure 8, ii) the nucleotide sequence containing nucleotides 14-1486 shown in Figure 8, iii) the nucleotide sequence of SEQ ID NO: 11, or iv) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24.

[0071] Other polynucleotides provided include, but are not limited to, polynucleotides encoding amino acid variants of therapeutic polypeptides that retain therapeutic protein binding activity, and which polynucleotides have a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence encoding the protein shown in Figures 3, 4, 5, 6, 7, or 8, or the nucleotide sequence of any of the provided polynucleotides.

[0072] Polynucleotides encoding amino acid variants of therapeutic polypeptides that retain binding activity to therapeutic proteins are also provided herein, which hybridize under stringent conditions to nucleotide sequences encoding proteins shown in Figures 3, 4, 5, 6, 7, or 8, or their complements, or to any nucleotide sequence of the provided polynucleotides. The term “stringent” is used to refer to conditions that are generally understood as stringent in the art. Hybridization stringency is determined primarily by temperature, ionic strength, and the concentration of denaturants such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65–68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd See Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989).

[0073] "Maintaining binding activity" is intended to mean, in this specification, that amino acid variants of a polynucleotide-encoded therapeutic protein compete for binding to heparin sulfate proteoglycans and β-dystroglycans with therapeutic proteins encoded by the nucleotide sequences shown in Figures 3, 4, 5, or 6, or for binding to heparin sulfate proteoglycans and α-dystroglycans with therapeutic proteins encoded by the nucleotide sequences shown in Figures 7 or 8, or for binding to heparin sulfate proteoglycans and α-dystroglycans with therapeutic proteins containing the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0074] Recombinant expression vectors containing one or more of the polynucleotides described herein are also provided. Recombinant AAV genomes containing the polynucleotides described herein are also provided.

[0075] In the expression vectors or recombinant AAV genomes described herein, the polynucleotide encoding the therapeutic protein is operably ligated to transcription factors (including, but not limited to, promoters, enhancers, and / or introns), particularly to transcription factors that function in the target cell of interest. For example, suitable promoters for use in mammalian host cells are well known and include, but are not limited to, those derived from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (such as adenovirus 2), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40). Other suitable mammalian promoters include heteromammalian promoters, such as heat shock promoters and actin promoters.Furthermore, for example, AAV delivery methods include those derived from actin and myosin gene families, such as the myoD gene family [see Weintraub et al., Science, 251:761-766 (1991)], muscle cell-specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol Cell Biol, 11:4854-4862 (1991)], human skeletal actin gene [Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)], muscle creatine kinase sequence factor [see Johnson et al., Mol Cell Biol, 9:3393-3399 (1989)], and regulatory factors derived from mouse creatine kinase enhancer (mCK) factor, regulatory factors derived from skeletal fast contraction troponin C gene, slow contraction cardiac troponin C gene, and slow contraction troponin I gene: hypoxia-inducible nuclear factor [Semenza et al.] Transduced muscle or hepatocytes may utilize muscle-specific transcription regulators, including but not limited to the following: [Mader and White, Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)], steroid-inducible factors and promoters including glucocorticoid response factors (GRE) [Mader and White, Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993)], tMCK promoter [see Wang et al., Gene Therapy, 15:1489-1499 (2008)], hybrid α-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7) promoter [Salva et al., Mol Ther, 15:320-329 (2007)], CK6 promoter [Wang et al., see above], and other regulatory factors. Therefore, an example of a muscle-specific transcription regulator is the tMCK promoter. An example of a liver-specific promoter is LSP [Wang and Verma, Proc. Natl. Acad. Sci. USA, 96, 3906-3910 (1999)].Another example of a promoter is a constitutive promoter, such as the cytomegalovirus (CMV) promoter, which is used for the production of therapeutic proteins in recombinant host cells. Another example is LAMA2.

[0076] For the expression of the therapeutic proteins described herein, expression systems and constructs in the form of plasmids, expression vectors, transcriptions or expression cassettes comprising at least one polynucleotide described herein, as well as host cells comprising such expression systems or constructs, are provided. As used herein, “vector” means any molecule or entity (e.g., polynucleotide, plasmid, bacteriophage, or virus) suitable for use in introducing protein-coding information into a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. Expression vectors, such as recombinant expression vectors, are useful for transforming host cells.

[0077] Host cells into which an expression vector, such as a recombinant expression vector, has been introduced are provided. The host cells can be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., yeast, insect, or mammalian cell (e.g., CHO cell)). The expression vector can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. For stable transfection of mammalian cells, a gene encoding a selectable marker (e.g., for antibiotic resistance) is generally introduced into the host cell along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. Cells stably transfected with the introduced polynucleotide can be identified, among other methods, by drug selection. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides into liposomes, mixing of nucleic acids with positively charged lipids, and direct microinjection of DNA into the nucleus.

[0078] The method selected is partly a function of the type of host cell used. These methods and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2001.

[0079] When host cells are cultured under appropriate conditions, they synthesize proteins, which can then be collected from the culture medium (if the host cells secrete the protein into the medium) or directly from the host cells producing it (if not lysed). The selection of appropriate host cells depends on various factors, including the desired expression level, polypeptide modifications desirable or required for activity (such as glycosylation or phosphorylation), and the ease of folding into biologically active molecules. As an example, Chinese hamster ovary cells overexpressing LARGE (CHO-LARGE cells) [Yoon et al., A Method to Produce The paper "and Purify Full-Length Recombinant Alpha Dystroglycan: Analysis of N- and O-Linked Monosaccharide Composition in CHO Cells with or without LARGE Overexpression, PLoS Curr. (2013 January 2)" is intended for use in the production of the glycosylated therapeutic proteins described herein.

[0080] Mammalian cell lines available as hosts for expression include, but are not limited to, those well known in the art, Chinese hamster ovary (CHO) cells, CHO-LARGE cells, HEK293 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and other standard cell lines in the art, as well as immortalized cell lines available from the American Type Culture Collection (ATCC).

[0081] The rAAV genomes provided herein lack AAV rep and cap DNA. The recombinant AAV genomes provided herein include polynucleotides encoding the therapeutic proteins described above, and one or more AAV ITRs flanking the polynucleotides. The AAV DNA within the rAAV genome may be from any AAV serotype from which the recombinant virus may originate, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. Other types of rAAV variants, such as rAAV with capsid mutations, are also intended. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background information section above, the nucleotide sequences of various AAV serotype genomes are known in the art. AAV1, AAV5, AAV6, AAV8, or AAV9 can be used to promote skeletal muscle-specific expression.

[0082] A DNA plasmid containing the rAAV genome is provided. The DNA plasmid is introduced into a cell tolerant of infection with an AAV helper virus (including, but not limited to, adenovirus, E1 deletion adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the AAV genome, rep and cap genes, and helper virus function to be packaged are provided to the cell, are standard in the art. The production of rAAV requires that the following components, the rAAV genome, the AAV rep and cap genes isolated from (i.e., not present in) the rAAV genome, and the helper virus function, be present in a single cell (referred to herein as the packaging cell). The AAV ITR and rep and cap genes may be from any AAV serotype from which the recombinant virus may originate, and may be from a different AAV serotype than the rAAV genome ITR, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. The generation of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety.

[0083] The method for generating packaging cells involves creating a cell line that stably expresses all the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes isolated from the rAAV genome, and selectable markers such as the neomycin resistance gene, is incorporated into the cell genome. The AAV genome is introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method is to use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0084] The general principles of rAAV production are reviewed, for example, in Carter, Current Opinions in Biotechnology, 1533-1539 (1992); and Muzyczka, Curr. Topics in Microbial. And Immunol., 158:97-129 (1992). Various approaches include Ratschin et al., Mol. Cell. Biol., 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Tratschin et al., Mo1. Cell. al., J.Virol.,62:1963(1988), ebkowski et al.,Mol.Cell.Biol.,7:349(1988), Samulski et al. al., J. Virol., 63:3822-3828 (1989); U.S. Patent No. 5,173,414, WO95 / 13365 and corresponding U.S. Patent No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO 99 / 11764; Perrin et al., Vaccine, 13:1244-1250 (1995); Paul et al. This information is found in al., Human Gene Therapy, 4:609-615 (1993), Clark et al., Gene Therapy 3:1124-1132 (1996), U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595, and McCarty, Mol. Ther., 16(10):1648-1656 (2008). The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on the portions relating to rAAV production.

[0085] Therefore, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 strain). In another embodiment, the packaging cells may be non-transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus macaque fetal lung cells).

[0086] In this specification, recombinant AAV, which is a replication-deficient, infectious, capsid-forming viral particle (rAAV), comprises an rAAV genome. rAAV encodes the therapeutic proteins described herein. The rAAV genome lacks AAV rep and cap DNA; that is, there is no AAV rep or cap DNA between the ITRs of the rAAV genome.

[0087] rAAV can be purified by methods standard in the art, for example, by column chromatography or a cesium chloride gradient. Methods for purifying rAAV vectors from helper viruses are known in the art and include, for example, the methods disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69:427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0088] In another embodiment, the present invention envisions a composition comprising rAAV or therapeutic protein as described herein. The composition of the present invention comprises rAAV or therapeutic protein on a pharmaceutically acceptable carrier. The composition may also contain other components such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are nontoxic to the recipient and preferably inactive at the dosage and concentration used, and include buffering agents such as phosphoric acid, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, Pluronic®, or polyethylene glycol (PEG).

[0089] The titer of rAAV administered by the method described herein will vary depending, for example, on the specific rAAV, mode of administration, therapeutic target, individual, and targeted cell type(s), and may be determined by standard methods in the art. The titer of rAAV is approximately 1 × 10⁶ per mL. 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 The above range may apply to DNase-resistant particles (DRPs). The dosage may be expressed in units of viral genome (vg) as understood in the art.

[0090] The present invention envisions a method for transducing rAAV into target cells in vivo or in vitro. The in vivo method comprises the step of administering an effective dose or multiple effective doses of a composition containing rAAV as described herein to an animal (including a human patient) in need thereof.

[0091] The dosage and frequency of administration of the therapeutic proteins described herein may vary depending on factors such as the route of administration, the specific therapeutic protein being administered, and the patient's size and general condition. Appropriate dosages can be determined by procedures known in the relevant art, for example, in clinical trials that may involve dose-escalation studies. Considering these factors, typical doses of the therapeutic proteins described herein may range from approximately 0.1 pg / kg to a maximum of approximately 30 mg / kg or more. Furthermore, doses may range from 0.1 pg / kg to a maximum of approximately 30 mg / kg, 1 pg / kg to a maximum of approximately 30 mg / kg, 10 pg / kg to a maximum of approximately 10 mg / kg, approximately 0.1 mg / kg to 5 mg / kg, or approximately 0.3 mg / kg to 3 mg / kg.

[0092] Therefore, a method for treating a patient with the therapeutic protein described herein is also provided. This method comprises the step of administering a composition comprising an effective dose or multiple effective doses of the therapeutic protein described herein to an animal (including a human patient) in need thereof.

[0093] If the dose is administered before the onset of the disorder / disease, the administration is prophylactic. If the dose is administered after the onset of the disorder / disease, the administration is therapeutic. "Effective dose" is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease condition being treated, a dose that slows or prevents progression to the disorder / disease condition, a dose that slows or prevents progression to the disorder / disease condition, a dose that reduces the severity of the disease, a dose that results in remission (partial or complete) of the disease, and / or a dose that prolongs survival. The methods described herein result in one or more of the following in treated patients: improved walking time, improved limb grip strength, reduced muscle pathology, and reduced neuropathology. Other endpoints achieved by the methods described herein are one or more of the following in treated patients: increased muscle fiber size, reduced number of small oxidized fibers, correction of muscle atrophy, increased muscle strength, and increased muscle regeneration. Dystroglycanopathy and laminin-deficient muscular dystrophy are intended for prevention or treatment by the methods of the present invention.

[0094] Combination therapies are also intended by this invention. Combination therapies as used herein include both concurrent and sequential treatments. Because they involve combinations with novel therapies, combinations of the methods described herein with standard drug therapies are particularly intended.

[0095] Administration of an effective dose of rAAV or a composition of therapeutic proteins may be via standard routes in the art, including but not limited to intramuscular, parenteral, intravenous, intrathecal, oral, oral cavity, nasal cavity, lung, intracranial, intraosseous, intraocular, rectal, or vaginal. The administration route(s) and serotype(s) of the AAV components of the rAAV of the present invention (specifically, AAV ITR and capsid protein) may be selected and / or adapted by those skilled in the art, taking into account the infectious disease and / or disease state being treated, as well as the target cells / tissues expressing the therapeutic proteins.

[0096] In particular, the actual administration of rAAV according to the present invention can be achieved by using any physical method for transporting the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, intramuscular injection, bloodstream injection, and / or direct injection into the liver. Simply resuspending rAAV in phosphate-buffered saline or Ringer's lactate solution has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on carriers or other components that may be co-administered with rAAV (however, DNA-degrading compositions should be avoided in the usual manner with rAAV). The capsid protein of rAAV may be modified so that rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or as a topical formulation delivered to muscle by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been developed to date and can be used in the implementation of the present invention. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0097] For intramuscular injection purposes, solutions of rAAV or therapeutic proteins can be used in adjuvants such as sesame oil or peanut oil, or in aqueous propylene glycol, and in sterile aqueous solutions. Such aqueous solutions can be buffered as needed, and the liquid diluent is first isotonicized with physiological saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacokinetically acceptable salt can be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used are readily available by standard techniques well known to those skilled in the art.

[0098] Suitable pharmaceutical forms of rAAV or therapeutic proteins for systemic (e.g., intravenous) injection include sterile aqueous solutions or dispersants, and sterile powders for the immediate preparation of sterile injection solutions or dispersants. In all cases, the form must be sterile and fluid enough to allow for easy injection. It must be stable under manufacturing and storage conditions and protected against microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants. Prevention of microbial action can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it would be preferable to include isotonic agents, such as sugars or sodium chloride. Long-term absorption of injectable compositions can be achieved by using absorption retarders, such as aluminum monostearate and gelatin.

[0099] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, along with various other components as needed, as listed above, and then sterilizing by filtration. Generally, dispersants are prepared by mixing the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired components from its previously sterilized filtered solution.

[0100] Transduction with rAAV can also be performed in vitro. In one embodiment, desired target muscle cells are isolated from the target, transduced with rAAV, and reintroduced into the target. Alternatively, syngeneic or heterologous muscle cells may be used if those cells do not produce an inappropriate immune response in the target.

[0101] Suitable methods for transduction into a target and reintroduction of transduced cells are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in a suitable medium and screening cells with the desired DNA using conventional techniques such as Southern blotting and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, which can be introduced into a target by various techniques, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth muscle and cardiac muscle using a catheter, for example.

[0102] Transduction of cells with rAAV according to the present invention results in sustained expression of the therapeutic protein described herein. Accordingly, the present invention provides methods for administering rAAV expressing the therapeutic protein described herein to a patient, preferably a human. These methods include transduction of one or more rAAVs of the present invention into tissue (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands).

[0103] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. This invention aims to achieve sustained expression of the therapeutic proteins described herein from transduced muscle cells.

[0104] "Muscle cells" or "muscle tissue" means cells or groups of cells derived from any type of muscle [e.g., skeletal muscle and smooth muscle (e.g., gastrointestinal tract, bladder, blood vessels, or cardiac tissue)]. Such muscle cells may be differentiated or undifferentiated, including myoblasts, myocytes, myotubes, cardiomyocytes, and cardiac muscle cells.

[0105] The term "transduction" is used to refer to the administration / delivery of a therapeutic protein to recipient cells, either in vivo or in vitro, via the rAAV of the present invention, resulting in the expression of the therapeutic protein by the recipient cells.

[0106] Accordingly, a method is provided herein for administering an effective dose (or a dose essentially administered simultaneously or at intervals) of rAAV encoding the therapeutic protein described herein to a patient in need thereof.

[0107] Methods for administering an effective dose (or a dose essentially administered simultaneously or at intervals) of the therapeutic protein described herein to a patient in need thereof are also provided herein. [Examples]

[0108] Aspects and embodiments of the present invention are illustrated by the following examples. Example 1 describes a construct encoding a therapeutic protein of the present disclosure. Example 2 describes recombinant expression of the therapeutic protein in cultured host cells. Example 3 describes an experiment demonstrating that the heparin-binding domain targets LAMA2(g1-G50) in the muscle of wild-type mice. Example 4 describes the dy W / dy W This document describes experiments to demonstrate the efficacy of AAV-mediated HBEGF-LAMA2(G1-5) expression in reducing symptoms and pathology in a mouse model. Example 5 describes experiments to demonstrate the efficacy of AAV-mediated HBEGF-DAG1(α) expression in reducing symptoms and pathology in a mouse model of dystroglycanopathy. Example 6 describes the properties of sHBEGF (domains) that are intended herein to be useful as linker domains in therapeutic proteins described herein and as nutrients in the various methods described herein.

[0109] Example 1 Constructs encoding therapeutic proteins Six exemplary DNA constructs encoding therapeutic proteins containing HBEGF and EGF domains were generated as follows: HB-EGF (terminates with heparin-binding domain)-LAMA2 G1-G5 (encoded by polynucleotides in Figure 3), HB-EGF (fully soluble)-LAMA2 G1-G5 (encoded by polynucleotides in Figure 4), HB-EGF (terminates with heparin-binding domain)-LAMA2 G3-G5 (encoded by the polynucleotide in Figure 5), HB-EGF (fully soluble)-LAMA2 G3-G5 (encoded by polynucleotides in Figure 6), HB-EGF (terminated with heparin-binding domain)-DAG1 (naturally processed alpha-DG gene) (encoded by polynucleotides in Figure 7), and HB-EGF (fully soluble)-DAG1 (naturally processed alpha-DG gene) (encoded by polynucleotides in Figure 8).

[0110] The constructs were expressed from plasmids in CHO cells. CHO cells were either transfected with a plasmid containing one of the constructs or pseudotransfected (-).

[0111] Transfected CHO cells were stained with antibodies against HB-EGF, dystroglycan, or laminin-α2 G5 domain. The results are shown in Figure 9A. Additionally, 48 hours after transfection, culture medium was collected from each plate and cell lysis was performed. Heparin-agarose pulldown was performed on both the cell lysates and culture medium, and these were loaded into Western blots along with the total cell lysates. The results are shown in Figure 9B.

[0112] Example 2 Recombinant expression of therapeutic proteins in cultured host cells The construct of Example 1 was subcloned into a plasmid to generate an AAV9 vector encoding a therapeutic protein.

[0113] An AAV vector carrying one of the therapeutic genes of Example 1 under the transcriptional control of the cytomegalovirus (CMV) promoter was generated.

[0114] The rAAV vector was generated by a modified cross-packaging approach that can package an AAV2 vector genome into multiple AAV capsid serotypes [Rabinowitz et al., J Virol. 76(2):791 - 801(2002)]. Generation was achieved using a standard three-plasmid DNA / CaPO4 precipitation method using HEK293 cells. HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin. The generation plasmids were (i) a plasmid encoding the therapeutic protein, (ii) a rep2-capX modified AAV helper plasmid encoding the cap serotype 9 isolate, and (iii) an adenovirus type 5 helper plasmid (pAdhelper) expressing the adenovirus E2A, E4 ORF6, and VA I / II RNA genes. A quantitative PCR-based titration method was used to determine the capsid-formed vector genome (vg) titer using a Prism 7500 Taqman detector system (PE Applied Biosystems). [Clark et al., Hum Gene Ther. 10(6):1031 - 1039(1999)]. The final titer (vg / ml -1 ) was determined by quantitative reverse transcriptase PCR using specific primers and probes using a Prism 7500 real-time detector system (PE Applied Biosystems, Grand Island, NY, USA). The aliquoted virus was stored at -80 °C until use.

[0115] The rAAV shown in Table 2 was used in the experiments described herein. [Table 2]

[0116] Example 3 The heparin-binding domain targets LAMA2 (G1-G5) in the muscles of wild-type mice. Wild-type mice contain 5x10 oz. containing HBEGF, HBEGF.LAMA2(G1-G5), HBEGF.LAMA2(G3-G5), HB.LAMA2(G1-G5), HB.LAMA2(G3-G5), or LAMA2(G1-G5) (see Table 2). 11 vg The pf rAAV9.CMV vector was intramuscularly injected into the gastrocnemius muscle. Cells were stained with antibodies specific to the recombinant G5 domain of human HB-EGF or LAMA2. Sham-injected mice (buffer only) are shown as negative controls. 4H8-2 is an anti-laminin antibody representing muscle cells in the section.

[0117] As previously shown in Figure 15, overexpression of HBEGF reduced muscle growth and / or induced mild muscle atrophy. However, IM injection of rAAV9.CMV.HB.LAMA2(G1-G5) led to the secretion and localization of LAMA2(G1-G5) protein in the extracellular matrix. In addition, muscles expressing HB.LAMA2(G1-G5) appeared to be larger than normal wild-type muscles. HB.LAMA2(G3-G5) showed lower overall protein staining than HB.LAMA2(G1-G5). The ECM targeting function of the HB domain of HBEGF enables the secretion and targeting of LAMA2(G1-G5) protein into the muscle extracellular matrix. In contrast, expression of LAMA2(G1-G5) without the HB domain resulted in very low protein production and no ECM localization was detected. Therefore, a construct containing only the HB domain, rather than the complete HB-EGF domain, achieves its objective of targeting LAMA2 (G1-G5) to the muscle ECM, and because this construct has the EGF domain removed from HBEGF, it successfully achieves this objective without adverse effects on EGF signaling.

[0118] The HBEGF prepropeptide portion still present in the HB construct may also improve LAMA2(G1-G5) protein folding and / or expression compared to LAMA2(G1-G5) containing only the signal peptide from HBEGF. Finally, HB.LAMA2(G1-G5), when properly localized, may improve muscle growth even in normal muscle.

[0119] Figure 16 shows the staining of HBEGF.LAMA2(G1-G5), HB.LAMA2(G1-G5), and LAMA2(G1-G5) using antibodies against human HBEGF protein and the G5 domain of human LAMA2. This data confirms that expression of LAMA2(G1-G5) alone significantly reduces protein synthesis in muscle, while the inclusion of the HB domain improves LAMA2(G1-G5) expression, which was visualized with anti-laminin and HBEGF antibodies.

[0120] Example 4 MDC1A's dy W / dy W Efficacy of AAV9-mediated HBEGF-LAMA2(G1-5) expression in reducing symptoms and pathology in mouse models dy W / dy W The mouse [Nonaka, Lab Anim Sci., 48(1):8-17(1998)] has a loss-of-function mutation in Lama2, causing impaired laminin-α2 production, similar to the pathogenesis of MDC1A. W / dy W These mice exhibit reduced size, grip strength, and lifespan compared to wild-type mice. By 3 months of age, they show muscle atrophy, dystrophy, and severe gait disturbances. Therefore, these mice are a suitable and robust model for testing MDC1A therapy.

[0121] To demonstrate the therapeutic effect of the LAMA2-expressing rAAV genome provided in Example 2, eight dy W / dy W For the offspring, a low dose of 10 11 Viral genome (vg) or high dose 10 12 Either vg, rAAV9.CMV.HBEGF.LAMA2(G1-G5), rAAV9.CMV.HB.LAMA2(G1-G5), or rAAV.CMV.HB.LAMA2(G3-G5) was administered intravenously via the facial vein. Control dy W / dy W We also administered a placebo injection of AAV buffer to the offspring.

[0122] Grip strength of the forelimb muscles was analyzed at 2 and 3 months post-injection (Figure 17). At 4 months of age, the mice were euthanized, and the limb muscles were dissected and recombinant protein expression was analyzed. As shown in Figure 17, both HB.LAMA2(G3-G5) and HB.LAMA2(G1-G5) prevented the decline in grip strength of dy / dy mice, showing a significant change from sham-treated dy / dy mice and bringing the strength values ​​within the range seen in untreated wild-type mice of the same age. Therefore, both HB-LAMA2(G3-G5) and HB-LAMA2(G1-G5) demonstrate therapeutic effects in the dy / dy model of MDC1A. In this experiment, HB.LAMA2(G3-G5) did not reach a significant improvement at 2 months compared to sham-treated dy / dy mice, while HB.LAMA2(G1-G5) did.

[0123] The role of transgene expression in preventing muscle pathology was also analyzed by comparing the percentage of muscle fibers with central nuclei, muscle fiber diameter and area, and variability in muscle fiber diameter in treated dy / dy mice. Using triceps muscle, muscle pathology in muscle fibers expressing the transgene was compared with the same pathological measurements in non-expressing muscle fibers. This experiment demonstrated that HB.LAMA2(G1-G5) expression increases muscle size. An example of staining showing such changes is shown in Figure 18. When quantified across all muscles, the mean transverse muscle area was 2328 mm for expressing muscle fibers. 2 1082 mm relative to non-expressing muscle fibers 2 (n=4 muscles, 400 muscle fibers analyzed per muscle) This represented a mean twofold increase in muscle size with treatment. The variance of the muscle fiber diameter index (diameter SD / mean × 1000) decreased from 620 in non-expressing muscles to 431 in expressing muscles (values ​​below 250 are considered normal), and the percentage of muscle fibers with a central nucleus, an indicator of the muscle degeneration and regeneration cycle, decreased from 28% in non-expressing muscle fibers to 14% in expressing muscle fibers (n=2 each).

[0124] While pathology is never reduced to zero, it is important to remember that some degree of pathology will occur in these animals before the expression of the therapeutic transgene occurs, as it takes 3–4 weeks for AAV to achieve maximum gene expression. In all such experiments, the mean level of muscle transformation was 26 ± 1% (analyzing n=4 triceps muscles, 400 fibers each). What we have gathered from these pathological measurements is that HB-LAMA2(G1–G5) appears to not only prevent dy / dy muscle damage but also revert to wild type and possibly increase muscle growth beyond that.

[0125] Example 5 Efficacy of AAV-mediated HBEGF-DAG1(α) expression in reducing symptoms and pathology in a mouse model of dystroglycanopathy Mice lacking fukutin, an α-dystroglycan-glycosylating enzyme encoded by the dystroglycan, or Fktn gene, are embryonically lethal and cannot be used in research on dystroglycanopathy therapy. Myf5Cre-Fktn is a variant where Fktn deletion is limited to skeletal muscle. loxP The efficacy will be demonstrated using mice [Kanagawa et al., Hum Mol Genet., 22(15):3003-3015 (2013)]. Myf5Cre-Fktn loxP Compared to wild-type mice, these mice exhibit reduced body weight, grip strength, and lifespan. They also show dystrophy of the muscles.

[0126] Myf5Cre-Fktn loxP To demonstrate the therapeutic effect of rAAV9.CMV.HBEGF-DAG1(α) in a mouse model, the same injection protocol and evaluation described in Example 3 were performed.

[0127] Large-vls mouse mutant (Lee et al., Mol. Cell. Neurosci. 30:160-172, 2005), another mouse model of dystroglycanopathy. Several Large-vls mice were subjected to 1 × 10¹⁶ sphincter-1412 The mice were injected with rAAV9.CMV.HB-DAG1IV via IM. Grip strength tests in these mice suggested potential improvement. Of the 4-7 animals analyzed per group at 2 months, forelimb grip strength decreased from 4.7±0.2 g / g in wild-type mice to 3.8±0.1 g / g in untreated Large vls mice (p=0.0005), while pAAV9.CMV.HB-DAG1(α)-treated Large vls mice showed a decrease of 1×10⁶ at P1. 12 vg (vs) to IV showed an improvement in grip strength to 4.4 ± 0.3 g / g (p=0.06 compared to Large vls in the sham treatment group). This data is very close to significance, and a statistically significant difference may be achieved with additional measurements.

[0128] Example 6 sHBEGF as a linker protein and nutrients The use of the heparin-binding domain and the EGF-like domain of soluble HBEGF (sHBEGF) in the various exemplary therapeutic proteins described herein offers a dual benefit to patients. Including both domains enhances muscle membrane stability when the heparin-binding domain is included with LAMA2(G1-5) or DAG1(α), and further stimulates muscle regeneration when the EGF-like domain is included. Figures 10, 11, and 12 show that sHBEGF activates the Akt kinase pathway in muscle, increasing the expression of muscle regeneration markers. Activated Akt kinase expression in muscle has been previously shown to stimulate significant muscle growth, similar to that observed with myostatin inhibitors. Therefore, the presence of the EGF-like domain of HBEGF in the various therapeutic proteins described herein adds further therapeutic effects to the treatment of the diseases described herein.

[0129] In the left calf muscle of a 5-week-old male C57BL / 6J mouse, near the midpoint of the muscle, 5 × 10 units of insulin were injected using a 0.3 mL insulin syringe with 50 μL of sterile PBS. 10The vector genome r(ds)AAV9.CMV.HB-EGF or r(ds)AAV9.CMV.sHB-EGF was injected. The same amount of sterile PBS was sham-injected into the contralateral (right) muscle of the mice. Four or twelve weeks after injection, the mice were sacrificed and dissected. The gastrocnemius muscle was embedded in OCT compound (Fisher Scientific, Pittsburgh, PA) and rapidly frozen with isopentane cooled with liquid nitrogen.

[0130] sHB-EGF expression was visualized using an antibody that recognizes sHB-EGF and co-stained with either an antibody against Galgt2 protein or CT glycan. sHB-EGF was expressed along the sascia membrane of skeletal muscle fibers analyzed 4 weeks after injection of r(ds)AAV9.CMV.sHB-EGF. Figure 9 shows that sHB-EGF can be secreted from muscle and adhere to the extracellular matrix, supporting its use as a linker protein.

[0131] Figure 10 shows that sHB-EGF induces the expression of therapeutic surrogate muscular dystrophy genes.

[0132] Figure 11 shows that sHB-EGF can induce the Akt tyrosine kinase cascade in skeletal muscle, stimulating muscle growth and regeneration.

[0133] Although the present invention has been described in relation to specific embodiments, those skilled in the art will understand that variations and modifications will occur. Therefore, only such limitations as those found in the claims should be imposed on the present invention.

[0134] All documents referenced in this application are incorporated herein by reference in their entirety. The present invention provides, for example, the following items. (Item 1) a) A first domain containing the heparin-binding domain of heparin-binding epidermal growth factor-like growth factor (HBEGF), and a second domain containing the G1-G5 domains of the human laminin alpha-2 (LAMA2) gene, b) A first domain comprising the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain comprising the G1-G5 domain of the human LAMA2 gene, c) A first domain of HBEGF comprising the heparin-binding domain, and a second domain comprising the G3-G5 domain of the human LAMA2 gene, or d) A polynucleotide encoding a protein comprising a first domain including the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain including the G3-G5 domain of the human LAMA2 gene. (Item 2) The polynucleotide according to item 1, wherein the first domain of the protein is encoded by the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 14. (Item 3) The polynucleotide according to item 1 or 2, wherein the second domain of the protein is encoded by the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 16. (Item 4) The polynucleotide according to any one of items 1 to 3, wherein the polynucleotide comprises the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 15. (Item 5) The polynucleotide according to any one of items 1 to 3, wherein the polynucleotide comprises the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 16. (Item 6) The polynucleotide according to any one of items 1 to 3, wherein the polynucleotide comprises the nucleotide sequences of SEQ ID NO: 14 and SEQ ID NO: 15. (Item 7) The polynucleotide according to any one of items 1 to 3, wherein the polynucleotide comprises the nucleotide sequences of SEQ ID NO: 14 and SEQ ID NO: 16. (Item 8) The polynucleotide described in item 1(a), comprising i) nucleotides 14-3235 shown in Figure 3, ii) the nucleotide sequence of SEQ ID NO: 1, or iii) the amino acid sequence of SEQ ID NO: 19. (Item 9) The polynucleotide described in item 1(b), comprising i) nucleotides 14-3361 shown in Figure 4, ii) the nucleotide sequence of SEQ ID NO: 3, or iii) the amino acid sequence of SEQ ID NO: 20. (Item 10) The polynucleotide described in item 1(c), comprising i) nucleotides 14-1930 shown in Figure 5, ii) the nucleotide sequence of SEQ ID NO: 5, or iii) the amino acid sequence of SEQ ID NO: 21. (Item 11) i) a polynucleotide as described in item 1(d), comprising nucleotides 14-2056 shown in Figure 6, the nucleotide sequence of SEQ ID NO: 7, or iii) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22. (Item 12) Recombinant adeno-associated virus (rAAV), wherein the genome of the rAAV contains a polynucleotide described in any one of items 1 to 11. (Item 13) Recombinant adeno-associated virus (rAAV) wherein the genome of the rAAV contains the polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. (Item 14) The rAAV described above, wherein the rAAV genome further comprises muscle-specific transcriptional regulators, as described in item 12 or 13. (Item 15) The rAAV described in item 14, wherein the muscle-specific transcription regulator is the CMV promoter. (Item 16) Recombinant adeno-associated virus (rAAV) wherein the genome of the rAAV contains nucleotides 3590-8215 of SEQ ID NO: 2, nucleotides 3590-8341 of SEQ ID NO: 4, nucleotides 3609-6929 of SEQ ID NO: 6, nucleotides 3590-7036 of SEQ ID NO: 8, or the nucleotide sequence shown in Figure 13. (Item 17) rAAVs as described in any one of items 12-16, including AAV9, AAV10, AAVrh74, AAV8, or AAV6 capsids. (Item 18) rAAV particles containing any one of the rAAVs described in items 12-17. (Item 19) Recombinant host cells containing any one of the polynucleotides described in items 1 through 11. (Item 20) Host cells as described in item 19, which are Chinese hamster ovary (CHO) cells or HEK293 cells. (Item 21) A protein encoded by a polynucleotide as described in any one of items 1 through 11. (Item 22) A protein containing the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22. (Item 23) A composition comprising a polynucleotide as described in any one of items 1 to 11, an rAAV as described in any one of items 12 to 17, an rAAV particle as described in item 18, or a protein as described in item 21 or 22. (Item 24) A method for treating laminin-deficient muscular dystrophy, comprising: a patient requiring the treatment, one polynucleotide from item 1 to 11, and one polynucleotide from item 12 to 17. A method comprising administering rAAV as described in item 18, rAAV particles as described in item 21 or 22, or a composition as described in item 23. (Item 25) A composition for use in providing treatment to a patient requiring treatment for laminin-deficient muscular dystrophy, wherein the composition comprises a polynucleotide as described in any one of items 1 to 11, an rAAV as described in any one of items 12 to 17, an rAAV particle as described in item 18, a protein as described in item 21 or 22, or a composition as described in item 23. (Item 26) Use of any polynucleotide described in any one of items 1 to 11, any rAAV described in any one of items 12 to 17, rAAV particles described in item 18, proteins described in item 21 or 22, or compositions described in item 23 for the preparation of drugs for the treatment of laminin-deficient muscular dystrophy. (Item 27) The method, composition, or use described in any one of items 24-26, wherein the laminin-deficient muscular dystrophy is MDC1A. (Item 28) a) A first domain containing the heparin-binding domain of HBEGF, and a second domain containing the processed native alpha chain (DAG1 alpha) of the human dystroglycan gene, or b) A polynucleotide encoding a protein comprising a first domain containing the heparin-binding domain of HBEGF and the EGF-like domain of HBEGF, and a second domain containing DAG1 alpha. (Item 29) The polynucleotide described in item 28, wherein the first domain of the protein is encoded by the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 14. (Item 30) The polynucleotide described in item 28 or 29, wherein the second domain of the protein is encoded by the nucleotide sequence of SEQ ID NO: 17. (Item 31) The polynucleotide according to any one of items 28 to 30, wherein the polypeptide comprises the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 17. (Item 32) The polynucleotide according to any one of items 28 to 30, wherein the polynucleotide comprises the nucleotide sequences of SEQ ID NO: 14 and SEQ ID NO: 17. (Item 33) The polynucleotide described in item 28(a), comprising i) nucleotides 14-1360 shown in Figure 7, ii) the nucleotide sequence of SEQ ID NO: 9, or iii) the amino acid sequence of SEQ ID NO: 23. (Item 34) The polynucleotide described in item 28(b), comprising i) nucleotides 14-1486 shown in Figure 8, ii) the nucleotide sequence of SEQ ID NO: 11, or iii) the amino acid sequence of SEQ ID NO: 24. (Item 35) Recombinant adeno-associated virus (rAAV) wherein the genome of the rAAV contains a polynucleotide described in any one of items 28 to 34. (Item 36) Recombinant adeno-associated virus (rAAV), wherein the genome of the rAAV contains the polynucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 11. S (rAAV). (Item 37) The rAAV according to item 35 or 36, wherein the genome of the rAAV further comprises muscle-specific transcriptional regulators. (Item 38) The rAAV described in item 37, wherein the muscle-specific transcription regulator is the CMV promoter. (Item 39) Recombinant adeno-associated virus (rAAV) wherein the genome of the rAAV contains nucleotides 3590-6340 of SEQ ID NO: 10, nucleotides 3590-6049 of SEQ ID NO: 12, or the nucleotide sequence shown in Figure 14. (Item 40) rAAVs as described in any one of items 35-39, including AAV9, AAV10, AAVrh74, AAV8, or AAV6 capsids. (Item 41) rAAV particles containing rAAV as described in any one of items 35-40. (Item 42) Recombinant host cells containing any one of the polynucleotides described in items 28-34. (Item 43) Host cells as described in item 42, which are Chinese hamster ovary (CHO) cells or HEK293 cells. (Item 44) A protein encoded by a polynucleotide as described in any one of items 28-34. (Item 45) A protein containing the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 25. (Item 46) A composition comprising a polynucleotide as described in any one of items 28-34, an rAAV as described in any one of items 35-40, an rAAV particle as described in item 41, or a protein as described in item 44 or 45. (Item 47) A method for treating dystroglycanopathy, comprising administering to a patient in need of the treatment a polynucleotide described in any one of items 28-34, an rAAV described in any one of items 35-40, an rAAV particle described in item 41, a protein described in item 44 or 45, or a composition described in item 46. (Item 48) A composition for use in providing treatment to a patient requiring treatment for dystroglycanopathy, wherein the composition comprises a polynucleotide as described in any one of items 28 to 34, an rAAV as described in any one of items 35 to 40, an rAAV particle as described in item 41, a protein as described in item 44 or 45, or a composition as described in item 46. (Item 49) Use of any polynucleotide described in any one of items 28-34, rAAV described in any one of items 35-40, rAAV particles described in item 41, proteins described in item 44 or 45, or compositions described in item 46 for the preparation of drugs for the treatment of dystroglycanopathy. (Item 50) The aforementioned dystroglycanopathy is known as Walker-Warburg syndrome, myophthalmoencephalopathy, or Fukuyama type. The method, composition, or use described in any one of items 47-49, relating to congenital muscular dystrophy, MDC1C, MDC1D, LGMD2I, LGMD2K, LGMD2M, LGMD2N, LGMD2O, LGMD2P, LGMD2T, or LGMD2U.

Claims

1. A polynucleotide that codes for a protein, i) The polynucleotide includes the nucleotide sequence of SEQ ID NO: 13 in the 5' to 3' direction, followed immediately by the nucleotide sequence of SEQ ID NO: 17, or ii) The polynucleotide includes the nucleotide sequence of SEQ ID NO: 14 in the direction from 5' to 3', followed immediately by the nucleotide sequence of SEQ ID NO:

17. Polynucleotide.

2. i) a nucleotide sequence encoding the nucleotide sequence of SEQ ID NO: 9, or ii) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23, according to claim 1.

3. The polynucleotide according to claim 1, comprising i) a nucleotide sequence encoding the nucleotide sequence of SEQ ID NO: 11, or ii) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

24.

4. Recombinant adeno-associated virus (rAAV), wherein the genome of the rAAV comprises the polynucleotide described in any one of claims 1 to 3.

5. Recombinant adeno-associated virus (rAAV), wherein the genome of the rAAV contains the polynucleotide sequence of SEQ ID NO: 9 or SEQ ID NO:

11.

6. The rAAV according to claim 4 or 5, wherein the genome of the rAAV further comprises a muscle-specific transcription factor.

7. The rAAV according to claim 6, wherein the muscle-specific transcription factor is a tMCK promoter.

8. The rAAV according to any one of claims 4 to 7, wherein the genome of the rAAV further comprises a CMV promoter.

9. Recombinant adeno-associated virus (rAAV), wherein the genome of the rAAV contains nucleotides 3590-6340 of SEQ ID NO: 10 or nucleotides 3590-6049 of SEQ ID NO:

12.

10. rAAV according to any one of claims 4 to 9, comprising AAV9, AAV10, AAVrh74, AAV8, or AAV6 capsid.

11. rAAV particles comprising rAAV according to any one of claims 4 to 10.

12. Recombinant host cells comprising the polynucleotide described in any one of claims 1 to 3.

13. The host cell according to claim 12, which is a Chinese hamster ovary (CHO) cell or a HEK293 cell.

14. A protein encoded by a polynucleotide according to any one of claims 1 to 3.

15. A protein containing the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO:

24.

16. A composition comprising a polynucleotide according to any one of claims 1 to 3, an rAAV according to any one of claims 4 to 10, an rAAV particle according to claim 11, or a protein according to claim 14 or 15.

17. A composition for use in administering dystroglycanopathy to a patient requiring treatment, wherein the composition comprises a polynucleotide according to any one of claims 1 to 3, an rAAV according to any one of claims 4 to 10, an rAAV particle according to claim 11, a protein according to claim 14 or 15, or the composition according to claim 16.

18. Use of a polynucleotide according to any one of claims 1 to 3, an rAAV according to any one of claims 4 to 10, an rAAV particle according to claim 11, a protein according to claim 14 or 15, or a composition according to claim 16 for the preparation of a pharmacopoeia for the treatment of dystroglycanopathy.

19. The composition according to claim 17, wherein the dystroglycanopathy is Walker-Warburg syndrome, myophthalmoencephalopathy, Fukuyama congenital muscular dystrophy, MDC1C, MDC1D, LGMD2I, LGMD2K, LGMD2M, LGMD2N, LGMD2O, LGMD2P, LGMD2T, or LGMD2U.

20. The use according to claim 18, wherein the dystroglycanopathy is Walker-Warburg syndrome, myophthalmoencephalopathy, Fukuyama congenital muscular dystrophy, MDC1C, MDC1D, LGMD2I, LGMD2K, LGMD2M, LGMD2N, LGMD2O, LGMD2P, LGMD2T, or LGMD2U.

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