Methods and materials for galgt2 gene therapy

Abstract

The present disclosure relates to recombinant adeno-associated virus (rAAV) delivery of GALGT2 polynucleotides. The present disclosure provides rAAVs and methods of using rAAVs for GALGT2 gene therapy of neuromuscular diseases. Exemplary neuromuscular diseases include, but are not limited to, muscular dystrophy, such as Duchenne muscular dystrophy, congenital muscular dystrophy type 1A, and limb girdle muscular dystrophy type 2D.

Description

[0001] This application is a divisional application of the invention patent application filed on September 16, 2016, with application number 201680054261.9 (international application number PCT / US2016 / 052051) and entitled "Method and Material for GALGT2 Gene Therapy".

[0002] Government Interest Statement This invention was made with government support under license numbers U54 NS055959 and R01 AR049722 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention.

[0003] Reference sequence lists are incorporated into this invention. This application contains, as a separate part of the disclosure, a sequence list submitted in computer-readable form (filename: 49885PCT_Seqlisting.txt; 14,467 bytes – ASCII text file; created on September 15, 2016), and the entire sequence list is incorporated herein by reference. Technical Field

[0004] This disclosure relates to the delivery of recombinant adeno-associated virus (rAAV) of GALGT2 polynucleotides. This disclosure provides rAAV and methods of using rAAV for GALGT2 gene therapy of neuromuscular diseases. Exemplary neuromuscular diseases include, but are not limited to, muscular dystrophy, such as Duchenne muscular dystrophy, congenital muscular dystrophy type 1A, and limb-girdle muscular dystrophy type 2D. Background Technology

[0005] Muscular dystrophy (MD) is a group of inherited disorders. This group is 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 appear later in life. These diseases differ in the distribution and severity of muscle weakness (and in some forms, the cardiac muscle), age of onset, rate of progression, and inheritance patterns.

[0006] Duchenne muscular dystrophy (DMD) is a type of MD and the most common severe childhood form of muscular dystrophy, affecting 1 in 5,000 newborn males. Its inheritance pattern is called X-linked recessive. DMD is caused by mutations in the DMD gene, resulting in a deficiency of dystrophin (427 kDa) in skeletal and cardiac muscle, as well as in the gastrointestinal tract and retina. Dystrophin not only protects the myofibrous membrane from eccentric contractions but also anchors many signaling proteins near the myofibrous membrane. Clinical signs of DMD are usually first noticed between 3 and 5 years of age, with gait changes and decreased motor skills typically leading to diagnostic evaluation. DMD is progressive, with patients losing the ability to walk by age twelve. Historically, patients died in the latter part of the second decade from respiratory complications, but improved supportive care, particularly the judicious use of nocturnal ventilation support, has extended life expectancy by nearly a decade. This extended lifespan reveals a general decline in cardiac function, accompanied by complications of dilated cardiomyopathy. This presents further clinical challenges and requires previously unmet cognitive and medical interventions. Non-progressive cognitive impairment may also be present in DMD. Despite hundreds of clinical trials conducted in DMD, treatment with corticosteroids remains the only consistently effective treatment. Current standards of care for DMD include the use of prednisone or defcoccus, which can extend walking time by several years, at the cost of significant side effects and limited evidence of any impact on survival.

[0007] Congenital muscular dystrophy type 1A (MCD1A) is another type of MD. MCD1A belongs to a group of neuromuscular disorders that appear at birth or infancy, characterized by hypotonia, muscle weakness, and muscle atrophy. MCD1A represents 30-40% of congenital muscular dystrophy, with some regional variation. The prevalence is estimated at 1 in 30,000. The disease appears at birth or in the first few months of life, accompanied by hypotonia and muscle weakness in the limbs and trunk. Breathing and eating disorders may also occur. Motor development is delayed and limited (sitting or standing can only be achieved with assistance). Early spinal stiffness, scoliosis, and respiratory insufficiency are present in infants. Typical elongated myopathy facies and facial involvement with oculomotor palsy may appear later. Seizures are possible, although they occur in less than one-third of subjects. Intellectual development is normal. MCD1A is caused by mutations in the LAMA2 gene encoding the laminin α-2 chain and is an autosomal recessive disorder. Current treatment is symptomatic and involves a multidisciplinary approach, including physical therapists, occupational therapists, and speech therapists, with the goal of optimizing each patient's abilities. Seizures or other neurological complications require specialized treatment. The prognosis for MDC1A is very severe; most affected children do not survive to adolescence. Currently, the prognosis can only be improved through dedicated multidisciplinary management, particularly in orthopedics and respiratory medicine.

[0008] Limb-girdle muscular dystrophy (LGMD) is another type of MD. LGMD is a rare condition, and its presentation varies from person to person depending on the time of onset, the area of ​​muscle weakness, cardiac and respiratory involvement, rate of progression, and severity. LGMD can occur in childhood, adolescence, young adulthood, or later. Men and women are equally likely to be affected. LGMD causes weakness in the shoulder and pelvic girdle, sometimes accompanied by weakness in the muscles near the thigh and arm over time. Leg weakness often precedes arm weakness. Facial muscles are usually unaffected. As the disease progresses, patients may experience walking difficulties and may eventually require a wheelchair. Involvement of the shoulder and arm muscles can lead to difficulty raising the arm overhead and lifting objects. In some types of LGMD, the heart and respiratory muscles may be involved.

[0009] There are at least nineteen forms of LGMD, and their forms are classified according to their associated genetic defects.

[0010] Type of inheritance pattern gene or chromosome LGMD1A is an autosomal dominant myotensin gene. LGMD1B is an autosomal dominant laminin A / C gene. LGMD1C autosomal dominant cavernin gene LGMD1D Autosomal dominant chromosome 7 LGMD1E is an autosomal dominant myodetin gene. LGMD1F Autosomal dominant chromosome 7 LGMD1G Autosomal dominant chromosome 4 LGMD1H Autosomal dominant chromosome 3 LGMD2A is an autosomal recessive calpain-3 gene. LGMD2B is an autosomal recessive Dysferlin gene. LGMD2C autosomal recessive γ-inositone gene LGMD2D autosomal recessive α-caryoga gene LGMD2E is an autosomal recessive β-inosinic gene. LGMD2F autosomal recessive δ-inositoid gene LGMD2G autosomal recessive resorcinolone gene LGMD2H Autosomal recessive TRIM32 LGMD2I is an autosomal recessive FKRP gene. LGMD2J autosomal recessive titin gene LGMD2K is an autosomal recessive gene associated with the POMT1 gene. LGMD2L is an autosomal recessive Fukutin gene. LGMD2M is an autosomal recessive Fukutin gene. LGMD2N is an autosomal recessive POMT2 gene. LGMD2O is an autosomal recessive gene associated with the POMGnT1 gene. LGMD2Q is an autosomal recessive plectin gene. LGMD's professional testing is now available through the National Debugging Group (NCG)'s national program diagnostics.

[0011] The GALGT2 gene (also known as B4GALNT2) encodes β1-4-N-acetyl-D-galactosamine (βGalNAc) glycosyltransferase. GALGT2 overexpression has been investigated in three different muscular dystrophy models: DMD, LGMD2D, and MDC1A [Xu et al., Am. J. Pathol, 175: 235-247 (2009); Xu et al., Am. J. Path., 171: 181-199 (2007); Xu et al., Neuromuscul. Disord., 17: 209-220 (2007); Martin et al., Am. J. Physiol. Cell. Physiol., 296: C476-488 (2009); and Nguyen et al., Proc. Natl. Acad. Sci. USA, 99: 5616-5621 (2002)]. It has been reported that GALGT2 overexpression in skeletal muscle can induce glycosylation of α-myodystrophy proteoglycans containing α1-4-N-acetyl-D-galactosamine (GalNAc) carbohydrates to generate CT glycoantigens (Neu5Ac / Gcα2-3[GalNAcβ1-4]Gal). α1-4GlcNAcβ-). The GALGT2 glycosyltransferase and CT carbohydrate produced are generally limited to the neuromuscular and tendon junctions in the skeletal muscle of studied adults, non-human primates, rodents and all other mammals [Martin et al., J. Neurocytol., 32: 915-929(2003)]. It has been reported that overexpression of GALGT2 in skeletal muscle stimulates ectopic glycosylation of the synaptic episynaptic membrane, which in turn stimulates ectopic overexpression of normal synaptic scaffold proteins that are orthologs or homologues of proteins missing in various forms of muscular dystrophy, including dystrophin substitutes (e.g., utrophin, reticulin 1) and laminin α2 substitutes (laminin α5 and agrin) [Xu et al. 2009, above; Xu et al., Am. J. Path. 2007, above; Xu et al., Neuromuscul. Disord. 2007, above; Nguyen et al., above; Chicoine et al., Mol. Ther, 22: 713-724. (2014). Overall, GALGT2 induction of these substitutes has been reported to enhance myofibril membrane integrity and prevent muscle damage in dystrophin-deficient and wild-type muscles [Martin et al., above]. GALGT2 overexpression in skeletal muscle has been reported to prevent muscle damage and inhibit muscle disease. This was observed in mdx mouse models of DMD [Xu et al., Neuromuscul. Disord. 2007, above; Martin et al. (2009), above; Nguyen et al., above], although only half of the fibers were transduced; it should be noted that the improvement was comparable to that of small dystrophin gene transfer [Martin et al. (2009), above]. Notably, GALGT2 gene transfer has also been reported in dystrophin type 1A dystrophin mice. W The model [Xu et al., Am. J. Path. 2007, above] and limb-girdle muscular dystrophy type 2D Sgca - / - In mouse models [Xu et al. 2009, above] it is preventative.

[0012] Adeno-associated virus (AAV) is a replication-defective parvovirus with a single-stranded DNA genome approximately 4.7 kb long, consisting of 145 inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank accession number NC_002077; the complete genome of AAV-2 is provided in GenBank accession number NC_001401 and in the literature [Srivastava et al., J. Virol., 45: 555-564 (1983)]; the complete genome of AAV-3 is provided in GenBank accession number NC_1829; the complete genome of AAV-4 is provided in GenBank accession number NC_001829; the genome of AAV-5 is provided in GenBank accession number AF085716; the complete genome of AAV-6 is provided in GenBank accession number NC_001862; at least a portion of the genomes of AAV-7 and AAV-8 are provided in GenBank accession numbers AX753246 and AX753249, respectively; and in the literature [Gao et al., J. Virol., 78:6381-6388]... The AAV-9 genome was provided in [Mol. Ther., 13(1): 67-76 (2006)]; the AAV-10 genome was provided in [Virology, 330(2): 375-383 (2004)]. Cis-acting sequences guiding viral DNA replication (rep), capsidation / packaging, and host cell chromosome integration are contained within the ITRs of AAVs. Three AAV promoters (named p5, p19, and p40 based on relative map localization) drive the expression of two AAV internal open reading frames to encode the rep and cap genes. Differential splicing of the two rep promoters (p5 and p19), plus a single AAV intron (nucleotides 2107 and 2227), results in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The Rep protein possesses multiple enzymatic properties ultimately responsible for replicating the viral genome. The cap gene, expressed by the p40 promoter, encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and a non-shared translation initiation site are responsible for generating these three associated capsid proteins. A single shared polyadenylation site is located at map position 95 of the AAV genome. For the AAV lifecycle and genetics, see [Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992)].

[0013] AAV possesses unique characteristics that make it attractive as a vector for delivering exogenous DNA into cells, such as in gene therapy. AAV infection of cultured cells is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cells, making it possible to target many different tissues in vivo. Additionally, AAV transduces slowly dividing and non-dividing cells and can essentially prolong the lifespan of these cells as a transcriptionally active nuclear appendage (extrachromosomal element). The AAV proviral genome is infectiously identical to the DNA cloned in the plasmid, enabling the construction of recombinant genomes. Moreover, because the signals guiding AAV replication, genome capsidation, and integration are contained within the ITRs of the AAV genome, some or all of the approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be able to replace the exogenous DNA. The rep and cap proteins can be trans-provided. Another remarkable characteristic of AAV is that it is a very stable and robust virus. It can easily withstand conditions used for inactivating adenoviruses (56 ºC to 65 ºC for several hours), and cold storage of AAV is less critical. AAV can even be freeze-dried. Finally, AAV-infected cells cannot resist superinfection.

[0014] AAV rh.74 has been used to deliver DNAs encoding various proteins. References [Xu et al., Neuromuscular Disorders, 17: 209-220 (2007)] and [Martin et al., Am. J. Physiol. Cell. Physiol., 296: 476-488 (2009)] address the expression of rh.74 in cytotoxic T-cell GalNAc transferases in Duchenne muscular dystrophy. Reference [Rodino-Klapac et al., Mol. Ther., 18(1): 109-117 (2010)] describes AAV rh.74 expression in a dystrophin small FLAG protein-tagged fusion following delivery of AAV rh.74 via vascular limb perfusion.

[0015] Muscular dystrophy is a group of diseases with no definitive cure, severely impacting individuals, families, and communities. The costs are unpredictable. Individuals suffer emotional stress, decreased quality of life, and loss of self-esteem. The extreme physical challenges resulting from limb dysfunction create difficulties in daily life. Family dynamics are challenged by financial hardship and interpersonal difficulties. Patients become estranged from their siblings, and spousal conflicts often lead to divorce, especially if the muscular dystrophy can be attributed to one parent. The burden of seeking treatment often becomes a lifelong, highly concentrated effort, diminishing and challenging every aspect of life. Beyond families, communities bear the financial burden, requiring increased facilities to accommodate muscular dystrophy patients for special education, special transportation, and to cover the costs of frequent hospitalizations for recurrent respiratory infections and cardiac complications. Financial responsibility should be shared by state and federal agencies, extending responsibility to taxpayer communities.

[0016] Therefore, there remains a need in this field to treat neuromuscular diseases, including but not limited to muscular dystrophy such as DMD, MDC1A and LGMD2D. Summary of the Invention

[0017] This invention provides a method for treating neuromuscular diseases in subjects in need, comprising administering recombinant adeno-associated virus (rAAV) such as rAAVrh74.MCK.GALGT2 to the subject, wherein the route of administration is intramuscular and the dose of rAAV administered is approximately 3 × 10⁻⁶. 11 VG / injection to approximately 5 × 10 12 vg / injection, administered via intramuscular route, with a dose of approximately 3 × 10⁻⁶ rAAV. 11 vg / injection, administered via intramuscular route, with a dose of approximately 1 × 10⁻⁶ rAAV. 12 vg / injection, administered via intramuscular route, with a dose of approximately 5 × 10⁻⁶ rAAV. 12 The administration route is intravenous perfusion, and the dose of rAAV is approximately 6 × 10⁻⁶. 12 vg / kg / limb to approximately 4.8×10 13 The dose of rAAV administered via interarterial limb perfusion (vg / kg / limb) is approximately 6 × 10⁻⁶. 12 The dose of rAAV administered via interarterial limb perfusion (vg / kg / limb) is approximately 1.2 × 10⁻⁶. 13 The dose of rAAV administered via interarterial limb perfusion (vg / kg / limb) is approximately 2.4 × 10⁻⁶. 13 The dose of rAAV administered via interarterial limb perfusion (vg / kg / limb) is approximately 4.8 × 10⁻⁶. 13The dose was administered via intravenous systemic administration, with a dose of approximately 2 × 10⁻⁶ g / kg / limb. 14 vg / kg to approximately 6×10 15 The dose was administered via intravenous systemic administration, and the dose of rAAV was approximately 4 × 10⁻⁶ g / kg. 14 vg / kg to approximately 6×10 15 The dose was administered via intravenous systemic administration, and the dose of rAAV was approximately 4 × 10⁻⁶ g / kg. 14 The dose was administered via intravenous systemic administration, and the dose of rAAV was approximately 8 × 10⁻⁶ g / kg. 14 The dose was administered via intravenous systemic administration, and the dose of rAAV was approximately 2 × 10⁻⁶ g / kg. 15 The dose of rAAV is approximately 6 × 10⁻⁶ g / kg or administered via systemic intravenous route. 15 vg / kg.

[0018] Treatment is anticipated for neuromuscular disorders such as Duchenne muscular dystrophy (DMD); Becker muscular dystrophy; congenital muscular dystrophy (MDC) types 1A, 1B, 1C, and 1D; limb-girdle muscular dystrophy (LGMD) types 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, and 2Q; Be Muscular dystrophy; Ullrich's congenital muscular dystrophy; myo-ocular-cerebrovascular disease; Fukuyama-type congenital muscular dystrophy; Walker-Warberg syndrome; myotonic dystrophy; myasthenic syndrome; congenital myasthenia gravis; inclusion body myopathy; inclusion body myositis; Emery-Dreifuss type muscular dystrophy; distal muscular dystrophy; dermatomyositis; central nucleus myopathy; facial-scapulothorax type muscular dystrophy; Myoshi type myopathy; mitochondrial myopathy; linear myopathy; Nonaka myopathy; myasthenia gravis and polymyositis. Therefore, muscular dystrophy is a likely neuromuscular disease.

[0019] This method resulted in improvements in subjects in the following areas: absolute muscle strength; strength decay during eccentric muscle contraction; serum CK levels; serum cardiac troponin levels; serum MMP9 levels; grip strength; limb torsional strength; limb range of motion or flexibility; walking; 6-minute walk test; knee flexor or extensor strength; maximum isometric voluntary muscle contraction; North Star movement assessment; muscle mass, fat loss, or edema as measured by T2-weighted MRI of the limbs; muscle contractures; limb joint angles; cardiac function (heart rate, cardiac output, shortening percentage, stroke volume); respiration (including respiratory rate, blood oxygen saturation, supplemental oxygen requirement); muscle necrosis; muscle regeneration; muscle atrophy; muscle inflammation; muscle calcification; muscle centronucleation; muscle size or muscle fiber size; lifespan; and / or expression of dystrophin or laminin α2 substitute proteins (myotrophic-associated proteins, reticulin 1, laminin α5, and aggregates).

[0020] The present invention also provides rAAV encoding the GALGT2 polypeptide, such as rAAVrh74.MCK.GALGT2. Detailed Implementation

[0021] This disclosure provides methods and products for treating neuromuscular diseases. The method involves delivering GALGT2 polynucleotides to muscle cells in a subject using an AAV as a gene delivery vector. Subjects include, but are not limited to, mammals such as dogs, cats, and humans. In some embodiments, the subject is a human patient. In some embodiments, the subject is a human pediatric patient.

[0022] In one aspect, a method for treating neuromuscular diseases is provided, comprising administering a recombinant adeno-associated virus (rAAV) encoding GALGT2 to a subject.

[0023] The neuromuscular diseases considered in this invention include, but are not limited to, muscular dystrophy (MD). The neuromuscular diseases considered in this invention also include neuromuscular diseases other than MD. Therefore, in some embodiments, the neuromuscular diseases considered in this invention include, but are not limited to, Duchenne muscular dystrophy (DMD); Becker muscular dystrophy; congenital muscular dystrophy (MDC) types 1A, 1B, 1C, and 1D; limb-girdle muscular dystrophy (LGMD) types 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, and... Type 2Q; Bethlem myopathy; Ullrich congenital muscular dystrophy; myo-ocular-cerebrovascular disease; Fukuyama type congenital muscular dystrophy; Walker-Warberg syndrome; myotonic dystrophy; myasthenia gravis syndrome; congenital myasthenia gravis; inclusion body myopathy; inclusion body myositis; Emery-Dreifuss type muscular dystrophy; distal muscular dystrophy; dermatomyositis; central nucleus myopathy; facioscapulohumeral type muscular dystrophy; Myoshi type myopathy; mitochondrial myopathy; linear myopathy; Nonaka myopathy; myasthenia gravis and polymyositis. In some embodiments, MD is DMD. In some embodiments, MD is MDC1A. In some embodiments, MD is LGMD2D.

[0024] In some embodiments of any method described in this invention, the subject is at least 1 year old (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years old) or older. In some embodiments of any method described in this invention, the subject is male. In some embodiments of any method described in this invention, the subject is female. In some embodiments of any method described in this invention, the subject is mobile during treatment. In some embodiments of any method described in this invention, the subject is immobile at the start of treatment. In some embodiments of any method described in this invention, the subject is a subject with a confirmed mutation in the DMD gene using clinically accepted techniques for defining mutations. Mutations in the dystrophin gene that produce the DMD phenotype are well known in the art, and methods for identifying them in subjects are also well known. See, for example, the Leiden Duchenne Muscular Dystrophy Mutation Database and literature [Aartsma-Rus et al., Hum. Mut., 30:293-299 (2009)].

[0025] In some embodiments of any of the methods described in this invention, the subject is shown by magnetic resonance imaging of the extensor digitorum brevis (EDB) muscle to preserve sufficient muscle mass to allow for transfection or gene transfer. In some embodiments of any of the methods described in this invention, the subject is receiving a stable dose of corticosteroid therapy (e.g., difflux, prednisone, or a general form thereof) for at least 4 weeks (e.g., 5, 6, 7, 8, 9, 8, 9, 10, 11, or 12 weeks) prior to the start of treatment. In some embodiments, the subject is receiving background steroid therapy (e.g., intermittent or chronic / continuous background steroid therapy). Those skilled in the art will understand that such a subject is one who is currently using steroids (or corticosteroids) on which another treatment, such as the gene therapy described in this invention, is applied.

[0026] In some embodiments of any method described in this invention, the subject is a subject without active viral infection. In some embodiments of any method described in this invention, the subject does not have a DMD mutation without functional impairment or loss of function. In some embodiments of any method described in this invention, the subject does not exhibit symptoms of cardiomyopathy, such as exertional dyspnea, foot edema, shortness of breath when lying supine, or lung rales. In some embodiments of any method described in this invention, the subject does not have an ejection fraction below approximately 40% as measured by echocardiography. In some embodiments of any method described in this invention, the subject is not a subject serologically proven to be undergoing treatment for HIV infection (because the subject may be in an immunocompromised state) or hepatitis A, B, or C (because the subject may have elevated transaminase levels).

[0027] In some embodiments of any method described in this invention, the subject has not been diagnosed with, and has not been treated for, an autoimmune disease. In some embodiments of any method described in this invention, the subject does not have persistent leukopenia or leukocytosis (white blood cell count ≤ 3.5 K / μL or ≥ 20.0 K / μL) or an absolute neutrophil count < 1.5 K / μL.

[0028] In some embodiments of any of the methods described in this invention, the subject does not have a concomitant disease (e.g., viral infection or autoimmune disease) or a requirement for chronic drug treatment that, in the opinion of a practicing physician, would pose an unnecessary risk of gene transfer.

[0029] In some embodiments of any method described in this invention, the subject does not have an rAAVrh74 binding antibody titer ≤ 1:400 as determined by ELISA. In some embodiments of any method described in this invention, the subject does not have an rAAVrh74 binding antibody titer ≤ 1:50 as determined by ELISA.

[0030] In some embodiments of any of the methods described in this invention, the subject does not have detectable circulating anti-Sda antibodies [Blood groups: P, I, Sda, and Pr. AABB (1991)], as determined, for example, by subject biosample ELISA immunoassay. Sda glycans are human blood group structures generated by GALGT2. The Sda blood group antigen is identical to the CT glycan structure [GalNAcb1,4[Neu5Aca2,3]Galb1,4GlcNAc-] generated by GALGT2 in mice. Although this disclosure is not bound by any particular theory or mechanism of action, it is anticipated that the presence or content of CT glycan structures may be enhanced in cells expressing GALGT2, and the inventors believe that subjects with such Sda antibodies, estimated to be 0.2% of the subject population, may be at risk of antibody-mediated tissue rejection after rAAVrh74.MCK.GALGT2 treatment.

[0031] Suitable biological samples used in the methods described in this invention include, for example, any biological fluid. Biological samples can be, for example, samples obtained from a subject (e.g., a mammal such as a human), or can be derived from such a subject. Biological samples can be derived from a muscle biopsy. Biological samples can also be biological fluids, such as urine, whole blood or portions thereof (e.g., plasma or serum), saliva, semen, sputum, cerebrospinal fluid, tears, or mucus. If desired, biological samples can be further fractionated into fractions containing specific analytes (e.g., proteins). For example, whole blood samples can be fractionated into serum or fractions containing specific types of proteins. If desired, biological samples can be combinations of different biological samples from a subject, such as a combination of two different bodily fluids.

[0032] Biological samples suitable for use in this invention can be fresh or frozen samples collected from subjects, or archival samples with a known history of diagnosis, treatment, and / or outcomes. Biological samples can be obtained from subjects, for example, those suspected of having or at risk of developing cancer or an infection (e.g., a viral infection). Any suitable method for obtaining biological samples can be employed, although exemplary methods include, for example, open muscle biopsy, venipuncture, swabs (e.g., oral swabs), irrigation, or fine-needle aspiration biopsy procedures. Biological samples can also be obtained from bone marrow or spleen.

[0033] In some embodiments, protein extracts can be prepared from biological samples. In some embodiments, the protein extract contains a total protein content. Protein extraction methods are well known in the art. See, for example, the literature [Roe, “Protein Purification Techniques: A Practical Approach”, 2nd ed., Oxford University Press (2001)]. Many different and universal kits are available for the extraction of proteins from body fluids and tissues, and these kits can be purchased from, for example, BioRad Laboratories (Hercules, CA), BD Biosciences Clontech (Mountain View, CA), Chemicon International, Inc. (Temecula, CA), Calbiochem (San Diego, CA), Pierce Biotechnology (Rockford, IL), and Invitrogen Corp. (Carlsbad, CA).

[0034] Methods for obtaining and / or preserving biological samples to preserve the viability or integrity of cells in the biological sample are well known to those skilled in the art. For example, the biological sample may be further contacted with one or more additional reagents, such as suitable buffers and / or inhibitors, including protease inhibitors, which are designed to maintain or minimize changes in protein structure (e.g., changes in osmotic pressure or pH). Such inhibitors include chelating agents such as ethylenediaminetetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (EGTA), and protease inhibitors such as benzyl sulfonyl fluoride (PMSF), aprotinin, and leucropeptide. Suitable buffers and storage conditions or manipulation of whole cells are described, for example, in the literature [Pollard and Walker, “Basic Cell Culture Protocols,” Methods in Molecular Biology, Vol. 75, Humana Press (1997); Masters, “Animal cell culture: apractical approach,” Practical Approach Series, Vol. 232, Oxford University Press (2000); and Jones, “Human cell culture protocols,” Methods in Molecular Medicine, Vol. 2, Humana Press (1996).].

[0035] The method of treating neuromuscular diseases disclosed herein results in the transduction of muscle cells (e.g., skeletal muscle, smooth muscle, or cardiomyocytes) with GALGT2 polynucleotides. The effective dose or multiple doses of the rAAV-containing composition disclosed herein for a subject are to prevent and slow the progression or improve (eliminate or reduce) the muscle pathology associated with the treated neuromuscular disorder. The impact on muscle pathology can be demonstrated through improvements to one or more measurement standards in the field, such as: absolute muscle strength; strength decay during eccentric muscle contraction; serum CK levels; serum cardiac troponin levels; serum MMP9 levels; grip strength; limb torque; limb range of motion or flexibility; walking; 6-minute walk test; knee flexor or extensor strength; maximal isometric voluntary muscle contraction; North Star movement assessment; muscle mass, fat loss, or edema as measured by T2-weighted MRI of the limbs; muscle contractures; limb joint angles; cardiac function (heart rate, cardiac output, shortening percentage, stroke volume); respiration (including respiratory rate, blood oxygenation, supplemental oxygen requirement); muscle necrosis; muscle regeneration; muscle atrophy; muscle inflammation; muscle calcification; muscle centronucleation; muscle size or muscle fiber size; lifespan; and expression of dystrophin or laminin α2 substitute proteins (myotrophic proteins, reticulin 1, laminin α5, and aggregates). See, for example, the literature [Forbes et al., Radiology, 269(1): 198-207 (2013); Govoni et al., Cell Mol. Life Sci., 70(23):4585-4602 (2013)]; and the literature [Chandrasekharan and Martin, Methods Enzymol., 479:291-322 (2010)]. If the dose is administered before the onset of neuromuscular disorder, the administration is prophylactic. If the dose is administered after the onset of neuromuscular disease, the administration is therapeutic. Therefore, treatment of the subject with the method described in this invention is considered to prevent, slow or prevent the progression of neuromuscular disease, reduce the severity of neuromuscular disease, alleviate (partial or total) neuromuscular disease, and / or prolong neuromuscular disease survival.

[0036] In some embodiments, any method described in this invention may further include detecting or measuring the expression level of GALGT2 in cells transduced with the GALGT2 transgene. Methods for measuring mRNA or protein expression are well known in the art (e.g., immunoassays, such as Western blotting).

[0037] In some embodiments, any method described in this invention may further include detecting or measuring the level of CT antigen expressed in cells transduced with GALGT2 polynucleotides [Chicoine et al.]. The above text ].

[0038] In some embodiments, any method described in this invention may further include detecting or measuring the levels of myotrophic-associated proteins expressed in cells transduced with GALGT2 polynucleotides [Chicoine et al.]. The above text ].

[0039] In some embodiments, any method described in this invention may further include detecting or measuring the number of fibers containing a central nucleus, said fibers being transduced with GALGT2 polynucleotides.

[0040] Therefore, the administration routes of rAAV considered in the above methods include, but are not limited to, intraperitoneal (IP), intramuscular (IM), and intravascular routes [including, for example, interarterial limb perfusion (ILP) and intravenous (IV)].

[0041] In the methods disclosed herein, the dose of rAAV to be administered will vary depending on, for example, the specific rAAV, the dosing regimen, the treatment target, the individual, and the targeted cell type, and can be determined by methods standard in the art. More than one dose may be administered, for example, once, twice, three times, or more. The dose titer of rAAV may be approximately 1 × 10⁻⁶ per milliliter. 6 Approximately 1×10 7 Approximately 1×10 8 Approximately 1×10 9 Approximately 1×10 10 Approximately 1×10 11 Approximately 1×10 12 Approximately 1×10 13 Approximately 1×10 14 Or approximately 1×10 15 More or more DNAase-resistant particles (DRPs). Dosage can also be expressed in viral genome (vg) units (i.e., 1 × 10⁻⁶). 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×10 14 vg, 1×10 15 vg). The method for AAV titration is described in the literature [Clark et al., Hum. Gene Ther., 10: 1031-1039(1999)].

[0042] In some implementations of the above method, the route of administration is the IM route, and the dose of rAAV administered is approximately 3 × 10⁻⁶. 11 Up to at least approximately 5×1012 vg / injection. (All ranges in this invention are intended to represent each individual value within a range, as well as the individual upper and lower limits of each range). In some embodiments of the method described above, the route of administration is the IM route, and the dose of rAAV administered is 3 × 10⁻⁶. 11 vg / injection. In some embodiments of the above method, the route of administration is the intramural route, and the dose of rAAV administered is 1×10 12 vg / injection. In some embodiments of the above method, the route of administration is the intramedullary route, and the dose of rAAV administered is 5 × 10⁻⁶. 12 vg / injection.

[0043] In some implementations of the above method, the route of administration is the ILP route, and the dose of rAAV administered is approximately 6 × 10⁻⁶. 12 Up to at least approximately 4.8 × 10 13 vg / kg. (All ranges in this invention are intended to represent each individual value within a range, as well as the individual upper and lower limits of each range). In some embodiments of the method described above, the route of administration is the ILP route, and the dose of rAAV administered is 6 × 10⁻⁶. 12 vg / kg / limb. In some embodiments of the above method, the route of administration is the ILP route, and the dose of rAAV administered is 1.2 × 10⁻⁶. 13 vg / kg / limb. In some embodiments of the above method, the route of administration is the ILP route, and the dose of rAAV administered is 2.4 × 10⁻⁶. 13 vg / kg / limb. In some implementations of the above method, the route of administration is the ILP route, and the dose of rAAV administered is 4.8 × 10⁻⁶. 13 vg / kg / limb.

[0044] In some implementations of the above methods, the route of administration is systemic IV, and the dose of rAAV administered is approximately 2 × 10⁻⁶. 14 Up to at least approximately 6×10 15 vg / kg. In some embodiments of the above method, the route of administration is systemic IV, and the dose of rAAV administered is approximately 4 × 10⁻⁶. 14 Up to at least approximately 6×10 15 vg / kg. (All ranges in this invention are intended to represent each individual value within a range, as well as the individual upper and lower limits of each range). In some embodiments of the above method, the route of administration is systemic IV, and the dose of rAAV administered is 4 × 10⁻⁶. 14 vg / kg. In some implementations of the above method, the route of administration is systemic IV, and the dose of rAAV administered is 8 × 10⁻⁶. 14vg / kg. In some implementations of the above method, the route of administration is systemic IV, and the dose of rAAV administered is 2 × 10⁻⁶. 15 vg / kg. In some implementations of the above method, the route of administration is systemic IV, and the dose of rAAV administered is 6 × 10⁻⁶. 15 vg / kg.

[0045] Human patients are subjects considered for treatment in this invention. Human patients are subjects considered for treatment via IM delivery in this invention. These patients include those who, for example: (i) are 9 years of age or older, (ii) are male, (iii) are ambly or non-ambly; (iv) have a confirmed mutation in the DMD gene using clinically accepted techniques for defining mutations; (v) maintain sufficient muscle mass to allow transfection or gene transfer as shown by magnetic resonance imaging of the extensor digitorum brevis (EDB) muscle; (vi) belong to any ethnicity; (vii) are capable of cooperating with all learning procedures; (viii) are sexually mature and / or sexually active, and willing to use reliable methods of contraception, if appropriate; and / or (ix) have received a stable dose of corticosteroids (e.g., defcodone, prednisone, or a general form thereof) for at least 12 weeks prior to gene transfer. Suitable patients may exclude, for example: (i) those with clinically observed active viral infection; (ii) those with DMD mutations without functional debilitating or loss of function; (iii) those exhibiting symptoms of cardiomyopathy, such as exertional dyspnea, foot edema, shortness of breath on supine position, or pulmonary rales; (iv) those with an ejection fraction of less than approximately 40% by echocardiography; (v) those with serological evidence of HIV infection or hepatitis A, B, or C; (vi) those who have or have been diagnosed with (or are receiving treatment for) an autoimmune disease; (vii) those with persistent leukopenia or leukocytosis (white blood cell count ≤ 3.5 K / L or ≥ 20.0 K / L) or an absolute neutrophil count < 1.5 K / L; and (viii) those with comorbidities (e.g., viral infection or autoimmune disease) or those requiring chronic drug treatment that, in the opinion of a practicing physician, poses an unnecessary risk of gene transfer. (ix) A titer of rAAVrh74-binding antibody ≥ 1:400 as determined by ELISA; or (x) the presence of circulating anti-Sda antibodies. In an exemplary clinical protocol, DMD patients received bilateral injections, with one extensor digitorum brevis (EDB) muscle in each patient receiving the vector rAAVrh74.MCK.GALGT2, and the other EDB muscle in each patient receiving saline alone. Subjects received 1 × 10 12 vg (total dose) is the carrier of the dose.

[0046] The cell transduction efficiency of the methods described above and below can be at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments involving IV limb perfusion delivery, transduction efficiency is increased by increasing the amount of the composition delivering rAAV, pre-rinsing before rAAV delivery, and / or increasing the residence time of said rAAV.

[0047] On the other hand, the present invention provides an rAAV genome. The given rAAV genome contains GALGT2 polynucleotides under the control of transcriptional control sequences. The rAAV genome lacks AAV rep and cap DNA. The AAV DNA in the rAAV genome may be derived from any AAV serotype from which recombinant viruses can be derived, 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, and AAVrh74. The nucleotide sequences of the genomes of these AAV serotypes are known in the art, as described in the background section above. In some embodiments, the AAV DNA in the rAAV genome is derived from AAV rh.74. The polynucleotide sequence of the AAV rh.74 genome is listed in SEQ ID NO: 1, where nucleotides 210-2147 are the Rep 78 gene open reading frame, 882-208 are the Rep52 open reading frame, 2079-2081 are the Rep78 terminator, 2145-2147 are the Rep78 terminator, 1797-1800 are the splice donor site, 2094-2097 are the splice acceptor site, 2121-2124 are the splice acceptor site, 174-181 are the p5 promoter +1 prediction, 145-151 are the p5 TATA box, 758-761 are the p19 promoter +1 prediction, 732-738 are the p19 TATA box, 1711-1716 are the p40 TATA box, 2098-4314 are the VP1 Cap gene open reading frame, and 2509- 2511 is the start of VP2, 2707-2709 is the start of VP3, and 4328-4333 is the polyA signal.

[0048] In some embodiments, the transcriptional control sequence of the rAAV genome is a muscle-specific control element, including but not limited to those derived from the actin and myosin gene families, such as from the myoD gene family [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)], control elements derived from the skeletal muscle actin gene [Muscat et al., Mol. Cell. Biol., 7: 4089-4099 (1987)], cardiac actin gene, muscle creatine kinase gene (MCK) promoter [Johnson et al., Mol. Cell. Biol., 9:3393-3399 (1989)] and MCK enhancer, MHCK7 promoter (a modified MCK promoter containing an enhancer of the myosin heavy chain [Salva et al., Mol.Ther., 15: 320-329 (2007)]), intermyolin promoters, regulators derived from the skeletal muscle fast-twitch troponin C gene, slow-twitch cardiac troponin C gene, and slow-twitch troponin I gene: hypoxia-inducible nuclear factor [Semenza et al., Proc. Natl. Acad. Sci. USA, 88: 5680-5684 (1991)], steroid-inducible elements, and promoters including glucocorticoid response elements (GRE) [Mader and White, Proc. Natl. Acad. Sci. USA, 90: 5603-5607 (1993)], and other control elements. In some embodiments, transcriptional regulatory elements include the MCK promoter. In some embodiments, transcriptional control elements include the MHCK7 promoter.

[0049] In some embodiments, the GALGT2 polynucleotide in the rAAV genome is the GALGT2 cDNA listed in GenBank accession number #AJ517771 (nucleotides 1002-2522 are listed as SEQ ID NO: 2). In some embodiments, the GALGT2 polynucleotide in the rAAV genome is the GALGT2 cDNA listed in GenBank accession number #AJ517771, or has sequence identity of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the GALGT2 cDNA. In some embodiments, the GALGT2 polypeptide encoded by the variant GALGT2 polynucleotide is identical to the polypeptide encoded by the GALGT2 cDNA listed in GenBank accession number #AJ517771. The amino acid sequence of the GALGT2 polypeptide encoded by the GALGT2 cDNA listed in GenBank accession number #AJ517771 is listed in SEQ ID NO: 3. In some embodiments, a variant GALGT2 polynucleotide encoding a variant GALGT2 polypeptide having at least one amino acid sequence variation is compared to the amino acid sequence of the polypeptide encoded by the GALGT2 cDNA listed in GenBank accession number #AJ517771 (SEQ ID NO: 3). Amino acid sequence changes can be, for example, substitutions, deletions, or insertions of one or more amino acids, preferably conserved substitutions. The variant GALGT2 polypeptide can have any combination of amino acid substitutions, deletions, or insertions that retain the glycosyltransferase activity of the polypeptide. On the one hand, the variant GALGT2 polypeptide can have many amino acid changes, such that its amino acid sequence has at least 60, 70, 80, 85, 90, 95, 97, 98, 99 or 99.5% identity with the GALGT2 cDNA encoding amino acid sequence (SEQ ID NO: 3) listed in Genbank accession number #AJ517771.

[0050] In some embodiments, the rAAV genome is the MCK.GALGT2 genome, the sequence of which is listed in SEQ ID NO: 2, and is accompanied by the following annotation. On the other hand, an isolated nucleic acid comprising the nucleotide sequence described in SEQ ID NO: 2 is provided. In some embodiments, the isolated nucleic acid consists of the nucleotide sequence described in SEQ ID NO: 2.

[0051] Also provided is an isolated nucleic acid comprising, from 5' to 3', in sequence: (i) a first AAV2 terminal inverted repeat (ITR); (ii) a muscle creatine kinase promoter sequence; (iii) a nucleotide sequence encoding a human GALGT2 polypeptide; and (iv) a second AAV2 terminal inverted repeat, wherein the human GALGT2 polypeptide has an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 3, 100% identical to that of SEQ ID NO: 3, or encoded by nucleotides 1002-2522 of SEQ ID NO: 2.

[0052] Consider recombinant AAV containing the above-mentioned nucleic acids and rAAV containing a nucleotide sequence that is at least 90% identical to the nucleotide sequence described in SEQ ID NO: 2.

[0053] A DNA plasmid containing the rAAV genome of this disclosure is provided. The DNA plasmid contains the rAAV genome as conceived in this invention. The rAAV genome is assembled into infectious viral particles by transferring the DNA plasmid into cells that allow infection with AAV helper viruses (e.g., adenovirus, E1-deficient adenovirus, or herpesvirus). Techniques for producing rAAV particles are standard in the art, wherein the AAV genome to be packaged, the rep and cap genes, and helper viral functions are provided to the cell. The production of rAAV requires the following components to be present within a single cell (referred to in this invention as a packaging cell): the rAAV genome, the AAV rep and cap genes isolated from the rAAV genome (i.e., not in the rAAV genome), and the helper viral functions. The AAVrep and cap genes can be derived from any AAV serotype from which recombinant viruses can be derived, and can be derived from AAV serotypes different from the rAAV genomic ITRs, 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, and AAVrh74. For example, the generation of pseudovirus rAAV is disclosed in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also considered. See, for example, the literature [Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014)].

[0054] The method for generating packaging cells involves producing cell lines that stably express all the components required 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 selection markers such as neomycin resistance genes are integrated into the cell's genome. The AAV genome is introduced into a bacterial plasmid via, for example, GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of a synthetic adapter 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 advantage of this method is that the cells are selectable and suitable for large-scale rAAV production. Other examples of suitable methods include introducing the rAAV genome and / or rep and cap genes into packaging cells using adenovirus or baculovirus instead of plasmids. Methods for generating rAAVs with their own complementary genomes are also known in the art.

[0055] The general principles of rAAV production are summarized in references such as [Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129]. Multiple methods have been described in the literature [Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828)]; US Patent No. 5,173,414; WO 95 / 13365 and corresponding US Patent Nos. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; References [Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615]; References [Clark et al. (1996) Gene Therapy [3:1124-1132]; described in U.S. Patent Nos. 5,786,211; 5,871,982; and 6,258,595. The foregoing documents are incorporated herein by reference in their entirety, with particular emphasis on those portions relating to the production of rAAV.

[0056] On the other hand, this disclosure therefore 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 (homogeneous 293 lineage). In another embodiment, the packaging cells are not cells transformed into cancer cells, such as low-passaged 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey embryonic lung cells).

[0057] The rAAV can be purified by standard methods in the art, such as column chromatography or cesium chloride gradient. Methods for purifying the rAAV vector from helper viruses are known in the art and include those disclosed, for example, in the literature [Clark et al., Hum. GeneTher., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002)] and U.S. Patent Nos. 6,566,118 and WO 98 / 09657.

[0058] Therefore, on the other hand, this disclosure contemplates rAAVs containing GALGT2 polynucleotides. In some embodiments, the rAAV contains an AAV rh74 capsid and a GALGT2 polynucleotide. In some embodiments, the rAAV genome lacks AAV rep and cap DNA. In some embodiments of the method, the rAAV is rAAVrh74.MCK.GALGT2. In some embodiments, the rAAV is a self-complementary genome.

[0059] On the other hand, this disclosure contemplates compositions comprising the rAAV described herein. The compositions of this disclosure comprise rAAV in a pharmaceutically acceptable carrier. The compositions may also contain other components such as diluents. Acceptable carriers and diluents are non-toxic to the recipient and preferably inert at the doses and concentrations used, including buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight peptides; 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 ions such as sodium; and / or nonionic surfactants such as Tween, Pluronic acid derivatives, or polyethylene glycol (PEG). In some embodiments, the rAAV is formulated in Tris, MgCl2, NaCl, and Pluronic acid F68. In some embodiments, the rAAV is prepared in 20 mM Tris (pH 8.0), 1 mM MgCl2 and 200 mM NaCl containing 0.001% Prönkel F68.

[0060] This invention also contemplates combination therapies. Combinations as used in this invention include concurrent or sequential treatment. Combinations of the methods disclosed herein with standard medical treatments (e.g., corticosteroids and / or immunosuppressive drugs) are particularly contemplated, as are combinations with novel treatments. In various embodiments, subjects are treated with the methods contemplated according to this invention before, during, or after corticosteroid treatment (or in any arrangement of two or more of the three possibilities).

[0061] Sterile injectable solutions are prepared by combining rAAV in a suitable solvent at the desired concentration with various other components listed above, and then sterilizing by filtration if necessary. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile carrier containing a basic dispersion medium and the desired other components listed above. In the case of sterile powders used to prepare sterile injectable solutions, vacuum drying and freeze-drying techniques are preferred methods. These methods produce a powder containing the active ingredient plus any other desired components from a previously sterile filtered solution.

[0062] Example Therefore, the aspects and implementation methods of the present invention will be illustrated by the following examples.

[0063] Example 1 A non-replicating rAAV vector named rAAVrh74.MCK.GALGT2 was generated. The rAAV vector contained complete human GALGT2 cDNA (Genbank accession number #AJ517771) under the control of the muscle creatine kinase gene promoter (MCK; muscle-specific promoter). Muscle-specific gene expression was driven using the MCK promoter / enhancer sequence, which consisted of a mouse MCK core enhancer (206 bp) fused to a 351 bp MCK core promoter (proximal). Following the core promoter, a 53 bp endogenous mouse MCK exon 1 (untranslated) was present for efficient transcription initiation, followed by an SV40 late 16S / 19S splicing signal (97 bp) and a small 5'UTR (61 bp). The introns and 5'UTR were derived from plasmid pCMVß (Clontech). The GALGT2 cassette contained a shared Kozak sequence prior to ATG initiation and a small 53 bp polyA signal for mRNA termination. The GALGT2 box mentioned above was previously described in the literature [Martin et al.] (2009), above The description is provided in SEQ ID NO: 2. The only viral sequence contained is the terminal inverted repeat (ITR) of AAV2, which is required for viral DNA replication and packaging. The pAAVrh74.MCK.GALGT2 plasmid contains a human GALGT2 cDNA expression cassette with a flanked AAV2 terminal inverted repeat (ITR). The cassette includes an MCK promoter, a chimeric intron with a Kozak sequence for optimizing gene expression, a human GALGT2 coding sequence, and a polyA signal. The sequence of the cassette with the flanked AAV ITR is listed in SEQ ID NO: 2. AAV vectors containing GALGT2 polynucleotides were generated via a modified cross-packaging method using adenovirus-free triple plasmid DNA transfection (CaPO4 precipitation) in HEK293 cells [Rabinowitz et al., J. Virol., 76:791–801 (2002)]. Vectors were also generated in a similar manner to those previously described by co-transfection of a plasmid containing GALGT2 polynucleotides with the AAV helper plasmid rep2-cap rh.74 and an adenovirus helper plasmid [Wang et al.]. Gene. Ther., 10 [1528–1534 (2003)]. The plasmid rep2-cap rh.74 encodes the wild-type AAV2 rep gene and the rh.74cap gene, and the adenovirus helper plasmid (pAdhelper) expresses adenovirus type 5 E2A, E4ORF6, and VA I / II RNA genes produced at high titers of rAAV.

[0064] Purification was performed using a sequential iodixanol gradient and linear NaCl salt gradient anion exchange column chromatography as described previously [Clark et al., Hum. Gene Ther The vector was purified from clarified 293 cell lysates [10:1031–1039 (1999)]. qPCR-based detection was performed using the previously described MCK-specific primer / probe set and Prism 7500 Taqman detector system (PE Applied Biosystems) (Clark et al., 10:1031–1039 (1999)). The above text The titer of the vector genome (vg) is measured using [a specific method / mechanism]. Vector titers range from 1 to 40 × 10⁻⁶. 12 Between vg / mL.

[0065] The carrier was prepared in 20 mM Tris (pH 8.0), 1 mM MgCl2, and 200 mM NaCl containing 0.001% Pronic F68. The carrier was provided as a frozen liquid, which was thawed prior to clinical administration.

[0066] Example 2 DMD Subject IM Delivery Human patients are subjects for whom this invention envisions treatment via IM delivery. In one exemplary clinical protocol, DMD patients received bilateral injections, with one extensor digitorum brevis (EDB) muscle in each patient receiving the carrier rAAVrh74.MCK.GALGT2, and the other EDB muscle in each patient receiving saline separately. Subjects in the first group received 3 × 10 11 The low-dose carrier (vg, total dose). Subjects in the second group received a higher dose of 1×10. 12 The carrier of vg (total dose).

[0067] Prepare an appropriate carrier diluent before transporting it to the clinical setting. Dilute the injectable diluent 1:1 with physiological saline. Keep the carrier on ice (do not freeze) until administration and administer to the subject within 8 hours of preparation. Handling of rAAVrh74.MCK.GALGT2 follows compliance standards for biosafety level 1 carriers. See www4.od.nih.gov / oba / RAC / guidelines_02 / APPENDIX_G.htm#_Toc7246561.

[0068] Clinical examination revealed myasthenia gravis in the subjects. The genetic diagnosis of DMD was established based on a DMD gene mutation consistent with the condition, within an appropriate clinical history. In this study, all subjects were immobile and had lost walking within the age range for DMD diagnosis (i.e., less than 12 years old without steroid treatment or less than 15 years old with long-term steroid treatment). Subjects received 12 weeks of stable-dose corticosteroid treatment (prednisone or defcoccus or their common forms) prior to treatment.

[0069] The vector or control was delivered directly intramuscularly to the extensor digitorum brevis (EDB) muscle of one foot of the subject, while the other foot received saline alone. Awake sedation was used in participants under 12 years of age. Patients 12 years of age and older may receive awake sedation or a sedative (such as chlorpheniramine) at least 1 hour prior to gene transfer. Additionally, the skin at the gene transfer site was pretreated with a lidocaine / prilocaine eutectic mixture in a cream matrix (EMLA cream) or cellulose disc (EMLA patch). Comparable cream-based anesthesia, such as lidocaine cream, may be used. The procedure was performed under sterile conditions. The injection site was cleaned three times consecutively with iodine-free surgical preparation swabs and covered with a disposable sterile drape. Standard clinical Doppler ultrasound and a sterile sheath around the transducer were used to maintain sterility of the injection area. To improve intramuscular injection accuracy by administering rAAVrh74.MCK.GALGT2 or placebo to the EDB carriers, a disposable MyoJect needle was used for simultaneous electromyography (EMG) recording and fluid injection. The anatomical midline of the muscle was identified on the skin, and 2–6 individual carrier injections were distributed throughout the muscle. The injections were performed 0.5 cm from the muscle surface. The total carrier dose was 3 × 10⁻⁶ in the low-dose group. 11 Vg per 1.5 ml, in the high-dose group it was 1×10 12 vg per 1.5 ml. The proximal and distal extent of vector delivery, as determined by ultrasound, was marked with a non-removable radiographic marker for reference in muscle biopsy after gene transfer.

[0070] Subjects underwent close monitoring of their vital signs. Post-injection monitoring and recording of the combined medication regimen were also conducted. Subjects were discharged two days after gene transfer (if no side effects were observed).

[0071] Subjects returned for follow-up. Muscle biopsies were performed on day 45 or 90. Immunological studies at days 45 and 75 post-transfer, and at 9, 12, 18, and 24 months, included detection of rAAVrh74-binding antibodies and GALGT2 antibodies, as well as ELISpot to detect T-cell responses to the capsid antigen. At the end of the first and second years, subjects underwent physical examinations, strength tests, and immunological studies.

[0072] Example 3 Efficacy Result Measurement Muscle biopsies were taken from EDB muscle. Samples were coded to maintain blinding in all subsequent analyses involving injection of rAAVrh74.MCK.GALGT2.

[0073] Efficacy assays included: GALGT2 expression confirmed by anti-CT epitope antibodies; GALGT2 protein expression quantified by Western blot and assessed by densitometry; transduction efficiency of vector genome expression measured by qPCR from muscle GALGT transgenes and normalized to single-copy genome controls; comparison of the number of fibers containing a central nucleus in muscle using paired t-tests; and analyses also included: expression of dystrophin proteins (with antibodies targeting the N-terminal, C-terminal, and rod domains), expression of myotrophic-associated proteins, and leukocyte markers including CD45, CD3, CD4, CD8, and MAC 387. The histological appearance of the muscle was examined. Antibodies against rAAVrh74 capsids and GALGT proteins were evaluated using rAAVrh74 and PBMCELISpots at different time points during the two-year study period. Muscle analyses of gene expression and inflammation were also performed without destroying blinded samples.

[0074] Transgenic expression was blindly compared between two EDB muscles from individual subjects and between biopsies from subjects at day 45 or 90. A vector-specific primer-probe set was used to amplify the unique 5' untranslated leader sequence of the transgene, which would distinguish transgene expressing GALGT2 protein from endogenous GALGT2. Protein quantification was performed using direct immunofluorescence (IF) and Western blotting (WB) studies of muscle tissue. CD4+ and CD8+ monocytes were quantified by immunostaining and reported as cell number / mm² area. MHCII and MHCII antigen expression was assessed on muscle sections. Muscle morphology measurements, including fiber size histograms and centrifugation quantification, were also performed. PCR analysis of viral DNA was also included in the analysis.

[0075] Immune response was assessed using IFN-γ ELISApots of GALGT2 and AAV capsid. Elevated IFN-γ levels > 2 SD were significant for both viruses and transgenes. AAV binding antibody assays were another measure of immune response.

[0076] Measurements used to improve one or more of the following indicators are considered: absolute muscle strength; force decay during eccentric muscle contraction; serum CK level; serum cardiac troponin level; serum MMP9 level; grip strength; limb torque; limb range of motion or flexibility; walking; 6-minute walk test; knee flexor or extensor strength; maximal isometric voluntary muscle contraction; North Star movement assessment; muscle mass, fat loss, or edema as measured by T2-weighted MRI of the limbs; muscle contractures; limb joint angles; cardiac function (heart rate, cardiac output, shortening percentage, stroke volume); respiration (including respiratory rate, blood oxygenation, supplemental oxygen requirement); muscle necrosis; muscle regeneration; muscle atrophy; muscle inflammation; muscle calcification; muscle centronucleation; muscle size or muscle fiber size; lifespan; and expression of dystrophin or laminin α2 substitute proteins (myotrophic proteins, reticulin 1, laminin α5, and aggregates). See, for example, the literature [Forbes et al.]. Radiology, 269 (1): 198-207(2013); Govoni et al. Cell Mol. Life Sci., 70 (23): 4585-4602 (2013); and Chandrasekharan and Martin, Methods Enzymol., 479 : 291-322 (2010)].

[0077] For each measurement, a paired t-test was used to perform statistical analysis based on the differences between the gene transfer examinations (clinical or muscle biopsy) before and after, with p-values ​​< 0.05 indicating significance.

[0078] Example 4 Vascular delivery of limb-isolated perfusion A vascular delivery route known as limb-isolated perfusion (ILP) is also being considered for the treatment of human patients. ILP can be delivered to multiple leg muscles via the femoral artery. This method allows for limb isolation from systemic circulation, increases transduction efficiency, and prevents viral escape into systemic circulation [Rodino-Klapac et al., Mol. Ther [., 18: 109-117 (2010)]. An exemplary clinical protocol is listed below.

[0079] rAAVrh74.MCK.GALGT2 was prepared as described in Examples 1 and 2.

[0080] The subjects received a stable dose of corticosteroids (prednisone or defcoccus or their general form) for 12 weeks prior to treatment. Prednisone treatment continued after gene transfer. Sedated and anesthetized subjects were secured to the operating table. Loose tourniquets were applied proximally and distally above the rhesus monkey's knee and below the gastrocnemius muscle. A small incision was made in the femoral triangle, the femoral artery was identified and freely dissected, and ligated proximally and distally to control bleeding and facilitate catheter introduction. Using a modified Seldinger method, a pre-flushed cannula was passed through the suture previously placed in the artery, and the femoral artery was inserted into a 3.0 Fr catheter sheath. The sheath was advanced only a few centimeters and secured with 3.0 braided sutures.

[0081] After the sheath is placed in the femoral artery and vein, administer 100-200 u / kg of ungraded heparin and allow it to circulate for 3-5 minutes. The Choice PT coronary guidewire is first placed in the right femoral vein and artery, then finally into the left femoral vein and artery. A 4 mm diameter Tyshak mini-balloon catheter is inserted through the right femoral artery into the femoral-iliac artery junction via a 3.3-French sheath. An 8 mm diameter, 2 cm long Tyshak mini-balloon catheter is inserted through a 4-French sheath to the appropriate position at the right femoral-iliac vein junction. A small hand injection of diluted control is performed to confirm proper occlusion of the left femoral artery and right femoral vein. If necessary, the sheath and balloon can be replaced with larger sizes. For example, the 4-French sheath in the right femoral vein can be replaced with a 6-French sheath, and a 12 mm × 2 cm long Tyshak II balloon catheter can be inserted through the Choice PT guidewire to the appropriate position. A small hand injection is performed through the sheath's lateral arm to confirm the location and complete occlusion of the femoral vein.

[0082] After inflating balloons in the right femoral artery and femoral vein, the right leg was isolated, and a pre-flush solution of 2 mL / kg Ringer's lactate heparinized solution was infused. After 1 minute, the 2 mL / kg Ringer's lactate flush was completed. Next, over 1–1 / 2 minutes, 2 × 10⁻⁶… 12 vg / kg / limb up to 4.8×10 13 The dose infusion volume was 8 mL / kg / limb, administered via the rAAVrh74.MCK.GALGT2 vector (bilateral limb perfusion resulted in a total patient dose of 4 × 10⁻⁶). 12 vg / kg and 9.6×10 13 (between vg / kg). After delivery of rAAVrh74.MCK.GALGT2, the residence time was 10 minutes, followed by infusion of 2 mL / kg heparinized Ringer's lactate through the right femoral artery sheath over 1 minute. The balloon was then deflated, and the catheter and guidewire were removed.

[0083] The left leg was then targeted with the same procedure. The left femoral artery was maintained in a 3.3-French sheath. Similarly, proper occlusion was demonstrated using a 4 mm diameter Tyshak miniballoon catheter in the left femoral artery via a Choice PT coronary guidewire and a 12 mm × 2 cm long Tyshak miniballoon catheter in a 5-French sheath at pressures up to 3 atmospheres through the left femoral vein. The infusion protocol was repeated, administering 2 mL / kg of heparinized Ringer's lactate over 1 minute, followed by 2 × 10⁻⁶ doses over 1 minute and 15 seconds. 12 vg / kg / limb up to 4.8×10 13 A dose of rAAVrh74.MCK.GALGT2 was administered at a dose of vg / kg / limb, with a residence time of 10 minutes. Finally, 2 mL / kg of heparinized Ringer's lactate was infused through a left femoral artery sheath, and the sheath was then removed from all four adjacent sites using compression hemostasis and HemCon patches.

[0084] As needed, variations of this protocol can be used to deliver rAAVrh74.MCK.GALGT2 to alternative muscle groups via other arteries. For example, delivery via the diaphragmatic artery, intercostal artery, and / or subcostal artery to supply the diaphragm, or delivery via the coronary artery to supply the heart, can be considered. Each procedure can be performed with similar dosages, and multiple procedures can be performed on a single patient or even at the time of admission.

[0085] Upon completion of administration, remove the tourniquet and catheter and apply direct pressure to the wound for 10 minutes to control bleeding. Close the wound with continuous subcutaneous 4.0 Vicryl sutures. Apply a pressure dressing to the site and hold it in place until the subject awakens from anesthesia.

[0086] Following the ILP delivery protocol, subject follow-up and efficacy outcome measurements / analyses were performed similarly to those for the IM treatment described above.

[0087] Example 5 Systemic vascular delivery Another route to deliver the rAAVrh74.MCK.GALGT2 carrier to muscle is systemic vascular delivery. An exemplary dose-escalation study to examine efficacy could be conducted as follows.

[0088] Determination of dosage range At 1 day of age, administer 1.4 × 10⁻⁶ mmol / L (via tail vein). 15vg / kg rAAVrh74.MCK.GALGT2IV resulted in over 90% transduction of all limb skeletal muscle in wild-type mice, including the tibialis anterior, gastrocnemius, quadriceps, and triceps, and similarly over 50% transduction of all cardiomyocytes in the wild-type mouse heart and over 70% of cardiomyocytes in the MDX model mouse heart. Notably, analysis of overall cardiac function in MDX mice after 3 months of treatment, compared to the treatment-mimicking MDX control animals, showed that cardiac output was almost doubled due to treatment with this dose of rAAVrh74.MCK.GALGT2, with or without dobutamine (a β-agonist that stimulates heart rate). Therefore, when the carrier was administered before the onset of disease-related cardiopathic events, rAAVrh74.MCK.GALGT2 treatment increased blood flow in dystrophin-deficient hearts by 80%. 5 × 10 15 vg / kg is considered the maximum therapeutic dose for transducing all cardiac and skeletal muscle cells throughout the body via intravenous injection. Therefore, approximately 5 × 10⁻⁶ is considered. 13 vg / kg to approximately 5×10 15 The vg / kg dosage range will cover the lowest and best effective doses for whole-patient treatment with rAAVrh74.MCK.GALGT2 in the clinical IV study.

[0089] plan rAAVrh74.MCK.GALGT2 was prepared as described in Examples 1 and 2.

[0090] One day prior to administration of rAAVrh74.MCK.GALGT2, the subjects began receiving prophylactic enteral prednisone (a glucocorticoid) (approximately 1 mg / kg / day). Prednisone treatment continued after gene transfer.

[0091] On the day of gene transfer (day 0) prior to the infusion of rAAVrh74.MCK.GALGT2, a physical examination was conducted to collect data from vital organs that sustain life.

[0092] If the subject is deemed inadequately hydrated by the PI, a pill containing 10-20 mL / kg of normal saline may be administered during the time between admission and gene transfer. Subjects should maintain a normal diet for 8 hours prior to gene transfer, after which they should not consume solid food. Clear fluid intake is permitted 2 hours prior to gene transfer, after which they will be completely fasted. Normal oral feeding will resume once they return to their pre-sedated baseline. Gene transfer will be performed under sterile conditions with mild to moderate sedation under the guidance of a qualified anesthesiologist. Sedation may vary, but subjects may be sedated with inhaled nitrous oxide and a continuous inhalation of sevoflurane or propofol infusion prior to propofol administration via intravenous injection. Sedation may be delayed for subjects in whom the PI deems sedation unnecessary for safe delivery of the carrier (in consultation with the anesthesiologist).

[0093] All subjects in the trial received intravenous injections of rAAVrh74.MCK.GALGT2 via peripheral limb veins. The considered dose range was 5 × 10⁻⁶. 13 vg / kg and 5×10 15 Between vg / kg.

[0094] As an example, each carrier dose was undiluted and aliquoted into 50 mL or less portions for use in 60 mL Becton Dickinson polypropylene syringes prepared by NCH Investigational Drug Pharmacy. The carrier saline solution was approximately 400 mOsmol / L. Infusion was administered using a Smiths Medical Medfusion 4000 syringe infusion pump equipped with the PharmGuard infusion management software suite, delivered via Smiths Medical MX563 infusion tubing. The infusion rate for any subject must not exceed 2 mL / kg / min. The infusion was administered over approximately 10 to 20 minutes. At the end of the infusion, the carrier was flushed from the infusion tubing with normal saline. It is considered that the carrier dose could be separated and administered separately as needed.

[0095] Closely monitor subjects for side effects during infusion, including continuous monitoring of heart rate, respiratory rate, and pulse oxygen saturation; and intermittent blood pressure monitoring. Heart rate, respiratory rate, pulse oxygen saturation, temperature, and blood pressure should be measured immediately before and after infusion, every five minutes during infusion, and repeated 15 minutes after infusion.

[0096] Subjects remained in the intensive care unit (ICU) bed after gene transfer and were hospitalized for 48 hours post-transfer. Vital signs were monitored hourly for the first 4 hours after injection, then every 4 hours until discharge. If the prognosticator (PI) was satisfactory, subjects could be transferred out of the ICU after the initial 24 hours of monitoring following infusion.

[0097] Following the delivery protocol, subject follow-up and efficacy outcome analysis were conducted similar to those for subjects receiving the IM treatment described above.

[0098] Although the invention has been described with reference to specific embodiments, it should be understood that many changes and modifications will occur to those skilled in the art. Therefore, only the limitations set forth in the claims should be applied to the invention.

[0099] All references cited in this application are incorporated herein by reference in their entirety, with particular attention paid to their contents. Furthermore, this application claims priority to U.S. Provisional Application No. 62 / 220,107, filed September 17, 2015; No. 62 / 221,068, filed September 20, 2015; and No. 62 / 301,260, filed February 2016; the entire contents of which are incorporated herein by reference.

Claims

1. A recombinant adeno-associated virus (rAAV) particle containing nucleic acid containing SEQ ID NO:

2.

2. The rAAV particle according to claim 1, wherein the rAAV is derived from serum types AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11 or AAVrh.

74.

3. The rAAV particle according to claim 2, wherein the rAAV is derived from AAVrh.

74.

4. The rAAV particle according to claim 3, wherein the polynucleotide sequence of the AAV rh.74 capsid is listed in SEQ ID NO:

1.

5. A composition comprising rAAV particles according to any one of claims 1-4 and a pharmaceutically acceptable carrier.

6. Use of the composition according to claim 5 in the preparation of a medicament for treating or preventing neuromuscular diseases.

7. Use of the composition according to claim 6, wherein the neuromuscular disease is Duchenne muscular dystrophy (DMD); Becker muscular dystrophy; congenital muscular dystrophy (MDC) types 1A, 1B, 1C, and 1D; limb-girdle muscular dystrophy (LGMD) types 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, and 2Q; Bethlem myopathy; Ullrich congenital muscular dystrophy; myo-ocular-encephalopathy; Fukuyama type congenital muscular dystrophy; Walker-Warberg syndrome; myotonic dystrophy; myasthenia gravis syndrome; congenital myasthenia gravis; inclusion body myopathy; inclusion body myositis; Emery-Dreifuss muscular dystrophy; distal muscular dystrophy; dermatomyositis; central nucleus myopathy; facial-scapular-humeral muscular dystrophy; Myoshi myopathy; mitochondrial myopathy; linear myopathy; Nonaka myopathy; myasthenia gravis; And polymyositis.

8. The use according to claim 6 or claim 7, wherein i) The composition is formulated for intramuscular (IM), limb-isolated infusion or perfusion (ILP), or intravenous (IV) administration. ii) The composition is formulated for IM administration. iii) The composition is formulated for intramural administration and contains approximately 3 × 10 11 Up to at least approximately 5×10 12 vg / injected rAAV dose, iv) The composition is formulated for infusion or ILP administration. v) The composition is formulated for infusion or ILP administration and contains approximately 2 × 10 12 Up to at least approximately 4.8 × 10 13 The rAAV dose is vg / kg / limb, or approximately 4 × 10⁻⁶. 12 Up to at least approximately 9.6 × 10 13 Total dose, vi) The composition is formulated for intravenous administration. vii) The composition is formulated for intravenous administration and contains approximately 2 × 10 14 Up to at least approximately 6×10 15 The rAAV dose of vg / kg, or viii) The composition contains approximately 5 × 10 13 vg / kg to approximately 5×10 15 rAAV dose of vg / kg.

Images