Materials and methods for the treatment of disorders associated with the IGHMBP2 gene

Abstract

The present disclosure provides gene therapy vectors, such as adeno-associated viruses (AAVs), designed for the treatment of immunoglobulin-μ binding protein 2 (IGHMBP2)-associated disorders. In one aspect, described herein is a polynucleotide comprising (a) one or more regulatory control elements and (b) a cDNA sequence for immunoglobulin-μ binding protein 2 (IGHMBP2). In some embodiments, the regulatory control element is a CBA promoter comprising the nucleotide sequence set forth in SEQ ID NO:3, or a P546 promoter comprising the nucleotide sequence set forth in SEQ ID NO:4, or a fragment thereof that retains regulatory control or promoter activity.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 62 / 939,270, filed on November 22, 2019, the entire contents of which are incorporated herein by reference.

[0002] Incorporation by reference of electronically submitted materials This application contains a computer-readable form of a sequence listing, identified as 54445_Seqlisting.txt, size: 73,110 bytes, created on November 23, 2019, which is incorporated herein by reference in its entirety as a separate part of this disclosure.

[0003] Field of the Invention The present disclosure provides gene therapy vectors, such as adeno-associated virus (AAV), designed for the treatment of disorders associated with mutations in the immunoglobulin - μ - binding protein 2 (IGHMBP2) gene. The disclosed rAAV provides wild-type IGHMBP2 cDNA, which results in the expression of the wild-type protein, to a subject in need thereof.

Background Art

[0004] The immunoglobulin - μ - binding protein 2 (IGHMBP2) gene encodes a component of the Upf1 - like group within the helicase superfamily 1 (SF1). This protein is known to have a helicase domain, an R3H domain, a zinc - finger domain, and a nuclear localization signal sequence. IGHMBP2 is ubiquitously expressed and contains 15 exons that encode 993 amino acids corresponding to a 110 kDa gene product. The exact role of the IGHMBP2 protein in disease development is unknown. Normal IGHMBP2 is known to play a role in ribosomal RNA maturation and translation, immunoglobulin class switching, mRNA precursor maturation, and transcriptional regulation either by DNA - binding activity or interaction with the TATA - binding protein. The IGHMPB2 protein is classified as a component of the Upf1 - like group within the helicase superfamily 1 (SF1) consisting of a helicase domain, an R3H domain, a zinc - finger domain, and a nuclear localization signal sequence. Autosomal recessive mutations in the IGHMPB2 gene are known to cause spinal muscular atrophy with respiratory distress type 1 (SMARD1) and Charcot - Marie - Tooth type 2S (CMT2S). Most of the patient mutations in the IGHMPB2 gene cluster within the helicase domain and are missense mutations.

[0005] SMARD1 is an autosomal recessive motor neuron disease characterized by early distal lower limb muscle atrophy following proximal muscle weakness and respiratory insufficiency. SMARD1 patients show diaphragmatic paralysis between 6 weeks and 13 months of age. Patients usually require ventilation before 13 months of age. Loss - of - function mutations in the IGHMBP2 gene are known to cause SMARD1.

[0006] Charcot-Marie-Tooth (CMT) neuropathy is the most commonly seen hereditary neuropathy. CMT2 is an axonal (non-demyelinating) peripheral neuropathy characterized by distal muscle weakness and atrophy, mild sensory loss, and normal or near-normal nerve conduction velocity. CMT2 is clinically similar to CMT1 but is typically less severe. Patients have slowly progressive distal muscle weakness with muscle atrophy in the upper and lower extremities. Subtypes of CMT2 are clinically similar and are distinguished only by molecular genetic findings. Most subtypes of CMT2 are inherited in an autosomal dominant pattern, although some are inherited in an autosomal recessive pattern. A loss-of-function mutation of the IGHMBP2 gene is known to cause CMT2, which is currently subclassified as CMT2S.

[0007] There is currently no treatment for CMT2S, and management involves treating the symptoms. Therefore, there is a need to develop gene replacement therapies for treating SMARD1 and CMT2S.

Summary of the Invention

[0008] In one aspect, a polynucleotide is described herein that comprises (a) one or more regulatory elements and (b) the cDNA sequence of immunoglobulin - μ - binding protein 2 (IGHMBP2). In some embodiments, the regulatory element is a CBA promoter comprising the nucleotide sequence set forth in SEQ ID NO: 3, or a P546 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 4, or a fragment thereof that retains regulatory control or promoter activity. In some embodiments, the vector comprises an SV40 intron having the nucleotide sequence of SEQ ID NO: 5 and a fragment of the SV40 intron. In some embodiments, the IGHMBP2 cDNA comprises the polynucleotide sequence set forth in SEQ ID NO: 1.

[0009] In one embodiment, the present disclosure provides an rAAV comprising a nucleotide sequence encoding a functional IGHMBP2 protein, wherein the nucleotide has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% sequence identity to SEQ ID NO: 1, more typically at least 90%, 91%, 92%, 93%, or 94%, even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and the protein retains IGHMBP2 activity. For example, the nucleotide sequence encoding the functional IGHMBP2 protein may include one or more base pair substitutions, deletions, or insertions that affect the function of IGHMBP2. Further, the nucleotide sequence encoding the functional IGHMBP2 protein may include one or more base pair substitutions, deletions, or insertions that may increase or decrease the expression of the IGHMBP2 protein, and this change in the expression pattern may be desired for the treatment of IGHMBP2-related disorders such as SMARD1 or CMT2S.

[0010] For example, the present disclosure provides an rAAV comprising a nucleotide sequence encoding a functional IGHMBP2 protein, wherein the protein comprises an amino acid sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% sequence identity to SEQ ID NO: 2, more typically at least 90%, 91%, 92%, 93%, or 94%, even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and the protein retains IGHMBP2 activity. For example, the nucleotide sequence encoding the functional IGHMBP2 protein may include one or more amino acid substitutions, deletions, or insertions that affect the function of the IGHMBP2 protein.

[0011] The terms "sequence identity", "percentage of sequence identity", or "percent identical" in the context of a nucleic acid sequence or an amino acid sequence refer to residues in two sequences that are the same when aligned to maximize correspondence. The length of the sequence identity comparison can be the full length of the genome, the full length of the gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides is desired. However, identity between smaller fragments can also be desired, such as at least about 9 nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides. The percentage of sequence identity can be determined by techniques known in the art. For example, homology can be determined by direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs such as ALIGN, ClustalW2, and BLAST. In one embodiment, when BLAST is used as the alignment tool, the following default parameters are used: genetic code = standard, filter = none, strand = both, cutoff = 60, expect = 10, matrix = BLOSUM62, descriptions = 50 sequences, sort = high score, database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR.

[0012] In another aspect, the present disclosure provides an rAAV construct contained in a plasmid comprising the nucleotide sequence of SEQ ID NO: 7. For example, the ssAAV9.CB.IGHMBP2 vector is within and includes the ITR of SEQ ID NO: 7 and comprises the nucleotide sequence shown in FIG. 13. The rAAV vector comprises a 5' ITR, CMV enhancer, CB promoter, modified SV40 intron sequence, coding sequence for the human IGHMBP2 gene, bGH polyA, and 3' ITR. In one embodiment, the vector comprises nucleotides 1 to 4397 of SEQ ID NO: 7. The nucleotides within the ITR can be in the forward or reverse orientation. For example, the CMV enhancer sequence, CB promoter sequence, SV40 sequence, human IGHMBP2 gene sequence, and bGH polyA sequence can be in the forward or reverse orientation. In another embodiment, the vector comprises a nucleotide sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to nucleotides 1 to 4397 of SEQ ID NO: 7. The plasmid described in SEQ ID NO: 7 further comprises kanamycin resistance and a pUC origin of replication.

[0013] In an exemplary embodiment, the present disclosure provides an rAAV construct contained in a plasmid comprising the nucleotide sequence of SEQ ID NO: 18. For example, the ssAAV9.CB.IGHMBP2-clinical vector is within and includes the ITR of SEQ ID NO: 18 and comprises the nucleotide sequence shown in FIG. 18. The rAAV vector comprises the 5’ ITR set forth in SEQ ID NO: 19. In addition, the rAAV vector comprises, in the reverse direction respectively, a CMV enhancer, a CB promoter, a modified SV40 intron sequence, a coding sequence for the human IGHMBP2 gene, bGH polyA, and the 3’ ITR set forth in SEQ ID NO: 12. In one embodiment, the vector comprises nucleotides 1 to 4386 of SEQ ID NO: 18. The nucleotides within the ITR can be in the forward or reverse direction. In another embodiment, the vector comprises a nucleotide sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to nucleotides 1 to 4386 of SEQ ID NO: 18. The plasmid set forth in SEQ ID NO: 18 further comprises a kanamycin resistance gene and a pUC origin of replication. The kanamycin resistance gene can be in the forward or reverse direction.

[0014] In a further aspect, the present disclosure provides an rAAV construct contained in a plasmid comprising the nucleotide sequence of SEQ ID NO: 8. For example, the ssAAV9.P546.IGHMBP2 vector is within and includes the ITR of SEQ ID NO: 8 and comprises the nucleotide sequence shown in FIG. 14. The rAAV vector comprises a 5' ITR, a P546 promoter, a modified SV40 intron sequence, a coding sequence for the human IGHMBP2 gene, bGH polyA, and a 3' ITR. In one embodiment, the vector comprises nucleotides 1 to 4375 of SEQ ID NO: 8. The nucleotides within the ITR can be in the forward or reverse orientation. For example, the P546 promoter sequence, the SV40 sequence, the human IGHMBP2 gene, and the bGH polyA sequence can be in the forward or reverse orientation. In another embodiment, the vector comprises a nucleotide sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to nucleotides 1 to 4397 of SEQ ID NO: 8. The plasmid described in SEQ ID NO: 8 further comprises kanamycin resistance and a pUC origin of replication.

[0015] In an exemplary embodiment, the present disclosure provides an rAAV construct contained in a plasmid comprising the nucleotide sequence of SEQ ID NO: 17. For example, the ssAAV9.P546.IGHMBP2-clinical vector is within and includes the ITR of SEQ ID NO: 17 and comprises the nucleotide sequence shown in FIG. 16. The rAAV vector comprises the 5' ITR set forth in SEQ ID NO: 19. In addition, the rAAV vector comprises, in the reverse direction, the P546 promoter sequence, a modified SV40 intron sequence, the coding sequence for the human IGHMBP2 gene, and bGH polyA, as well as the 3' ITR set forth in SEQ ID NO: 12. In one embodiment, the vector comprises nucleotides 1 to 4364 of SEQ ID NO: 17. The nucleotides within the ITR can be in the forward or reverse direction. In another embodiment, the vector comprises a nucleotide sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to nucleotides 1 to 4364 of SEQ ID NO: 17. The plasmid set forth in SEQ ID NO: 17 further comprises a kanamycin resistance gene and a pUC origin of replication. The kanamycin resistance gene can be in the forward or reverse direction.

[0016] In another aspect, a recombinant adeno-associated virus (rAAV) having a genome comprising the polynucleotide sequences described herein is described herein. In some embodiments, the rAAV is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, or Anc80, AAV7m8, and derivatives thereof. In some embodiments, the genome of the rAAV comprises a promoter fragment and IGHMBP2 cDNA.

[0017] In some embodiments, the genome of the rAAV comprises a CBA promoter and an IGHMBP2 cDNA. Exemplary genomes include a CBA promoter and an IGHMBP2 cDNA such as rAAV described as nucleotides 1 to 4397 of SEQ ID NO: 7, or rAAV described as nucleotides 1 to 4386 of SEQ ID NO: 18, ssAAV9.CB.IGHMBP2.

[0018] In some embodiments, the genome of the rAAV comprises a P546 promoter and an IGHMBP2 cDNA. Exemplary genomes include a P546 promoter and an IGHMBP2 cDNA such as ssAAV9.P546.IGHMBP2, and rAAV described as nucleotides 1 to 4375 of SEQ ID NO: 8, or rAAV described as nucleotides 1 to 4364 of SEQ ID NO: 17.

[0019] In some embodiments, the genome of the rAAV comprises a fragment of the CBA promoter or a fragment of the P546 promoter and an IGHMBP2 cDNA, and the fragment of the promoter retains promoter activity.

[0020] In another aspect, rAAV particles comprising the rAAV described herein are described herein.

[0021] A composition comprising any of the rAAV described herein or any of the viral particles described herein. In some embodiments, the composition further comprises an agent that increases the viscosity and / or density of the composition. For example, in some embodiments, the agent is a contrast agent. The contrast agent can be a non-ionic low-osmolar compound or contrast agent of about 20-40%, or about 25%-about 35% of a non-ionic low-osmolar compound such as iohexol. The disclosed compositions can be formulated for any delivery means such as direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery, or intravenous delivery.

[0022] In some embodiments, the composition comprises an agent that increases the viscosity of the composition by about 0.05%, or about 1%, or 1.5%, or about 2%, or about 2.5%, or about 3% or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%. In some embodiments, the agent increases the viscosity of the composition by about 1% to about 5%, or about 2% to 12%, or about 5% to about 10%, or about 1% to about 20%, or about 10% to about 20%, or about 10% to about 30%, or about 20% to about 40%, or about 20% to about 50%, or about 10% to about 50%, or about 1% to about 50%.

[0023] In some embodiments, the composition comprises an agent that increases the density of the composition by about 0.05%, or about 1%, or 1.5%, or about 2%, or about 2.5%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%. In some embodiments, the agent increases the density of the composition by about 1% to about 5%, or about 2% to 12%, or about 5% to about 10%, or about 1% to about 20% or about 10% to about 20%, or about 10% to about 30%, or about 20% to about 40% or about 20% to about 50%, or about 10% to about 50%, or about 1% to about 50%.

[0024] For example, the disclosed composition is formulated for intrathecal delivery and comprises a dose of about 1e13 vg per patient to about 1e15 vg per patient of rAAV or rAAV particles.

[0025] In addition, the disclosed composition is formulated for intravenous delivery and comprises a dose of about 1e13 vg / kg to about 2e14 vg / kg of rAAV or rAAV particles.

[0026] Particularly contemplated is a method of treating an IGHMBP2-related disorder in a subject in need thereof, comprising administering an rAAV or rAAV particle as described herein. In some embodiments, the method further comprises administering an immunosuppressive agent before, after, or simultaneously with the rAAV or rAAV particle. IGHMPB2-related disorders include disorders or diseases caused by mutations that result in loss of function of the IGHMPB2 protein or cause a reduction in the expression of the IGHMPB2 protein. An IGHMPB2-related disorder can be any disease or disorder associated with a reduction in the expression or activity of the IGHMPB2 protein, despite the cause of the reduction in expression or activity. The present disclosure contemplates an IGHMPB2-related disorder in a subject who is homozygous for a mutation in the IGHMPB2 gene or heterozygous for a mutation in the IGHMPB2 gene. For example, an IGHMBP2-related disorder is a neurological disorder associated with the presence of a mutation in the IGHMBP2 gene such as SMARD1 or CMT2S. IGHMBP2-related disorders also include disorders in which the patient has a mixed phenotype, such that the severity of the neurological disorder is between that observed in patients affected by SMARD1 and CMT2S.

[0027] In any of the methods, the subject has a mutation in the IGHMBP2 gene. These mutations include those currently known, such as those shown in Table 1 or 2 herein, or mutations in the IGHMBP2 gene that will be identified in the future and are associated with neurological disorders.

[0028] As used herein, "subject" can be any animal and can also be referred to as a patient. Preferably, the subject is a vertebrate, and more preferably, the subject is a mammal such as a farm livestock (e.g., cows, horses, pigs) or a pet (e.g., dogs, cats). In some embodiments, the subject is a human. In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is a pediatric subject such as, for example, a subject ranging in age from 1 to 10 years. In some embodiments, the subject is 4 to 15 years old. In one embodiment, the subject is an adolescent subject such as, for example, a subject ranging in age from 10 to 19 years. In other embodiments, the subject is an adult (18 years or older). In any of the disclosed methods, rAAV or viral particles are delivered by direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery, or intravenous delivery. For example, in any of the methods, a dose of rAAV or rAAV particles from about 1e13 vg per patient to about 1e15 vg per patient is administered to the subject by intrathecal delivery. Additionally, in any of the disclosed methods, a dose of rAAV or rAAV particles from about 1e13 vg / kg to about 2e14 vg / kg is administered to the subject by intravenous delivery.

[0029] In another aspect, the use of the rAAV or rAAV particles described herein in the preparation of a medicament for the treatment of an IGHMBP2-related disorder such as SMARD1 or CMT2S is described herein. For example, any of the disclosed medicaments are formulated for direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery, or intravenous delivery. For example, the medicament contains a dose of rAAV or rAAV particles from about 1e13 vg per patient to about 1e15 vg per patient and is administered to the subject by intrathecal delivery. Additionally, the medicament contains a dose of rAAV or rAAV particles from about 1e13 vg / kg to about 2e14 vg / kg and is administered to the subject by intravenous delivery. In some embodiments, the medicament is administered before, after, or simultaneously with the administration of an immunosuppressant.

[0030] In another aspect, compositions comprising the rAAV or rAAV particles described herein for the treatment of IGHMBP2-related disorders such as SMARD1 or CMT2S are described herein. For example, any of the disclosed compositions are formulated for direct injection into cerebrospinal fluid, intraventricular delivery, intrathecal delivery, or intravenous delivery. For example, the composition comprises a dose of about 1e13 vg to about 1e15 vg of rAAV or rAAV particles per patient and is administered to a subject by intrathecal delivery. In addition, the composition comprises a dose of about 1e13 vg / kg to about 2e14 vg / kg of rAAV or rAAV particles and is administered to a subject by intravenous delivery. In some embodiments, the composition is administered before, after, or simultaneously with the administration of an immunosuppressant. In another embodiment, the composition further comprises an immunosuppressant.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0032] The immunoglobulin - μ - binding protein 2 (IGHMBP2) gene encodes a protein that is a component of the Upf1 - like group within the helicase superfamily 1 (SF1), which consists of a helicase domain, an R3H domain, a zinc finger domain, and a nuclear localization signal sequence. Mutations in the IGHMPB2 gene are known to cause spinal muscular atrophy associated with spinal muscular atrophy with respiratory distress type 1 (SMARD1) and Charcot - Marie - Tooth disease type 2S (CMT2S). Most of the patient mutations in the IGHMPB2 gene cluster within the helicase domain and are missense mutations.

[0033] IGHMPB2 mutations The wild-type cDNA sequence of IGHMPB2 is set forth in SEQ ID NO: 1 (Genbank NM_002180.2), and the IGHMPB2 protein, also known as DNA-binding protein SMUB-2, is set forth in SEQ ID NO: 2 (Genbank NP_002171.2). The wild-type gene product is a 993-amino acid protein with seven putative helicase motifs and a DEAD box-like motif typical of RNA helicases. Mutations can lead to dysfunction of helicase activity. The IGHMPB2 gene is known to have 15 exons. Mutations in the IGHMPB2 gene have been found to be associated with SMARD1 and CMT2S. There are approximately 26 known IGHMPB2 mutations that cause SMARD1. The mutations include recessive missense mutations, nonsense mutations, frameshifts, in-frame deletions, frameshift insertions, and splice donor site mutations, and span 15 exons of the IGHMPB2 gene (Luan et al., Brain & Dev. 28:685-689, 2016, incorporated herein by reference). Exemplary mutations known to cause SMARD1 are summarized below in Table 1. The disclosed gene therapy vectors and treatment methods are not limited to disorders caused by the mutations provided in Table 1 or those known at the time of filing, as other mutations in IGHMPB2 that cause SMARD1 may be identified in the future. Table 1

Table 1

[0034] Known IGHMPB2 mutations that cause CMT2S are autosomal recessive mutations that cause axonal neuropathy (Cottenie et al., Am J Hum Genet. 2014;95:590-601, Schottmann et al., Neurology. 2015;84:523-31, both incorporated herein by reference). Exemplary mutations known to cause CMT2S are summarized below in Table 2. The disclosed gene therapy vectors and treatment methods are not limited to the disorders caused by the mutations provided in Table 2 or those known at the time of filing, as other mutations in IGHMPB2 that cause CMT2S may be identified in the future.

Table 2-1

[0035] Diagnosis and Progression of SMARD1 SMARD1 is also known as autosomal recessive distal spinal muscular atrophy 1, distal hereditary motor neuropathy type VI (dHMN6 or HMN6), or distal myopathy type 1 (DSMA-1). This disorder is a variant of infantile SMA. The most prominent symptoms of SMARD1 are severe respiratory distress due to diaphragmatic paralysis with visceroptosis shown on chest X-ray, low birth weight of less than 3%, inability to wean, and progressive muscle weakness in the upper limbs, and the distal muscles are also affected. Additional symptoms include low motor nerve conduction velocity and reduction in the size of myelinated fibers on sural nerve biopsy. Sensory and autonomic nerves are also affected in some patients, as evidenced by reduced pain perception, hyperhidrosis, constipation, and bladder incontinence. Clinical features include intrauterine fetal growth retardation, prematurity, weak cry, and foot deformities. Symptoms usually appear between 1 month and 6 months of age.

[0036] Diagnosis and Progression of Charcot-Marie-Tooth Hereditary Neuropathy 2 (CMT2) CMT2 is a progressive peripheral motor and sensory neuropathy, and is generally diagnosed by measuring one or more of: i) nerve conduction velocity (NCV) that is within the normal range (>40 - 50 m / sec), although sometimes within a mildly abnormal range (>30 - 40 m / sec); ii) EMG findings consistent with axonal neuropathy, such as positive waves, polyphasic potentials, or fibrillation, and reduced amplitude of the evoked motor and sensory responses; and iii) substantial reduction of compound muscle action potential (CMAP) and / or (not always) a family history typically consistent with an autosomal recessive pattern.

[0037] Nerve biopsy is not required for diagnosis but can be used to monitor progression or confirm diagnosis. Nerve biopsy shows loss of myelinated fibers with signs of regeneration, axonal sprouting, and atrophic axons with neurofilaments, and larger internodal gaps and shorter internodal lengths compared to controls, suggesting abnormal internodal formation.

[0038] CMT2S more prominently affects motor than sensory nerves, although both are involved. Affected individuals typically have slowly progressive weakness and atrophy of the distal muscles of the feet and / or hands, usually associated with reduced tendon reflexes and mild or no sensory loss. Affected individuals usually become symptomatic between 5 and 25 years of age, although onset can range from infancy with delayed walking to 30 years later. The typical presenting symptom is weakness of the feet and ankles. The first physical finding is reduced or absent tendon reflexes associated with weakness of dorsiflexion of the foot at the ankle.

[0039] Adult patients with CMT2S typically have bilateral foot drop, symmetric atrophy of the muscles below the knees (stork leg appearance), and absent tendon reflexes in the lower extremities. Active tendon reflexes and extensor plantar responses, as well as asymmetric muscle atrophy, have also been reported in up to 15% of affected individuals. Involvement of the vocal cords or phrenic nerve resulting in difficulty with speech or breathing has been observed. In addition, restless leg syndrome and sleep apnea have also been observed.

[0040] AAV gene therapy The present disclosure provides a gene therapy vector that expresses IGHMPB2 cDNA, such as an rAAV vector, and a method of treating an IGHMPB2-related disorder. IGHMPB2-related disorders include disorders caused by mutations that cause loss of function of the IGHMPB2 protein or that cause reduced expression of the IGHMPB2 protein. Further, any disease or disorder associated with reduced expression or activity of the IGHMPB2 protein, regardless of the cause of the reduced expression or activity.

[0041] As used herein, the term "AAV" is a common abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which a specific function is provided by a coinfecting helper virus. Currently, 13 serotypes of AAV exist, and they have been characterized in general information and reviews of AAV that can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, since it is well known that the various serotypes are very closely related both structurally and functionally even at the genetic level, it is fully anticipated that these same principles will apply to additional AAV serotypes. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R. Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes exhibit very similar replication properties mediated by homologous rep genes, and they all have three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis that reveals extensive cross-hybridization between serotypes along the length of the genome and the presence of similar self-annealing segments at the termini corresponding to the "inverted terminal repeats" (ITRs). Similar infectious patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0042] As used herein, an "AAV vector" refers to one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious virus particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0043] As used herein, an "AAV virion," "AAV virus particle," or "AAV vector particle" refers to a virus particle consisting of at least one AAV capsid protein and a polynucleotide AAV vector encapsulated within the capsid. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, since such a vector is contained within an AAV vector particle, production of an AAV vector particle necessarily includes production of an AAV vector.

[0044] Adeno-associated virus (AAV) is a replication-defective parvovirus, and its single-stranded DNA genome is approximately 4.7 kb in length and contains inverted terminal repeats (ITRs). Exemplary ITR sequences can be 130 base pairs in length or 141 base pairs in length, such as the ITR sequences set forth in SEQ ID NOs: 11, 12, and 19. There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45:555-564 (1983), as corrected by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As another example, the complete genome of AAV-1 is provided by GenBank accession number NC_002077, the complete genome of AAV-3 is provided by GenBank accession number NC_1829, the complete genome of AAV-4 is provided by GenBank accession number NC_001829, the AAV-5 genome is provided by GenBank accession number AF085716, the complete genome of AAV-6 is provided by GenBank accession number NC_001862, at least a portion of the genomes of AAV-7 and AAV-8 are provided by GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 regarding AAV-8), the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004), the AAV-10 genome is presented in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45 (2007). Cis-acting sequences that direct viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration are contained within the ITRs.Three AAV promoters (named p5, p19, and p40 relative to their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. In combination with differential splicing of a single AAV intron (e.g., at nucleotides 2107 and 2227 of AAV2), the two rep promoters (p5 and p19) result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. Rep proteins possess multiple enzymatic properties that ultimately participate in viral genome replication. 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 reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0045] AAV has unique features that make it attractive as a vector for delivering foreign DNA into cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV allows the possibility of infecting many mammalian cells and targeting many different tissues in vivo. Additionally, AAV can transduce both slowly dividing and non-dividing cells and persist essentially throughout the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in a plasmid that enables the construction of recombinant genomes. Further, since the signals that direct AAV replication, genome capsid formation, and integration are contained within the ITRs of the AAV genome, some or all of the approximately 4.3 kb internal to the genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is a very stable and robust virus. This easily withstands the conditions (several hours at 56 °C to 65 °C) used to inactivate adenovirus and reduces the importance of cryopreservation of AAV. AAV can be lyophilized. Finally, AAV-infected cells do not show resistance to superinfection.

[0046] Multiple studies have demonstrated long-term (greater than 1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997), Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996), and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000), and Chao et al., Mol Ther, 4:217-222 (2001). Additionally, because muscle is highly vascularized, recombinant AAV transduction has led to the appearance of transgene products in the systemic circulation following intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997), and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). Further, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cytokines for proper antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics.

[0047] The recombinant AAV genome of the present disclosure includes the nucleic acid molecule of the present disclosure and one or more AAV ITRs adjacent to the nucleic acid molecule. The AAV DNA in the rAAV genome can be derived from any AAV serotype from which a recombinant virus can be derived, including but not limited to AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, or Anc80, AAV7m8, and derivatives thereof). The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background art section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0048] The provided recombinant AAV (i.e., infectious encapsidated rAAV particles) includes an rAAV genome. The term "rAAV genome" refers to a polynucleotide sequence derived from a modified native AAV genome. In some embodiments, the rAAV genome is modified to remove the native cap and rep genes. In some embodiments, the rAAV genome includes endogenous 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the rAAV genome includes ITRs from an AAV serotype different from the AAV serotype from which the AAV genome is derived. In some embodiments, the rAAV genome includes a transgene of interest flanked by inverted terminal repeats (ITRs) at the 5' and 3' termini. In some embodiments, the genome of rAAV includes a "gene cassette". In an exemplary embodiment, both rAAV genomes lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the genomic ITRs.

[0049] The rAAV genomes provided herein, in some embodiments, include one or more AAV ITRs adjacent to a transgene polynucleotide sequence. The transgene polynucleotide sequence is operably linked to transcriptional control elements (including, but not limited to, promoters, enhancers, and / or polyadenylation signal sequences) that are functional in a target cell to form a gene cassette. Examples of promoters are the pIRF promoter, the chicken β-actin promoter (CBA) comprising the polynucleotide sequence set forth in SEQ ID NO: 3, and the P546 promoter comprising the polynucleotide sequence set forth in SEQ ID NO: 4. Additional promoters are contemplated herein, including, but not limited to, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as the actin promoter, the myosin promoter, the elongation factor-1a promoter, the hemoglobin promoter, and the creatine kinase promoter.

[0050] In addition, provided herein are promoter sequences that are at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequences of the CB promoter sequence, the P546 promoter sequence, and the CBA (SEQ ID NO: 3) or P546 (SEQ ID NO: 4) sequences that exhibit transcriptional enhancing activity.

[0051] Other examples of transcriptional control elements are tissue-specific control elements, such as promoters that enable specific expression within neurons or specifically within astrocytes. Examples include the neuron-specific enolase and glial fibrillary acidic protein promoters. Inducible promoters are also contemplated. Non-limiting examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline-regulated promoter. The gene cassette may also include intron sequences to facilitate processing of the transgene RNA transcript when expressed in mammalian cells. An example of such an intron is the SV40 intron.

[0052] The rAAV genomes provided herein include a polynucleotide (SEQ ID NO: 1) encoding the IGHMPB2 protein. In some embodiments, the rAAV genomes provided herein include a polynucleotide encoding a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the IGHMPB2 cDNA (SEQ ID NO: 1).

[0053] The rAAV genomes provided herein include nucleotides 1 to 4397 of SEQ ID NO: 7 or nucleotides 1 to 4375 of SEQ ID NO: 8. In some embodiments, the rAAV genomes provided herein include a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 1 to 4397 of SEQ ID NO: 7 or nucleotides 1 to 4375 of SEQ ID NO: 7 or 8.

[0054] In some embodiments, the rAAV genomes provided herein encode the IGHMPB2 protein and hybridize under stringent conditions to the polynucleotide sequence set forth in SEQ ID NO: 1 or its complement.

[0055] The DNA plasmids of the present disclosure contain the rAAV genomes of the present disclosure. The DNA plasmids are transferred to cells permissive for infection by an AAV helper virus (e.g., an adenovirus, an E1-deleted adenovirus, or a herpes virus) for assembly of the rAAV genome into infectious virus particles. Techniques for producing rAAV particles in which the AAV genome to be packaged, the rep and cap genes, and the helper virus functions are provided to the cells are standard in the art. Production of rAAV requires that the following components, the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not present therein), and the helper virus functions, be present within a single cell (referred to herein as a packaging cell). The AAV rep and cap genes can be derived from any AAV serotype from which the recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-9, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-10, AAV-11, AAV-12, and AAV-13, and can be derived from AAV serotypes different from the rAAV genome ITR. Production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety.

[0056] A method for generating packaging cells is to create a cell line that stably expresses all the components necessary for the production of AAV particles. For example, a plasmid (or plasmids) containing an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes isolated from the rAAV genome, and a selectable marker such as the neomycin resistance gene is integrated into the cell's genome. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081), addition of synthetic linkers 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 production of rAAV. Another example of a suitable method is to employ an adenovirus or baculovirus rather than a plasmid for introducing the rAAV genome and / or the rep gene and the cap gene into the packaging cells.

[0057] The general principles of rAAV production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in 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. Biol. 5:3251 (1985), McLaughlin et al., J. Virol., 62:1963 (1988), and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et 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), WO 97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. Vaccine 13:1244-1250 (1995), Paul et al. Human Gene Therapy 4:609-615 (1993), Clark et al. Gene Therapy 3:1124-1132 (1996), U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety, and the portions of the documents relating to rAAV production are particularly emphasized.

[0058] Accordingly, the present disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (a homologous 293 strain). In another embodiment, the packaging cells are not 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 (African green monkey fetal lung cells).

[0059] rAAV can be purified by standard methods in the art, such as by column chromatography or 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.

[0060] The compositions provided herein comprise rAAV and one or more pharmaceutically acceptable excipients. Acceptable excipients are non-toxic to the recipient, preferably inert at the dosages and concentrations employed, and include buffers such as phosphates (e.g., phosphate buffered saline (PBS)), citrates, 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®, copolymers such as poloxamer 188, Pluronics (e.g., Pluronic F68), or polyethylene glycol (PEG), but are not limited thereto. The compositions provided herein can include a pharmaceutically acceptable aqueous excipient containing a nonionic low osmolality compound such as iodixanol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, or ioxilan, and the aqueous excipient containing the nonionic low osmolality compound can have one or more of the following properties: an osmolality of about 180 mOsm / kg water by vapor pressure osmometry, an osmolality of about 273 mOsm / L, an absolute viscosity of about 2.3 cp at 20 °C and about 1.5 cp at 37 °C, and a specific gravity of about 1.164 at 37 °C.

[0061] Exemplary compositions include an agent for increasing the viscosity and / or density of the composition. For example, the composition includes a contrast agent for increasing the viscosity and / or density of the composition. Exemplary compositions include from about 20 to 40% nonionic low osmolality compound or contrast agent, or from about 25% to about 35% nonionic low osmolality compound. Exemplary compositions include 20 mM Tris (pH 8.0), 1 mM MgCl 2It contains scAAV or rAAV viral particles formulated in 200 mM NaCl, 0.001% poloxamer 188, and about 25% to about 35% nonionic hypotonic compounds. Another exemplary composition contains scAAV formulated in 1×PBS and 0.001% Pluronic® F68.

[0062] The dosage of rAAV administered by the methods of the present disclosure can vary, for example, depending on the specific rAAV, mode of administration, timing of administration, treatment goal, individual, and cell type targeted, and can be determined by standard methods in the art. The dosage can be expressed in units of viral genome (vg). Dosages contemplated herein are 1×10 7 、1×10 8 、1×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、1×10 11 、about 1×10 12 、about 1×10 13 、about 1.1×10 13 、about 1.2×10 13 、about 1.3×10 13 、about 1.5×10 13 、about 2×10 13 、about 2.5×10 13 、about 3×10 13 、about 3.5×10 13 、about 4×10 13 、about 4.5×10 13 、about 5×10 13 、about 6×10 13 、about 1×10 14 、about 2×10 14 、about 3×10 14 、about 4×10 14 、about 5×10 14 、about 1×10 15 、about 1×10 16comprising up to or more than the total viral genome.

[0063] about 1×10 9 ~ about 1×10 10 , about 5×10 9 ~ about 5×10 10 , about 1×10 10 ~ about 1×10 11 , about 1×10 11 ~ about 1×10 15 vg, about 1×10 12 ~ about 1×10 15 vg, about 1×10 12 ~ about 1×10 14 vg, about 1×10 13 ~ about 6×10 14 vg, about 1×10 13 ~ about 1×10 15 vg, and about 6×10 13 ~ about 1.0×10 14 vg doses are also contemplated. One dose exemplified herein is 1x10 13 vg administered via intrathecal delivery. Another dose exemplified herein is 1.5×10 13 vg.

[0064] Doses can also be expressed in units of vg / kg. Doses contemplated herein are about 1×10 7 vg / kg, 1×10 8 vg / kg, 1×10 9 vg / kg, 5×10 9 vg / kg, 6×10 9 vg / kg, 7×10 9 vg / kg, 8×10 9 vg / kg, 9×10 9 vg / kg, 1×10 10 vg / kg, 2x10 10 vg / kg 10 , 3×10 10 vg / kg, 4×10 10 vg / kg, 5×10 10 vg / kg, 1×10 11 vg / kg, about 1×10 12 vg / kg, about 1×10 13 vg / kg, about 1.1×1013 vg / kg, about 1.2×10 13 vg / kg, about 1.3×10 13 vg / kg, about 1.5×10 13 vg / kg, about 2×10 13 vg / kg, about 2.5×10 13 vg / kg, about 3×10 13 vg / kg, about 3.5×10 13 vg / kg, about 4×10 13 vg / kg, about 4.5×10 13 vg / kg, about 5×10 13 vg / kg, about 6×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg, about 5×10 14 vg / kg, about 1×10 15 vg / kg, about 1×10 16 including up to vg / kg.

[0065] about 1×10 9 vg / kg~about 1×10 10 vg / kg, about 45×10 9 vg / kg~about 5×10 10 vg / kg, about 1×10 10 vg / kg~about 1×10 11 vg / kg, about 1×10 11 vg / kg~about 1×10 15 vg / kg, about 1×10 12 vg / kg~about 1×10 15 vg / kg, about 1×10 12 vg / kg~about 1×10 14 vg / kg, about 1×10 13 vg / kg~about 2×10 14 vg / kg, about 1×10 13 vg / kg~about 1×10 15 vg / kg, and about 6×10 13 vg / kg~about 1.0×10 14 vg / kg doses are also contemplated. One dose exemplified herein is 1x10 administered via intravenous delivery 13It is vg / g. Another dosage exemplified in this specification is 2.5×10 14 vg / kg, which is administered via intravenous delivery.

[0066] Methods of transducing target cells with rAAV, either in vivo or in vitro, are contemplated by the present disclosure. In vivo methods include administering to an animal (including a human) in need thereof a composition comprising an effective dosage or effective multiple dosages of the rAAV of the present disclosure. When the dosage is administered prior to the onset of a disorder / disease, the administration is prophylactic. When the dosage is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present disclosure, an effective dosage is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease state, reduces the severity of the disease, results in remission (partial or complete) of the disease, and / or extends survival. Examples of diseases for which prevention or treatment by the methods of the present disclosure are contemplated are SMARD1 and CMT2S.

[0067] Combination therapies are also contemplated by the present disclosure. Combinations as used herein include both concurrent treatment and sequential treatment. Combinations of the methods of the present disclosure with standard medical care are particularly contemplated as combinations with novel therapies. In some embodiments, combination therapy includes administering an immunosuppressive agent in combination with the gene therapy disclosed herein.

[0068] Administration of an effective dosage of the composition can be by standard routes in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route of administration and serotype of the AAV components of the rAAV of the present disclosure (in particular, AAV ITR and capsid proteins) can be selected and / or adapted by one of ordinary skill in the art considering the infection and / or disease state being treated, as well as the target cells / tissues expressing the wild-type IGHMPB2 protein.

[0069] The present disclosure provides for local and systemic administration of effective dosages of the rAAV and compositions of the present disclosure. For example, systemic administration refers to administration to the circulatory system such that the entire body is affected. Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration through injection, infusion, or transplantation.

[0070] Transduction of cells by the rAAV of the present disclosure results in the sustained expression of the IGHMPB2 protein. The present disclosure thus provides methods for administering / delivering rAAV expressing the IGHMPB2 protein to animals, preferably humans. These methods include transducing cells with one or more rAAV of the present disclosure.

[0071] The term "transduction" is used to refer to the administration / delivery of the coding region of IGHMPB2 to recipient cells, either in vivo or in vitro, via the replication-deficient rAAV of the present disclosure that results in the expression of IGHMPB2 by the recipient cells.

[0072] Immunosuppressive agent The immunosuppressive agent can be administered before or after the initiation of an immune response to rAAV in a subject after administration of gene therapy. In addition, the immunosuppressive agent can be administered concurrently with gene therapy or protein replacement therapy. The immune response in a subject includes a harmful immune response or inflammatory reaction that follows or is caused by administration of rAAV to the subject. The immune response can be the production of antibodies in a subject in response to the administered rAAV.

[0073] Exemplary immunosuppressive agents include glucocorticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, purine analogs, methotrexate, and cell cycle inhibitors such as cyclophosphamide, inosine monophosphate dehydrogenase (IMDH) inhibitors, biologic agents such as monoclonal antibodies or fusion proteins and polypeptides, and dipeptide boronic acid molecules such as bortezomib.

[0074] An immunosuppressant can be an anti-inflammatory steroid that reduces inflammation and suppresses or modulates the immune system of a subject. Exemplary anti-inflammatory steroids are glucocorticoids such as prednisone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, budesonide, or prednisone.

[0075] A Janus kinase inhibitor is an inhibitor of the JAK / STAT signaling pathway by targeting one or more of the enzymes of the Janus kinase family. Exemplary Janus kinase inhibitors include tofacitinib, baricitinib, upadacitinib, peficitinib, and oclacitinib.

[0076] A calcineurin inhibitor binds to cyclophilin and inhibits the activity of calcineurin. Exemplary calcineurin inhibitors include cyclosporine, tacrolimus, and pimecrolimus.

[0077] An mTOR inhibitor reduces or inhibits the serine / threonine-specific protein kinase mTOR. Exemplary mTOR inhibitors include sirolimus, everolimus, and temsirolimus.

[0078] Immunosuppressants include immunosuppressive macrolides. The term "immunosuppressive macrolide" refers to a macrolide agent that suppresses or modulates the immune system of a subject. A macrolide is a class of drugs that contain a large macrocyclic lactone ring to which one or more deoxy sugars such as cladinose or desosamine are attached. The lactone ring is usually 14, 15, or 16 members. Macrolides belong to the polyketide class of drugs and can be natural products. Examples of immunosuppressive macrolides include tacrolimus, pimecrolimus, and sirolimus.

[0079] Purine analogs block nucleotide synthesis and include IMDH inhibitors. Exemplary purine analogs include azathioprine, mycophenolic acid, and leflunomide.

[0080] Exemplary immunosuppressive biologic agents include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, isekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, belatacept, and daclizumab.

[0081] In particular, the immunosuppressant is an anti-CD20 antibody. The term anti-CD20 specific antibody refers to an antibody that specifically binds to CD20 or inhibits or reduces the expression or activity of CD20. Exemplary anti-CD20 antibodies include rituximab, ocrelizumab, or ofatumumab.

[0082] Additional examples of immunosuppressive antibodies include anti-CD25 antibodies (or anti-IL2 antibodies or anti-TAC antibodies) such as basiliximab and daclizumab, as well as anti-CD3 antibodies such as muromonab-CD3, otelixizumab, teplyzumab, and visilizumab, and anti-CD52 antibodies such as alemtuzumab.

[0083] The following examples are provided by way of illustration and not limitation. The numerical ranges described include each integer value within each range and the minimum and maximum of the integers described.

Example

[0084] Example 1 - Gene Therapy Construct Encoding IGHMBP2 The AAV genomic construct encoding IGHMBP2 was generated as described in Figure 1, depicting an AAV9 vector design using the full-length transcript of IGHMBP2 cDNA under the control of a ubiquitous promoter. The promoters contemplated for inclusion in these constructs are either i) the cmv-enhancer chicken beta-actin promoter (CBA, SEQ ID NO: 3), or the synthetic truncated methyl CpG-binding protein 2 (MeCp2) promoter designated P546 (SEQ ID NO: 4) or 546.

[0085] The human GFP cDNA clone was obtained from Origene, Rockville, MD. Only the IGHMBP2 cDNA was further subcloned into the self-complementary AAV9 genome under the control of one or more of either i) the P546 promoter or v) the hybrid chicken β-actin promoter (CB). The plasmid constructs also included introns such as the simian virus 40 (SV40) chimeric intron, and the bovine growth hormone (BGH) polyadenylation signal (BGH polyA). The constructs were packaged into either AAV9 genome.

[0086] The map for plasmid ssAAV.CB.IGHMBP2.Kan.-Fw (with the kanamycin resistance gene in the forward direction) is described in Figure 2, and the sequence of the entire plasmid is provided as SEQ ID NO: 7. The ssAAV.CB.IGHMBP2 vector is within and includes the ITR of SEQ ID NO: 7 and contains the nucleotide sequence as shown in Figure 13. The rAAV vector contains the 5’ AAV2 ITR, CMV enhancer, CBA promoter, modified SV40 intron sequence, coding sequence for the IGHMBP2 gene, bGH polyA, and 3’ AAV2 ITR. The plasmid described in SEQ ID NO: 7 further includes the pUC origin of replication and kanamycin resistance. Plasmid ssAAV.CB.IGHMBP2.Kan.-Rv (with the kanamycin resistance gene in the reverse direction) is provided as SEQ ID NO: 9.

[0087] Table 2 shows the molecular characteristics of plasmid ssAAV.CB.IGHMBP2.Kan.-Fw (SEQ ID NO: 7), the range of which refers to the nucleotides in SEQ ID NO: 7,

Number

Table 2-2

[0088] A map for plasmid ssAAV.P546.IGHMBP2.Kan.-Fw (with the kanamycin resistance gene in the forward direction) is described in FIG. 3, and the sequence of the entire plasmid is provided in SEQ ID NO: 8. The ssAAV.P546.IGHMBP2 vector is within and includes the ITRs of SEQ ID NO: 8 and contains the nucleotide sequence as shown in FIG. 14. The rAAV vector includes the 5’ AAV2 ITR, the P546 promoter (also referred to herein as the MeCp2 promoter or the P546 promoter), a modified SV40 intron sequence, the coding sequence for the IGHMBP2 gene, bGH polyA, and the 3’ AAV2 ITR. The plasmid described in SEQ ID NO: 8 further includes kanamycin resistance along with the pUC origin of replication. Plasmid ssAAV.P546.IGHMBP2.Kan.-Rv (with the kanamycin gene in the reverse direction) is provided as SEQ ID NO: 10.

[0089] Table 3 shows the molecular characteristics of plasmid ssAAV.P546.IGHMBP2.Kan.-Fw (SEQ ID NO: 8), the range of which refers to the nucleotides in SEQ ID NO: 8,

Number

Table 3

[0090] A map for plasmid ssAAV.P546.IGHMBP2.Kan-clinical (where the P546 promoter sequence, IGHMBP2 cDNA sequence, SV40 intron, and bGH polyadenylation sequence are in the reverse orientation) is described in Figure 15, and the sequence of the entire plasmid is provided in SEQ ID NO: 17. The ssAAV.P546.IGHMBP2-clinical vector is within and includes the ITRs and contains the nucleotide sequence as shown in Figure 16. The rAAV vector contains the 5’ AAV2 ITR (SEQ ID NO: 19), P546 promoter, modified SV40 intron sequence, coding sequence for the IGHMBP2 gene, bGH polyA, and 3’ AAV2 ITR (SEQ ID NO: 12). The plasmid described in SEQ ID NO: 17 further includes a kanamycin resistance along with the pUC origin of replication. The kanamycin resistance gene is in the forward orientation in Figure 15, but plasmids with the kanamycin resistance gene in the reverse orientation are also contemplated.

[0091] Table 4 shows the molecular characteristics of plasmid ssAAV.P546.IGHMBP2.Kan-clinical (SEQ ID NO: 17), the range of which refers to the nucleotides in SEQ ID NO: 17,

No.

No.

Table 4

[0092] A map for plasmid ssAAV.CB.IGHMBP2.Kan-clinical (where the CMV enhancer sequence, CB promoter sequence, IGHMBP2 cDNA sequence, SV40 intron, and bGH polyadenylation sequence are in the reverse orientation) is described in Figure 17, and the sequence of the entire plasmid is provided in SEQ ID NO: 18. The ssAAV.CB.IGHMBP2-clinical vector is within and includes the ITRs and contains a nucleotide sequence as shown in Figure 18. The rAAV vector contains the 5’ AAV2 ITR (SEQ ID NO: 19), CMV enhancer, CB promoter, modified SV40 intron sequence, coding sequence for the IGHMBP2 gene, bGH polyA, and the 3’ AAV2 ITR (SEQ ID NO: 12). The plasmid described in SEQ ID NO: 18 further includes kanamycin resistance along with the pUC origin of replication. The kanamycin resistance gene is in the forward orientation in Figure 17, although plasmids with the kanamycin resistance gene in the reverse orientation are also contemplated.

[0093] Table 5 shows the molecular characteristics of plasmid ssAAV.CB.IGHMBP2.Kan-clinical (SEQ ID NO: 18), the range of which refers to the nucleotides in SEQ ID NO: 18,

Number

Number

Table 5

[0094] Example 2 - CSF Delivery of the IGHMBP2 Gene Therapy Vector in Mice Mouse models of SMARD1 and CMT2S were used to compare the effects of CSF delivery of AAV expressing IGHMBP2. Table 4 below provides different mouse models that can be used to investigate the efficacy of IGHMBP2 gene therapy vectors. In this study, two different mouse models representing the very severe end (em3) and moderate disease form (nmd-2J) of the disease spectrum were used in this study.

Table 6

[0095] Em3 mouse model For the first study, nmd em3 / em3 Homozygous mice were administered an IGHMBP2 gene therapy vector or a control via intracerebroventricular injection (ICV) into the cerebrospinal fluid (CSF). The "nmd" mouse mutation causes progressive degeneration and muscle atrophy of spinal motor neurons (Cox et al., Neuron 21:1327-1337, 1998). The nmd mutation was identified as a putative transcriptional activator and an ATPase / DNA helicase previously described as Smbp2 or Catf1. The nmd phenotype is attenuated in a semi-dominant manner by a major locus on mouse chromosome 13.

[0096] Nmd em3 / em3 Homozygous mice received a single intracerebroventricular injection of 5e10 viral genomes (vg) per animal of either ssAAV9.CB.IGHMBP2 (virus A) or ssAAV9.P546.IGHMBP2 (virus C) or empty viral particles (virus B) formulated in 1×PBS and 0.001% Pluronic F68 (represented as PBS / F68). Strength tests were performed starting 3 weeks after administration and survival was also monitored. Figure 3 demonstrates that treatment with virus A and virus C appeared to restore paralysis, but these mice remained smaller than the controls.

[0097] Figure 4 provides representative images of nmd homozygous mice next to control mice at 2 - 3 weeks after AAV administration. Mice treated with virus A or virus C showed improvement in clasping. In addition, mice treated with virus B (empty virus particles) were unable to spread their hindlimbs compared to mice treated with viruses A and C. em3 / em3 Mice treated with virus A or virus C showed improvement in clasping. In addition, mice treated with virus B (empty virus particles) were unable to spread their hindlimbs compared to mice treated with viruses A and C.

[0098] Figure 5 provides survival analysis of nmd mice. Treatment with virus A or virus C restored the survival period of the mice (one died early in each case). However, virus B (empty virus particles) did not seem to recover. There were occasional survivors in the untreated and virus B - treated groups, but these mice were very small and weak. As shown in Figure 6, treatment with virus A or virus C improved the body weight of nmd homozygous mice, but the body weight did not fully recover when measured 8 weeks after treatment. em3 / em3 Treatment with virus A or virus C restored the survival period of the mice (one died early in each case). However, virus B (empty virus particles) did not seem to recover. There were occasional survivors in the untreated and virus B - treated groups, but these mice were very small and weak. As shown in Figure 6, treatment with virus A or virus C improved the body weight of nmd em3 / em3 homozygous mice, but the body weight did not fully recover when measured 8 weeks after treatment.

[0099] For the strength test, the treated mice were inverted on the lid of a wire cage for up to 60 seconds, and the time when they fell or when the 60 - second endpoint was reached was noted. The longest time of three trials was recorded. If the mouse failed the first trial of lasting 60 seconds, the first failed trial was retested to confirm whether it was due to A) not wanting to participate deliberately, B) slipping, or C) being unable to complete the 60 - second wire suspension. If it failed the second time, a break of about 3 - 5 minutes was given and it was retested again. The numbers in the data / graph of Figure 7 reflect the longest trial. Figure 7 provides the time after treatment compared to the waiting time until the mouse falls when suspended from the wire. Treatment with virus A showed an initial improvement, followed by a continued decrease in strength, and later recovered to wild - type levels 8 weeks after injection. Treatment with virus C improved the strength such that the homozygous mice were similar to normal mice (nmd + / + ) in terms of strength.

[0100] In addition, the treated nmd em3 / em3 The nerves of homozygous mice were extracted and evaluated. Both the left and right phrenic nerves, femoral motor nerves, and femoral sensory nerves were extracted and fixed overnight by electron microscopy (EM) fixation. The nerves were then washed three times with 1×PBS buffer and stored at 4°C until sent to our histological core. The axon number was determined using the semi-automated "WEKA Trainable Segmentation" plugin of ImageJ. The treated nmd em3 / em3 Cross-sections of the femoral nerves of homozygous mice are shown in Figure 8A, and the axon area is provided in Figure 8B. All groups were significantly different from each other by the Kolmogorov-Smirnov test. Treatment with virus A or virus C significantly increased the axon area.

[0101] The treated nmd 8 weeks after treatment em3 / em3 The muscle area of the intercostal muscles of homozygous mice was also investigated. In the treated mice, 300 muscle fibers were manually traced in ImageJ from hematoxylin and eosin cross-sections of individual tissues of each animal. The measurements were provided in pixel units, and the graph in Figure 8D represents the cumulative frequency showing the range of the muscle fiber area.

[0102] Cross-sections of the hindlimb muscles are shown in Figure 8C. For cross-sectional images from the hindlimbs, two incisions were added to the patella and calcaneus to define the leg lengths at the knee and ankle. A third incision was added at a point equidistant from the patella and calcaneus. The bone and muscle morphology were examined to select images from the same region among all animals. Mice treated with virus A or virus C showed an increase in muscle area, with virus C showing the highest performance.

[0103] In addition, treatment with virus A or virus B was performed on nmd em3 / em3The neuromuscular junctions in the hindlimbs of homozygous mice were partially restored. Figures 9A and 9B are representative photographs showing the immunocytochemistry of the MG and Sol muscles obtained from the hindlimbs 8 weeks after treatment with rAAV. The MG and Sol muscles were weighed and the muscles were fixed with 4% PFA. The muscles were stained with antibodies that detect neurofilament (green) as a marker for presynaptic nerves and bungarotoxin (red) as a marker for postsynaptic Ach receptors. The neuromuscular junctions (NMJs) were photographed via an SP8 Leica confocal microscope. Examples of each type of NMJ occupancy are provided in Figure 9A. Occupancy was determined manually as either fully innervated (the red BTX acetylcholine channels and the green neurofilament channels completely overlap), partially innervated (the red and green channels partially overlap), or completely denervated (one channel is completely absent). Untreated nmd em3 / em3 There was little innervation of the neuromuscular junctions in the hindlimbs of homozygous mice. Virus A and C treated nmd em3 / em3 In homozygous mice, a mixture of fully innervated, fragmented, and non-innervated NMJs was found. The graph provides a count of NMJs on the medial gastrocnemius and soleus muscles, showing an increase in fully innervated NMJs with both Virus A and Virus C, with Virus C showing a higher percentage of fully innervated NMJs.

[0104] Nmd-2J mouse model In addition, nmd-2J mice were administered an IGHMBP2 gene therapy vector or a control via intracerebroventricular injection (ICV) into the cerebrospinal fluid (CSF). nmd-2J mice have moderate SMARD1-like paralysis and tend to die at approximately 60 - 100 days. These mice received a single intracerebroventricular injection of 5e10 viral genomes (vg) per animal of either ssAAV9.CB.IGHMBP2 (virus A) or ssAAV9.P546.IGHMBP2 (virus C) or empty viral particles (virus B) formulated in 1×PBS and 0.001% Pluronic F68 (represented as PBS / F68). As described above, FIG. 10 provides a survival plot demonstrating that viruses A and C improved the survival of nmd-2J mice.

[0105] Electromyography (EMG) is a potential clinical biomarker for efficacy. Electrophysiological results include compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE) recorded from the hindlimb muscles following sciatic nerve stimulation as described in Arnold et al, Annals of clinical and translational neurology, 1(1), 34 - 44, 2014. CMAP measures the strength of innervation and MUNE provides an estimate of the number of neurons innervating the muscle.

[0106] Briefly, two thin ring electrodes were placed on an anesthetized mouse to record electrophysiological results. The active (E1) ring electrode was placed on the skin over the proximal portion of the gastrocnemius muscle of the hindlimb at the knee joint, and the reference (E2) ring electrode was placed over the area of the central portion of the metatarsal bone of the foot. The skin under the ring electrodes was coated with gel to reduce impedance. The sciatic CMAP response was obtained using a maximal supramaximal stimulation (up to approximately 120%) of the sciatic nerve (square wave pulses of 0.1 millisecond duration and intensity 1 - 10 mA). The mean single motor unit potential (SMUP) size and MUNE were calculated by delivering a maximal submaximal stimulation of 0.1 millisecond duration at a frequency of 1 Hz while increasing the intensity in 0.026 mA steps to obtain a minimal all - or - none response, and recording the incremental responses. Ten increments were averaged to provide the mean single motor unit potential SMUP amplitude. MUNE was calculated as follows: MUNE = CMAP / mean SMUP. Peak - to - peak measurements were used for the amplitudes of CMAP and SMUP. As shown in FIGS. 11A - D, there were significant increases in CMAP and MUNE in mice treated with virus A or virus C, but CMAP did not differ between mice treated with virus A and mice treated with virus C. FIGS. 11A - D demonstrate a clear difference between mice treated with the IGHMBP2 gene therapy vector compared to untreated mice.

[0107] Em5 mouse model The em5 mouse model was also used to investigate the effect of the IGHMBP2 gene therapy vector administered via intracerebroventricular injection (ICV) into the cerebrospinal fluid (CSF). em5 mice have a CMT2S phenotype of sensory and motor neuropathy and have a lifespan of approximately 10 months. These mice received a single intracerebroventricular injection of either 5e10 viral genomes (vg) per animal of ssAAV9.CB.IGHMBP2 (virus A) or ssAAV9.P546.IGHMBP2 (virus C) or empty viral particles (virus B) formulated in 1×PBS and 0.001% Pluronic F68 (represented as PBS / F68).

[0108] Figure 12A provides the results of the hanging wire test in healthy mice and Em5 mice treated with virus A and virus C. Healthy mice and mice treated with virus A and virus C showed significantly better grip strength compared to untreated em5 mice. There was no significant difference between healthy mice and mice treated with virus A or virus C. Figure 12B shows the weight of the medial gastrocnemius muscle (MG) in treated and untreated mice. Figure 12B shows an increase in the muscle mass of MG relative to the total body weight in healthy mice and Em5 mice treated with virus A and virus C compared to untreated em5 mice. There was no difference between treated animals and healthy animals.

[0109] Example 3 - Clinical Trials in Humans ssAAV9.CB.IGHMBP2 (Virus A) or ssAAV9.P546.IGHMBP2 (Virus C) is administered intrathecally to human patients suffering from IGHMBP2-related disorders such as SMARD1 or CMT2S. The scAAV for clinical trials is produced using a triple transfection method of HEK293 cells under cGMP conditions.

[0110] Patients selected for participation will be between 1 and 20 years old, diagnosed with an IGHMBP2-related disorder such as SMARD1 or CMT2S as determined by genotype. Patients will receive a single gene transfer dose of ssAAV per patient. The ssAAV is formulated in 20 mM of 1 mM MgCl 2 , 200 mM of NaCl, 0.001% poloxamer 188 Tris (pH 8.0) and will be delivered only once through intrathecal injection. Safety is evaluated based on clinical evidence and by review of the safety label. There is a minimum of 3 - 4 weeks between the enrollment of each subject to allow for scrutiny of the safety data on day 30 after gene transfer. Disease progression is measured and the impact of treatment on quality of life and the likelihood of long-term survival is evaluated. In certain embodiments, for example, the following are provided: (Item 1) A polynucleotide comprising: (a) one or more regulatory elements; and (b) a cDNA sequence of immunoglobulin - μ - binding protein 2 (IGHMBP2). (Item 2) The polynucleotide according to Item 1, wherein the regulatory element is a CBA promoter or a P546 promoter, or a fragment thereof. (Item 3) The polynucleotide according to Item 1 or 2, wherein the IGHMBP2 cDNA comprises a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 1, or comprises the polynucleotide sequence set forth in SEQ ID NO: 1. (Item 4) The polynucleotide according to any one of Items 1 to 3, comprising the nucleotide sequence of SEQ ID NO: 3 or 4. (Item 5) A recombinant adeno - associated virus (rAAV) having a genome comprising the polynucleotide sequence according to any one of Items 1 to 4. (Item 6) The rAAV according to Item 5, wherein the genome comprises a P546 promoter and an IGHMBP2 cDNA. (Item 7) The rAAV according to Item 5, wherein the genome comprises a CBA promoter and an IGHMBP2 cDNA. (Item 8) The rAAV according to any one of Items 5 to 7, wherein the rAAV is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13 or Anc80, AAV7m8, and derivatives thereof. (Item 9) An rAAV particle comprising the rAAV according to any one of Items 5 to 8. (Item 10) A composition comprising the rAAV according to any one of Items 5 to 8 or the viral particle according to Item 9. (Item 11) The composition according to Item 10, further comprising an agent that increases the viscosity or density of the composition. (Item 12) The composition according to Item 10, wherein the agent is a contrast agent. (Item 13) The composition according to any one of items 9 to 12, which is formulated for direct injection into cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery, or intravenous delivery. (Item 14) The composition according to any one of items 10 to 13, wherein the composition is formulated for intrathecal delivery and contains a dose of rAAV or rAAV particles of about 1e13 vg per patient to about 1e15 vg per patient. (Item 15) The composition according to any one of items 10 to 13, wherein the composition is formulated for intravenous delivery and contains a dose of rAAV or rAAV particles of about 1e13 vg / kg to about 2e14 vg / kg. (Item 16) A method of treating an IGHMBP2-related disorder in a subject in need of treatment for an IGHMBP2-related disorder, comprising administering an rAAV according to any one of items 5 to 8, an rAAV particle according to item 9, or a composition according to any one of items 10 to 15. (Item 17) The method according to item 16, wherein the disorder is SMARD1 or CMT2S. (Item 18) The method according to item 16 or 17, wherein the subject has a mutation in the IGHMBP2 gene. (Item 19) The method according to any one of items 16 to 18, wherein the rAAV or the rAAV particle is administered by direct injection into cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery, or intravenous delivery. (Item 20) The method according to any one of items 16 to 19, wherein a dose of rAAV or rAAV particles of about 1e13 vg per patient to about 1e15 vg per patient is administered to the subject by intrathecal delivery. (Item 21) The method according to any one of items 16 to 20, wherein a dose of rAAV or rAAV particles of a dose of about 1e13 vg / kg to about 2e14 vg / kg is administered to the subject by intravenous delivery. (Item 22) The method according to any one of items 16 to 21, further comprising the step of administering an immunosuppressant. (Item 23) Use of an rAAV according to any one of items 5 to 8, an rAAV particle according to item 9, or a composition according to any one of items 10 to 15 in the preparation of a medicament for the treatment of an IGHMBP2-related disorder. (Item 24) The use according to item 23, wherein the disorder is SMARD1 or CMT2S. (Item 25) The use according to item 23 or 24, wherein the medicament is formulated for direct injection into cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery, or intravenous delivery. (Item 26) The use according to any one of items 23 to 25, wherein the agent comprises a dose of rAAV or rAAV particles of about 1e13 vg per patient to about 1e15 vg per patient, and the agent is formulated for intrathecal delivery to the subject. (Item 27) The use according to any one of items 23 to 25, wherein the agent comprises a dose of rAAV or rAAV particles of about 1e13 vg / kg to about 2e14 vg / kg, and the agent is formulated for intravenous delivery to the subject. (Item 28) A composition comprising an rAAV according to any one of items 5 to 8, an rAAV particle according to item 9, or a composition according to any one of items 10 to 15 for the treatment of an IGHMBP2-related disorder. (Item 29) The composition according to item 28, wherein the disorder is SMARD1 or CMT2S. (Item 30) The composition according to item 28 or 29, wherein the composition is formulated for direct injection into cerebrospinal fluid, intraventricular delivery, intrathecal delivery or intravenous delivery. (Item 31) The composition according to any one of items 28 to 30, wherein the composition comprises a dose of rAAV or rAAV particles of about 1e13 vg per patient to about 1e15 vg per patient, and the composition is formulated for intrathecal delivery. (Item 32) The composition according to any one of items 28 to 30, wherein the composition comprises a dose of rAAV or rAAV particles of about 1e13 vg / kg to about 2e14 vg / kg, and the composition is formulated for intravenous delivery. (Item 33) The composition according to any one of items 28 to 32, wherein the composition further comprises an immunosuppressant.

Claims

**Claim 1** A recombinant adeno-associated virus (rAAV) having a genome comprising a nucleic acid consisting of nucleotides 1 to 4397 of SEQ ID NO: 7, nucleotides 1 to 4386 of SEQ ID NO: 18, nucleotides 1 to 4375 of SEQ ID NO: 8, or nucleotides 1 to 4364 of SEQ ID NO:

17. **Claim 2** The rAAV according to claim 1, wherein the rAAV is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh74, AAV11, AAV12, AAV13 or Anc80, AAV7m8, and derivatives thereof. **Claim 3** rAAV particles comprising the rAAV according to claim 1 or 2. **Claim 4** A composition comprising the rAAV according to claim 1 or 2 or the viral particles according to claim 3. **Claim 5** The composition according to claim 4, further comprising an agent for increasing the viscosity or density of the composition. **Claim 6** The composition according to claim 5, wherein the agent for increasing the viscosity or density of the composition is a contrast agent. **Claim 7** The composition according to claim 4 or 5, wherein the composition is formulated for direct injection into cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery, or intravenous delivery. **Claim 8** The composition is a) formulated for intrathecal delivery and contains a dose of rAAV or rAAV particles from 1e13 vg per patient to 1e15 vg per patient, or b) formulated for intravenous delivery and contains a dose of rAAV or rAAV particles from 1e13 vg / kg to 2e14 vg / kg, the composition according to any one of claims 4 to 7. **Claim 9** A composition for the treatment of an IGHMBP2-related disorder, comprising the rAAV according to claim 1 or 2, the rAAV particles according to claim 3, or the composition according to any one of claims 4 to 8. **Claim 10** The composition according to claim 9, wherein the disorder is SMARD1 or CMT2S. **Claim 11** The composition according to claim 9 or 10, wherein the composition is formulated for direct injection into cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery or intravenous delivery. **Claim 12** The composition according to any one of claims 9 to 11, further comprising an immunosuppressant.

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