Materials and methods for treatment of disorders associated with ighmbp2 gene

Abstract

To provide gene therapy vectors, such as adeno-associated virus (AAV), designed for treatment of an immunoglobulin-μ binding protein 2 (IGHMB P2)-related disorder.SOLUTION: In one aspect, described herein is a polynucleotide comprising (a) one or more regulatory control elements, and (b) a cDNA sequence of immunoglobulin-μ binding protein 2 (IGHMBP2). In some embodiments, the regulatory control element is a CBA promoter comprising a specific nucleotide sequence, or a P546 promoter comprising a specific nucleotide sequence, or a fragment thereof which retains regulatory control or promoter activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 939,270, filed November 22, 2019, which is incorporated herein in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference in its entirety as a separate part of this disclosure and identified as 54445_Seqlisting.txt, size: 73,110 bytes, created date: November 23, 2019.

[0003] FIELD OF THE INVENTION The present disclosure provides gene therapy vectors, such as adeno-associated viruses (AAVs), designed for the treatment of disorders associated with mutations in the immunoglobulin-mu binding protein 2 (IGHMBP2) gene. The disclosed rAAVs deliver wild-type IGHMBP2 cDNA to a subject in need thereof, resulting in expression of the wild-type protein. [Background technology]

[0004] The immunoglobulin-mu binding protein 2 (IGHMBP2) gene encodes a member of the Upf1-like group within helicase superfamily 1 (SF1). This protein is known to possess a helicase domain, an R3H domain, a zinc finger domain, and a nuclear localization signal sequence. IGHMBP2 is ubiquitously expressed and contains 15 exons encoding 993 amino acids corresponding to a 110 kDa gene product. The exact role of the IGHMBP2 protein in disease pathogenesis is unknown. Normal IGHMBP2 is known to play a role in ribosomal RNA maturation and translation, immunoglobulin class switching, pre-mRNA maturation, and transcriptional regulation through either DNA-binding activity or interaction with TATA-binding protein. IGHMBP2 is classified as a member of the Upf1-like group within helicase superfamily 1 (SF1), which consists 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 patient mutations in the IGHMPB2 gene are missense mutations clustered within the helicase domain.

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

[0006] Charcot-Marie-Tooth (CMT) neuropathy is the most common 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 velocities. CMT2 is clinically similar to CMT1 but is typically less severe. Patients have slowly progressive distal muscle weakness accompanied by muscle atrophy of the upper and lower extremities. CMT2 subtypes are clinically similar and are distinguished only by molecular genetic findings. Most CMT2 subtypes are inherited in an autosomal dominant manner, but some are inherited in an autosomal recessive manner. Recessive loss-of-function mutations in the IGHMBP2 gene are known to cause CMT2, which is now subclassified as CMT2S.

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

[0008] In one aspect, described herein is a polynucleotide comprising (a) one or more regulatory control elements and (b) a cDNA sequence of 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. 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%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, and the protein retains IGHMBP2 activity. For example, the nucleotide sequence encoding the functional IGHMBP2 protein may contain one or more base pair substitutions, deletions, or insertions that affect the function of IGHMBP2. Additionally, a nucleotide sequence encoding a functional IGHMBP2 protein may contain one or more base pair substitutions, deletions, or insertions that may increase or decrease expression of the IGHMBP2 protein, and this change in expression pattern may be desirable for the treatment of an IGHMBP2-related disorder 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%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, wherein 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," "percent sequence identity," or "percent identity" in the context of nucleic acid or amino acid sequences refer to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison can be the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 500-5000 nucleotides is desirable. However, identity between smaller fragments, such as at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, or at least about 36 or more nucleotides, is also desirable. Percent sequence identity can be determined by techniques known in the art. For example, homology can be determined by direct comparison of 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, prediction=10, matrix=BLOSUM62, explanation=50 sequences, sort=high score, database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss protein+Spupdate+PIR.

[0012] In another aspect, the 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 Figure 13. The rAAV vector comprises a 5' ITR, a CMV enhancer, a CB promoter, a modified SV40 intron sequence, a coding sequence for the human IGHMBP2 gene, a bGH polyA, and a 3' ITR. In one embodiment, the vector comprises nucleotides 1-4397 of SEQ ID NO:7. The nucleotides within the ITR can be in the forward or reverse orientation. For example, the CMV enhancer sequence, the CB promoter sequence, the SV40 sequence, the human IGHMBP2 gene sequence, 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 1-4397 of SEQ ID NO: 7. The plasmid set forth in SEQ ID NO: 7 further comprises kanamycin resistance and a pUC origin of replication.

[0013] In an exemplary embodiment, the 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 Figure 18. The rAAV vector comprises the 5' ITR set forth in SEQ ID NO: 19. In addition, the rAAV vector contains, in reverse orientation, a CMV enhancer, a CB promoter, a modified SV40 intron sequence, a coding sequence for the human IGHMBP2 gene, a bGH polyA, and a 3' ITR set forth in SEQ ID NO: 12. In one embodiment, the vector comprises nucleotides 1-4386 of SEQ ID NO: 18. The nucleotides within the ITR 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 1-4386 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 a forward or reverse orientation.

[0014] In a further aspect, the 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 Figure 14. The rAAV vector comprises a 5' ITR, a P546 promoter, a modified SV40 intron sequence, a coding sequence for the human IGHMBP2 gene, a bGH polyA, and a 3' ITR. In one embodiment, the vector comprises nucleotides 1-4375 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 1-4397 of SEQ ID NO: 8. The plasmid set forth in SEQ ID NO: 8 further comprises kanamycin resistance and a pUC origin of replication.

[0015] In an exemplary embodiment, the 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 Figure 16. The rAAV vector comprises the 5' ITR set forth in SEQ ID NO: 19. In addition, the rAAV vector contains, in reverse orientation, a P546 promoter sequence, a modified SV40 intron sequence, a coding sequence for the human IGHMBP2 gene, and a bGH polyA, and a 3' ITR set forth in SEQ ID NO: 12. In one embodiment, the vector comprises nucleotides 1-4364 of SEQ ID NO: 17. The nucleotides within the ITR 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 1-4364 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 a forward or reverse orientation.

[0016] In another aspect, the present invention describes a recombinant adeno-associated virus (rAAV) having a genome comprising the polynucleotide sequence 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 their derivatives.In some embodiments, the genome of 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 ssAAV9.CB.IGHMBP2, the rAAV set forth as nucleotides 1-4397 of SEQ ID NO:7, or the rAAV set forth as nucleotides 1-4386 of SEQ ID NO:18.

[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 an rAAV set forth as nucleotides 1-4375 of SEQ ID NO:8, or an rAAV set forth as nucleotides 1-4364 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, wherein the promoter fragment retains promoter activity.

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

[0021] A composition comprising any of the rAAVs 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 an imaging agent. The imaging agent can be about 20% to 40% of a non-ionic low-osmolar compound or contrast agent, or about 25% to about 35% of a non-ionic low-osmolar compound such as iohexol. The disclosed compositions can be formulated for any means of delivery, such as direct injection into the cerebrospinal fluid, intraventricular delivery, intrathecal delivery, or intravenous delivery.

[0022] In some embodiments, the composition includes 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 includes 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 compositions are formulated for intrathecal delivery and include a dose of rAAV or rAAV particles of about 1e13vg per patient to about 1e15vg per patient.

[0025] Additionally, the disclosed compositions are formulated for intravenous delivery and include a dose of rAAV or rAAV particles of about 1e13vg / kg to about 2e14vg / kg.

[0026] Particularly contemplated is a method for treating an IGHMBP2-related disorder in a subject in need of such treatment, comprising administering the rAAV or rAAV particles described herein. In some embodiments, the method further comprises administering an immunosuppressant before, after, or simultaneously with the rAAV or rAAV particles. IGHMPB2-related disorders include disorders or diseases caused by mutations that result in the loss of function of IGHMPB2 protein or cause reduced expression of IGHMPB2 protein. IGHMPB2-related disorders can be any disease or disorder associated with reduced expression or activity of IGHMPB2 protein, regardless of the cause of the reduced expression or activity. The present disclosure contemplates IGHMPB2-related disorders in subjects who are homozygous for a mutation in the IGHMPB2 gene or heterozygous for a mutation in the IGHMPB2 gene. For example, the IGHMBP2-related disorder is a neurological disorder associated with the presence of a mutation in the IGHMBP2 gene, such as SMARD1 or CMT2S. IGHMBP2-associated disorders also include disorders in which patients have a mixed phenotype, such that the severity of neurological impairment 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, including currently known mutations, such as those shown in Tables 1 or 2 herein, or future identified mutations in the IGHMBP2 gene that are associated with a neurological disorder.

[0028] As used herein, a "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 animal (e.g., a cow, a horse, a pig) or a pet (e.g., a dog, a cat). 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 old. In other embodiments, the subject is an adult (18 years of age or older). In any of the disclosed methods, the rAAV or viral particles are delivered by direct injection into the cerebrospinal fluid, intraventricular delivery, intrathecal delivery, or intravenous delivery. For example, in any of the methods, 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. Additionally, in any of the disclosed methods, a dose of about 1e13 vg / kg to about 2e14 vg / kg of rAAV or rAAV particles is administered to the subject via intravenous delivery.

[0029] In another aspect, described herein is the use of the rAAV or rAAV particles described herein in the preparation of a medicament for the treatment of an IGHMBP2-associated disorder, such as SMARD1 or CMT2S. For example, any of the disclosed medicaments is formulated for direct injection into cerebrospinal fluid, intraventricular delivery, intrathecal delivery, or intravenous delivery. For example, the medicament comprises a dose of rAAV or rAAV particles of about 1e13vg per patient to about 1e15vg per patient and is administered to a subject by intrathecal delivery. Additionally, the medicament comprises a dose of rAAV or rAAV particles of about 1e13vg / kg to about 2e14vg / kg and is administered to a subject by intravenous delivery. In some embodiments, the medicament is administered before or after, or simultaneously with, the administration of an immunosuppressant.

[0030] In another aspect, described herein are compositions comprising the rAAV or rAAV particles described herein for the treatment of IGHMBP2-associated disorders, such as SMARD1 or CMT2S. For example, any of the disclosed compositions is formulated for direct injection into the cerebrospinal fluid, intraventricular delivery, intrathecal delivery, or intravenous delivery. For example, the composition comprises a dose of rAAV or rAAV particles of about 1e13 vg per patient to about 1e15 vg per patient and is administered to a subject via intrathecal delivery. Additionally, the composition comprises a dose of rAAV or rAAV particles of about 1e13 vg / kg to about 2e14 vg / kg and is administered to a subject via 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 explanation of the drawings]

[0031] [Figure 1] Figure 1 provides a schematic diagram of AAV9.CB.IGHMBP2 (Promoter 1 = CB promoter) and AAV9.P546.IGHMBP2 (Promoter 2 = P546 promoter). [Figure 2] Figure 2 provides a plasmid map of ssAAV.CB.IGHMBP2.Kan-Fw (SEQ ID NO: 7). [Figure 3] Figure 3 provides a plasmid map of ssAAV.P546.IGHMBP2.Kan-Fw (SEQ ID NO: 8). This vector is identical to ssAAV.P546.IGHMBP2.Kan described herein. The P5465 promoter (SEQ ID NO: 4) is also referred to herein as MeCp2 or 546, and these terms may be used interchangeably. [Figure 4] Figure 4 provides representative photographs of nmdem3 / em3 homozygous mice treated with virus A (AAV9.CB.IGHMBP2), virus B (empty AAV9 capsid), and virus C (AAV9.P546.IGHMBP2). All images were taken 2–3 weeks after treatment. [Figure 5]Figure 5 provides a survival analysis of nmdem3 / em3 homozygous mice treated with virus A, virus B, and virus C up to 8 weeks post-injection, when the animals were sacrificed for histology. Virus A and virus C largely restore survival to these mice, while virus B (empty virus particles) has no effect. [Figure 6A] 6A-6B provide graphs showing weight gain in nmdem3 / em3 homozygous mice following treatment with Virus A, Virus B, or Virus C. [Figure 6B] 6A-6B provide graphs showing weight gain in nmdem3 / em3 homozygous mice following treatment with Virus A, Virus B, or Virus C. [Figure 7] Figure 7 provides a graph depicting strength testing of nmdem3 / em3 homozygous mice using the hanging wire test, measuring the time mice can hold on to the wire before falling after treatment with virus A, virus B, or virus C. [Figure 8A-8B] Figures 8A-8D provide photographs demonstrating that Virus A and Virus C had a strong effect on nerve and muscle area. [Figure 8C-8D] Figures 8A-8D provide photographs demonstrating that Virus A and Virus C had a strong effect on nerve and muscle area. [Figure 9A] Figures 9A-9C: Figures 9A and 9B provide representative photographs of fully innervated, denervated, and partially innervated neuromuscular junctions (NMJs) in the medial gastrocnemius (MG) muscle 8 weeks after treatment with Virus A or Virus C. Wild-type mice had fully innervated NMJs, while untreated mice had little innervation at the NMJs after 2-3 weeks of age. Treated mice had a mixture of fully innervated, fragmented, and denervated NMJs 8 weeks after treatment. The middle panel stains for neurofilament, which represents presynaptic nerves, and the right panel stains for postsynaptic acetylcholine receptors. The graph in Figure 9C provides counts of NMJs on the medial gastrocnemius and soleus muscles. [Figure 9B] Figures 9A-9C: Figures 9A and 9B provide representative photographs of fully innervated, denervated, and partially innervated neuromuscular junctions (NMJs) in the medial gastrocnemius (MG) muscle 8 weeks after treatment with Virus A or Virus C. Wild-type mice had fully innervated NMJs, while untreated mice had little innervation at the NMJs after 2-3 weeks of age. Treated mice had a mixture of fully innervated, fragmented, and denervated NMJs 8 weeks after treatment. The middle panel stains for neurofilament, which represents presynaptic nerves, and the right panel stains for postsynaptic acetylcholine receptors. The graph in Figure 9C provides counts of NMJs on the medial gastrocnemius and soleus muscles. [Figure 9C] Figures 9A-9C: Figures 9A and 9B provide representative photographs of fully innervated, denervated, and partially innervated neuromuscular junctions (NMJs) in the medial gastrocnemius (MG) muscle 8 weeks after treatment with Virus A or Virus C. Wild-type mice had fully innervated NMJs, while untreated mice had little innervation at the NMJs after 2-3 weeks of age. Treated mice had a mixture of fully innervated, fragmented, and denervated NMJs 8 weeks after treatment. The middle panel stains for neurofilament, which represents presynaptic nerves, and the right panel stains for postsynaptic acetylcholine receptors. The graph in Figure 9C provides counts of NMJs on the medial gastrocnemius and soleus muscles. [Figure 10] FIG. 10 provides survival plots demonstrating that Virus A and Virus C improve survival in the nmd-2J mouse model. [Figure 11]Figures 11A-11D provide plots of the electrophysiological results, namely, compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE). There is a significant increase in CMAP and MUNE with both Virus A and Virus C. Viruses A and C did not differ from each other. CMAP: Measures the strength of innervation. MUNE: Estimates the number of neurons innervating the muscle. [Figure 12] Figures 12A-12C provide results from the hanging wire test in healthy mice and Em5 mice treated with virus A and virus C. Figure B shows the increase in muscle mass in healthy mice and Em5 mice treated with virus A and virus C, and Figure 12C provides the weight of the gastrocnemius muscle in relation to total body weight. [Figure 13-1] Figure 13 provides the annotated sequence of plasmid ssAAV.CB.IGHMBP2.Kan-Fw (SEQ ID NO: 7). [Figure 13-2] Figure 13 provides the annotated sequence of plasmid ssAAV.CB.IGHMBP2.Kan-Fw (SEQ ID NO: 7). [Figure 14-1] Figure 14 provides the annotated sequence of plasmid ss.AAV.P546.IGHMBP2.Kan-Fw (SEQ ID NO: 8). [Figure 14-2] Figure 14 provides the annotated sequence of plasmid ss.AAV.P546.IGHMBP2.Kan-Fw (SEQ ID NO: 8). [Figure 15] Figure 15 provides a plasmid map of ssAAV.P546.IGHMBP2.Kan-Clinical (SEQ ID NO: 17). The P546 promoter (SEQ ID NO: 4) is also referred to herein as MeCp2 or 546, and these terms may be used interchangeably. In this plasmid, the P546 promoter sequence, SV40 intron sequence, and IGHMBP2 cDNA sequence are in reverse orientation. [Figure 16-1] Figure 16 provides the annotated sequence of plasmid ssAAV.P546.IGHMBP2.Kan-Clinical (SEQ ID NO: 17). [Figure 16-2] Figure 16 provides the annotated sequence of plasmid ssAAV.P546.IGHMBP2.Kan-Clinical (SEQ ID NO: 17). [Figure 16-3] Figure 16 provides the annotated sequence of plasmid ssAAV.P546.IGHMBP2.Kan-Clinical (SEQ ID NO: 17). [Figure 17] Figure 17 provides a plasmid map of ssAAV.CB.IGHMBP2.Kan-Clinical (SEQ ID NO: 18), in which the CMV enhancer sequence, CB promoter sequence, SV40 intron sequence, and IGHMBP2 cDNA sequence are in reverse orientation. [Figure 18-1] Figure 18 provides the annotated sequence of plasmid ssAAV.CB.IGHMBP2.Kan-Clinical (SEQ ID NO: 18). [Figure 18-2] Figure 18 provides the annotated sequence of plasmid ssAAV.CB.IGHMBP2.Kan-Clinical (SEQ ID NO: 18). [Figure 18-3] Figure 18 provides the annotated sequence of plasmid ssAAV.CB.IGHMBP2.Kan-Clinical (SEQ ID NO: 18). DETAILED DESCRIPTION OF THE INVENTION

[0032] The immunoglobulin-mu binding protein 2 (IGHMBP2) gene encodes a protein that is a member 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. Mutations in the IGHMPB2 gene are known to cause spinal muscular atrophy with respiratory distress type 1 (SMARD1) and Charcot-Marie-Tooth disease type 2S (CMT2S). The majority of patient mutations in the IGHMPB2 gene are missense mutations, clustered within the helicase domain.

[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 the 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, spanning 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 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] SMARD1 Diagnosis and Progression SMARD1 is also known as autosomal recessive distal spinal muscular atrophy 1 (dHMN6 or HMN6) or distal hereditary motor neuropathy type VI (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 evisceration shown on chest radiographs, low birth weight below 3%, failure to wean, and progressive muscle weakness in the upper limbs, although distal muscles are also affected. Additional symptoms include low motor nerve conduction velocity and reduced size of myelinated fibers on sural nerve biopsy. Sensory and autonomic nerves are also affected in some patients, as evidenced by decreased pain perception, excessive sweating, constipation, and bladder incontinence. Clinical features include intrauterine growth restriction, prematurity, weak cry, and foot deformities. Symptoms usually appear between 1 and 6 months of age.

[0036] Diagnosis and progression of Charcot-Marie-Tooth hereditary neuropathy type 2 (CMT2) CMT2 is a progressive peripheral motor and sensory neuropathy generally diagnosed by one or more of the following measurements: i) nerve conduction velocity (NCV) within the normal range (>40-50 m / s) but occasionally within a mildly abnormal range (>30-40 m / s); ii) EMG showing evidence of axonal neuropathy with findings such as positive waves, polyphasic potentials, or fibrillations and reduced amplitude of evoked motor and sensory responses; iii) a significantly reduced compound motor action potential (CMAP) and / or a family history typically (but not always) consistent with a recessive pattern.

[0037] Nerve biopsy is not required for diagnosis but can be used as a method to monitor progression or confirm the diagnosis. Nerve biopsy shows loss of myelinated fibers with signs of regeneration, axonal sprouting, and atrophic axons with neurofilaments, as well as larger internodal gaps and shorter internodal lengths than controls, suggesting developmental abnormalities in internodal formation.

[0038] CMT2S involves both motor and sensory nerves more prominently, although both can be involved. Affected individuals typically have slowly progressive weakness and atrophy of the distal muscles of the feet and / or hands, usually associated with diminished tendon reflexes and mild or no sensory loss. Affected individuals usually become symptomatic between the ages of 5 and 25, but onset ranges from infancy with delayed walking to later in life. The typical presenting symptom is foot and ankle weakness. The initial physical examination findings are diminished or absent tendon reflexes with weakness of the foot dorsiflexion at the ankle.

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

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

[0041] As used herein, the term "AAV" is a general abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells where certain functions are provided by a co-infecting helper virus. Currently, there are 13 serotypes of AAV, which are characterized in the general information and overview of AAV, which 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 various serotypes are very closely related both structurally and functionally, even at the genetic level, it is fully expected that these same principles can be applied to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R.P.Tattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974).) For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes, and they all possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome and the presence of similar self-annealing segments at the ends corresponding to the "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

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

[0043] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and a polynucleotide AAV vector enclosed in the capsid.When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to mammalian cells), it is typically referred to as an "AAV vector particle" or simply an "AAV vector".Therefore, since such a vector is contained within the AAV vector particle, the production of AAV vector particles necessarily includes the production of AAV vectors.

[0044] Adeno-associated virus (AAV) is a replication-deficient parvovirus, and its single-stranded DNA genome is about 4.7 kb long, including inverted terminal repeats (ITRs).Exemplary ITR sequences can be 130 base pairs long or 141 base pairs long, 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), revised by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077, the complete genome of AAV-3 is provided under GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829, the AAV-5 genome is provided under GenBank Accession No. AF085716, the complete genome of AAV-6 is provided under GenBank Accession No. NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are provided under GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 regarding AAV-8), and the AAV-9 genome is disclosed in Gao et al. The AAV-10 genome is provided 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), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs.Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Coupled with differential splicing of a single AAV intron (e.g., at nucleotides 2107 and 2227 in 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. The Rep proteins possess multiple enzymatic properties ultimately involved 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 responsible for 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 to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, allowing the potential for targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially throughout the lifespan of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, 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 an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less important. AAV can be lyophilized. Finally, AAV-infected cells do not tolerate superinfection.

[0046] Several studies have demonstrated long-term (greater than 1.5 years) recombinant AAV-mediated protein expression in muscle. Clark et al., Hum Gene Ther, 8:659-669 (1997), Kessler et al., Proc Nat. Acad. Sc. USA, 93:14082-14087 (1996), and Xiao See, e.g., 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). Furthermore, because muscle is highly vascularized, recombinant AAV transduction resulted in the appearance of the transgene product 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). Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cellular factors for correct 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 comprises the nucleic acid molecule of the present disclosure and one or more AAV ITRs flanking 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 their derivatives). The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV mutants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background 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) comprise an rAAV genome. The term "rAAV genome" refers to a polynucleotide sequence derived from a native AAV genome that has been modified. In some embodiments, the rAAV genome has been modified to remove the native cap and rep genes. In some embodiments, the rAAV genome comprises endogenous 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the rAAV genome comprises ITRs from an AAV serotype different from the AAV serotype from which the AAV genome was derived. In some embodiments, the rAAV genome comprises a transgene of interest flanked on the 5' and 3' ends by inverted terminal repeats (ITRs). In some embodiments, the genome of an rAAV comprises a "gene cassette." In exemplary embodiments, both rAAV genomes lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome.

[0049] In some embodiments, the rAAV genome provided herein comprises one or more AAV ITRs flanking the 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 target cells to form a gene cassette. Examples of promoters include 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, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1a promoter, the hemoglobin promoter, and the creatine kinase promoter.

[0050] Additionally, 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 sequence 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 transactivation activity.

[0051] Other examples of transcriptional control elements are tissue-specific control elements, such as promoters that allow expression specifically in neurons or 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 an intron sequence to facilitate processing of the transgene RNA transcript when expressed in mammalian cells. One example of such an intron is the SV40 intron.

[0052] The rAAV genomes provided herein comprise a polynucleotide encoding the IGHMPB2 protein (SEQ ID NO: 1). In some embodiments, the rAAV genomes provided herein comprise a polynucleotide encoding a polypeptide comprising 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 comprise nucleotides 1-4397 of SEQ ID NO: 7 or nucleotides 1-4375 of SEQ ID NO: 8. In some embodiments, the rAAV genomes provided herein comprise a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 1-4397 of SEQ ID NO: 7 or nucleotides 1-4375 of SEQ ID NO: 7 or 8.

[0054] Provided herein is an rAAV genome, in some embodiments, a polynucleotide sequence that encodes an IGHMPB2 protein and hybridizes under stringent conditions to the polynucleotide sequence set forth in SEQ ID NO: 1 or its complement.

[0055] The DNA plasmid of the present disclosure contains the rAAV genome of the present disclosure. The DNA plasmid is transferred to a cell permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided in the cell, are standard in the art. rAAV production requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and helper virus functions. The AAV rep and cap genes can be derived from any AAV serotype from which a recombinant virus can be derived, and can be derived from an AAV serotype different from the rAAV genomic ITRs, 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. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0056] The 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 multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a 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. S6. USA, 79:2077-2081), the addition of a synthetic linker containing a restriction endonuclease cleavage site (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 use adenovirus or baculovirus instead of a plasmid to introduce the rAAV genome and / or rep and cap genes into the packaging cell.

[0057] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches include Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984), Tratschin et al., Mo 1. Cell. Biol. al., J. Virol., 62:1963 (1988), and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al. al., J. Virol., 63:3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365, and corresponding U.S. Pat. Nos. 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. 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 incorporated herein by reference in their entireties, with particular emphasis placed on the portions of the documents relating to rAAV production.

[0058] Thus, 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 (allogeneic 293 cells). In another embodiment, the packaging cells are non-transformed cancer cells such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus 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, including, for example, those 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 nontoxic to recipients and preferably inert at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate (e.g., phosphate-buffered saline (PBS)), citrate, or other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight polypeptides, 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 dextrins; 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). The compositions provided herein can include a pharmaceutically acceptable aqueous excipient containing a non-ionic low-osmolar compound, such as iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, or ioxilan, and the aqueous excipient containing the non-ionic low-osmolar compound can have one or more of the following properties: an osmolality of about 180 mgI / mL, about 322 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 about 20-40% non-ionic hypoosmolar compound or contrast agent, or about 25% to about 35% non-ionic hypoosmolar compound. An exemplary composition includes scAAV or rAAV viral particles formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188, and about 25% to about 35% non-ionic hypoosmolar compound. Another exemplary composition includes scAAV formulated in 1x PBS and 0.001% Pluronic® F68.

[0062] The dosage of rAAV administered in the methods of the present disclosure varies depending, for example, on the specific rAAV, mode of administration, time of administration, therapeutic goal, individual, and targeted cell type, and can be determined by standard methods in the art. Dosages 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 5×1013 , 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 16 It contains up to or more of the total viral genome.

[0063] Approximately 1×10 9 ~Approx. 1×10 10 , about 5×10 9 ~Approx. 5×10 10 , about 1×10 10 ~Approx. 1×10 11 , about 1×10 11 ~Approx. 1×10 15 vg, approx. 1×10 12 ~Approx. 1×10 15 vg, approx. 1×10 12 ~Approx. 1×10 14 vg, approx. 1×10 13 ~about 6×10 14 vg, approx. 1×10 13 ~Approx. 1×10 15 vg, and approximately 6 × 10 13 ~Approx. 1.0×10 14 Doses of 100 mg / kg are also contemplated. One dose exemplified herein is 1 x 10 mg administered via intrathecal delivery. 13 vg. Another dose exemplified herein is 1.5 x 10 13 vg.

[0064] Doses may also be expressed in units of vg / kg. Doses contemplated herein are approximately 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 / kg10 , 3×10 10 vg / kg, 4 × 10 10 vg / kg, 5 × 10 10 vg / kg, 1 × 10 11 vg / kg, approximately 1×10 12 vg / kg, approximately 1×10 13 vg / kg, approximately 1.1×10 13 vg / kg, approximately 1.2×10 13 vg / kg, approximately 1.3×10 13 vg / kg, approximately 1.5×10 13 vg / kg, approx. 2×10 13 vg / kg, approximately 2.5×10 13 vg / kg, approx. 3×10 13 vg / kg, approximately 3.5×10 13 vg / kg, approx. 4×10 13 vg / kg, approximately 4.5×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 2×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 1×10 15 vg / kg, approximately 1×10 16 vg / kg.

[0065] Approximately 1×10 9 vg / kg ~ approx. 1×10 10 vg / kg, approx. 45×10 9 vg / kg ~ approx. 5×10 10 vg / kg, approximately 1×10 10 vg / kg ~ approx. 1×10 11 vg / kg, approximately 1×10 11 vg / kg ~ approx. 1×10 15 vg / kg, approximately 1×10 12 vg / kg ~ approx. 1×10 15 vg / kg, approximately 1×10 12 vg / kg ~ approx. 1×10 14 vg / kg, approximately 1×10 13 vg / kg ~ approx. 2×10 14vg / kg, approximately 1×10 13 vg / kg ~ approx. 1×10 15 vg / kg, and approximately 6 × 10 13 vg / kg ~ approx. 1.0×10 14 Doses of 100 mg / kg are also contemplated. One dose exemplified herein is 1 x 10 mg administered via intravenous delivery. 13 vg / g. Another dose exemplified herein is 2.5 x 10 administered via intravenous delivery. 14 vg / kg.

[0066] Methods for transducing target cells with rAAV in vivo or in vitro are contemplated by the present disclosure. In vivo methods include administering an effective dose or effective multiple doses of a composition comprising an rAAV of the present disclosure to an animal (including a human) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present disclosure, an effective dose 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 extent of the disease, causes remission (partial or complete) of the disease, and / or prolongs survival. Examples of diseases contemplated for prevention or treatment by the methods of the present disclosure are SMARD1 and CMT2S.

[0067] Combination therapy is also contemplated by the present disclosure.As used herein, combination includes both simultaneous treatment and sequential treatment.As a combination with new therapy, the combination of the method of the present disclosure with standard medical care is particularly contemplated.In some embodiments, combination therapy includes administering an immunosuppressant in combination with the gene therapy disclosed herein.

[0068] Administration of an effective dose of the composition can be by any route standard 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 (particularly the AAV ITRs and capsid proteins) can be selected and / or adapted by those skilled in the art, taking into account the infection and / or disease state to be treated and the target cells / tissues expressing wild-type IGHMPB2 protein.

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

[0070] The transduction of cells by the rAAV of the present disclosure results in the sustained expression of IGHMPB2 protein.The present disclosure therefore provides the method of administering / delivering the rAAV that expresses 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 for IGHMPB2 to recipient cells either in vivo or in vitro via the replication-deficient rAAV of the present disclosure, resulting in expression of IGHMPB2 by the recipient cells.

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

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

[0074] The immunosuppressant may be an anti-inflammatory steroid, which is a steroid that reduces inflammation and suppresses or modulates the subject's immune system. Exemplary anti-inflammatory steroids are glucocorticoids such as prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, budesonide, or prednisone.

[0075] Janus kinase inhibitors are inhibitors 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] Calcineurin inhibitors bind to cyclophilin and inhibit the activity of calcineurin. Exemplary calcineurin inhibitors include cyclosporine, tacrolimus, and picecrolimus.

[0077] mTOR inhibitors reduce or inhibit the serine / threonine-specific protein kinase mTOR. Exemplary mTOR inhibitors include sirolimus, everolimus, and temsirolimus.

[0078] Immunosuppressants include immunosuppressant macrolides. The term "immunosuppressant macrolide" refers to a macrolide drug that suppresses or modulates a subject's immune system. Macrolides are a class of drugs that contain a large macrocyclic lactone ring to which one or more deoxysugars, such as cladinose or desaminoglycans, 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 immunosuppressant macrolides include tacrolimus, pimecrolimus, and sirolimus.

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

[0080] Exemplary immunosuppressive biologics include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, 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 or anti-TAC antibodies) such as basiliximab and daclizumab, as well as anti-CD3 antibodies such as muromonab-CD3, otelixizumab, teplizumab, and visilizumab, and anti-CD52 antibodies such as alemtuzumab.

[0083] The following examples are offered by way of illustration and not by way of limitation: The numerical ranges listed include each integer value within each range, including the stated integer minimum and maximum. [Example]

[0084] Example 1 - Gene therapy constructs encoding IGHMBP2 AAV genomic constructs encoding IGHMBP2 were generated as described in Figure 1, which depicts the AAV9 vector design with the full-length transcript of the 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] A human GFP cDNA clone was obtained from Origene, Rockville, MD. The IGHMBP2 cDNA alone was further subcloned into a self-complementary AAV9 genome under the control of one or more of the following: i) the P546 promoter; or v) the hybrid chicken β-actin promoter (CB). The plasmid construct also contained an intron, such as the simian virus 40 (SV40) chimeric intron, and a bovine growth hormone (BGH) polyadenylation signal (BGH polyA). The construct was packaged into either AAV9 genome.

[0086] A map for plasmid ssAAV.CB.IGHMBP2.Kan.-Fw (with the kanamycin resistance gene in the forward orientation) is set forth in Figure 2, and the sequence of the entire plasmid is provided in SEQ ID NO:7. The ssAAV.CB.IGHMBP2 vector is within and includes the ITRs of SEQ ID NO:7 and contains the nucleotide sequence as shown in Figure 13. The rAAV vector contains the 5' AAV2 ITR, a CMV enhancer, a CBA promoter, a modified SV40 intron sequence, a coding sequence for the IGHMBP2 gene, bGH polyA, and the 3' AAV2 ITR. The plasmid set forth in SEQ ID NO:7 further contains kanamycin resistance along with a pUC origin of replication. Plasmid ssAAV.CB.IGHMBP2.Kan.-Rv (with the kanamycin resistance gene in the reverse orientation) 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), where ranges refer to nucleotides in SEQ ID NO: 7.

number

[0088] A map for plasmid ssAAV.P546.IGHMBP2.Kan.-Fw (with the kanamycin resistance gene in the forward orientation) is set forth in Figure 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 Figure 14. The rAAV vector contains the 5'AAV2 ITR, the P546 promoter (also referred to herein as the MeCp2 promoter or P546 promoter), a modified SV40 intron sequence, a coding sequence for the IGHMBP2 gene, bGH polyA, and the 3'AAV2 ITR. The plasmid set forth in SEQ ID NO:8 further contains kanamycin resistance along with a pUC origin of replication. Plasmid ssAAV.P546.IGHMBP2.Kan.-Rv (with the kanamycin gene in the reverse orientation) 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), where the ranges refer to nucleotides in SEQ ID NO: 8.

number

[0090] A map for plasmid ssAAV.P546.IGHMBP2.Kan-clinical (with the P546 promoter sequence, IGHMBP2 cDNA sequence, SV40 intron, and bGH polyadenylation sequence in reverse orientation) is set forth in Figure 15, and the sequence of the entire plasmid is provided in SEQ ID NO: 17. The ssAAV.P546.IGHMBP2-clinical vector contains the nucleotide sequence within and including the ITRs 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 set forth in SEQ ID NO: 17 further contains kanamycin resistance along with a pUC origin of replication. While the kanamycin resistance gene is in the forward orientation in Figure 15, 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), where the ranges refer to nucleotides in SEQ ID NO: 17.

number

number

[0092] A map for plasmid ssAAV.CB.IGHMBP2.Kan-clinical (CMV enhancer sequence, CB promoter sequence, IGHMBP2 cDNA sequence, SV40 intron, and bGH polyadenylation sequence in reverse orientation) is set forth in Figure 17, and the sequence of the entire plasmid is provided in SEQ ID NO: 18. The ssAAV.CB.IGHMBP2-clinical vector contains the nucleotide sequence within and including the ITRs 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 3' AAV2 ITR (SEQ ID NO: 12). The plasmid set forth in SEQ ID NO: 18 further contains kanamycin resistance along with a pUC origin of replication. The kanamycin resistance gene is in the forward orientation in Figure 17, but 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), where ranges refer to nucleotides in SEQ ID NO: 18.

number

number

[0094] Example 2 - CSF delivery of IGHMBP2 gene therapy vector in mice The SMARD1 and CMT2S mouse models were used to compare the efficacy 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. Two different mouse models representing the very severe end of the disease spectrum (em3) and the moderate disease form (nmd-2J) were used in this study. [Table 6]

[0095] Em3 mouse model For the first study, nmd em3 / em3 Homozygous mice received either the IGHMBP2 gene therapy vector or a control via intracerebroventricular injection (ICV) into the cerebrospinal fluid (CSF). The "nmd" mouse mutation causes progressive degeneration of spinal motor neurons and muscle atrophy (Cox et al., Neuron 21:1327-1337, 1998). The nmd mutation was identified in a putative transcriptional activator and ATPase / DNA helicase previously described as Smbp2 or Catf1. The nmd phenotype is attenuated in a semidominant manner by a major genetic 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 1x PBS and 0.001% Pluronic F68 (represented as PBS / F68). Strength testing was performed starting 3 weeks after administration, and survival rates were also monitored. Figure 3 demonstrates that treatment with virus A and virus C appeared to reverse paralysis, but these mice remained smaller than controls.

[0097] Figure 4 shows nmd next to control mice 2–3 weeks after AAV administration. em3 / em3 Representative images of homozygous mice are provided. Mice treated with virus A or virus C showed improved clasping. In addition, mice treated with virus B (empty virus particles) were unable to spread their hind limbs compared to mice treated with viruses A and C.

[0098] Figure 5 shows the nmd em3 / em3 A survival analysis of mice is provided. Treatment with virus A or virus C restored survival time for mice (one mouse died early from each). However, virus B (empty virus particles) did not appear to restore survival. Untreated and virus B-treated mice occasionally survived, but these mice were very small and weak. As shown in Figure 6, treatment with virus A or virus C significantly reduced nmd em3 / em3 Homozygous mice had improved body weight but did not fully recover when measured 8 weeks after treatment.

[0099] For strength testing, treated mice were inverted on a wire cage lid for up to 60 seconds, and the time when they fell or reached the 60-second end point was noted. The longest time of three attempts was recorded. If a mouse failed the first attempt, which lasted 60 seconds, it was tested again to determine whether the first failed attempt was due to A) a deliberate unwillingness to participate, B) slipping, or C) an inability to complete the 60-second wire suspension. If the mouse failed the second attempt, it was given a break of approximately 3–5 minutes and tested again. The numbers in the data / graph in Figure 7 reflect the longest attempt. Figure 7 provides the time after treatment relative to the latency to fall when the mouse was suspended from the wire. Treatment with virus A showed an initial improvement, followed by a decline in strength, which later recovered to wild-type levels 8 weeks after injection. Treatment with virus C demonstrated that homozygous mice exhibited similar strength to normal mice (nmd). + / + ) and improved the strength.

[0100] In addition, treated nmd em3 / em3 Nerves from homozygous mice were extracted and evaluated. Both left and right phrenic nerves, femoral motor nerves, and femoral sensory nerves were extracted and fixed overnight in electron microscope (EM) fixative. The nerves were then washed three times with 1x PBS buffer and stored at 4°C until sent to our histology center. Axon number was determined using the semi-automated "WEKA Trainable Program" plugin in ImageJ. Treated nmd em3 / em3 A cross section of the femoral nerve from a homozygous mouse is shown in Figure 8A, and the axon area is provided in Figure 8B. All groups were significantly different from each other by Kolmogorov-Smirnov test. Treatment with virus A or virus C significantly increased the axon area.

[0101] Treated nmd 8 weeks after treatment em3 / em3 The intercostal muscle area of ​​homozygous mice was also investigated. In treated mice, 300 muscle fibers were manually traced in ImageJ from hematoxylin and eosin sections of individual tissues from each animal. Measurements are provided in pixels, and the graph in Figure 8D represents the cumulative frequency indicating the extent of muscle fiber area.

[0102] Cross sections of hindlimb muscles are shown in Figure 8C. For cross-sectional images from the hindlimb, two incisions were made at the patella and calcaneus to define leg length at the knee and ankle. A third incision was made equidistant from the patella and calcaneus. Bone and muscle morphology was examined to select images from the same region across all animals. Mice treated with virus A or virus C showed an increase in muscle area, with virus C performing best.

[0103] In addition, treatment with virus A or virus B inhibited nmd em3 / em3We partially restored neuromuscular junctions in the hindlimbs of homozygous mice. Figures 9A and 9B are representative photographs showing immunocytochemistry of MG and Sol muscles obtained from the hindlimbs 8 weeks after treatment with rAAV. MG and Sol muscles were weighed and fixed in 4% PFA. Muscles were stained with antibodies detecting neurofilament (green) as a marker for presynaptic neurons and bungarotoxin (red) as a marker for postsynaptic ACh receptors. Neuromuscular junctions (NMJs) were photographed using an SP8 Leica confocal microscope. Examples of each type of NMJ occupancy are provided in Figure 9A. Occupancy was manually determined as either fully innervated (full overlap of the red BTX acetylcholine channel and the green neurofilament channel), partially innervated (partial overlap of the red and green channels), or completely denervated (complete absence of one channel). Untreated NMJs em3 / em3 The hind limbs of homozygous mice had little innervation of the neuromuscular junction. em3 / em3 In homozygous mice, a mixture of fully innervated, fragmented, and non-innervated NMJs was found. The graph provides counts 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 received either 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 1x PBS and 0.001% Pluronic F68 (represented as PBS / F68). As described above, Figure 10 provides survival plots demonstrating that viruses A and C improved survival in nmd-2J mice.

[0105] Electromyography (EMG) is a potential clinical biomarker of efficacy. Electrophysiological results include compound muscle action potentials (CMAP), single motor unit potentials (SMUP), and motor unit number estimates (MUNE), recorded from 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 a muscle.

[0106] Briefly, two thin ring electrodes were placed on anesthetized mice to record electrophysiological results. The active (E1) ring electrode was placed on the skin over the proximal part of the gastrocnemius muscle of the hind leg at the knee joint, and the reference (E2) ring electrode was placed over the mid-metatarsal region of the foot. The skin under the ring electrodes was coated with gel to reduce impedance. Sciatic CMAP responses were obtained using supramaximal stimulation (approximately 120% of maximum) of the sciatic nerve (square-wave pulses of 0.1 ms duration and 1-10 mA intensity). Average single motor unit potential (SMUP) size and MUNE were calculated by recording incremental responses by delivering submaximal stimuli of 0.1 ms duration at a frequency of 1 Hz, while increasing the intensity in 0.026 mA steps to obtain a minimally induced arrhythmic response. Ten increments were averaged to provide the average single motor unit potential SMUP amplitude. MUNE was calculated as follows: MUNE = CMAP / mean SMUP. Peak-to-peak measurements were used for CMAP and SMUP amplitude. As shown in Figures 11A-D, there was a significant increase 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. Figures 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 effects of IGHMBP2 gene therapy vectors administered via intracerebroventricular injection (ICV) into the cerebrospinal fluid (CSF). em5 mice have a CMT2S phenotype, a sensory and motor neuron disorder, and have a survival time of approximately 10 months. 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 1x 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 greater 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 (MG) in treated and untreated mice. Figure 12B shows an increase in MG muscle mass in relation to 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 and healthy animals.

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

[0110] Patients selected for participation will be between 1 and 20 years of age and diagnosed with an IGHMBP2-related disorder, such as SMARD1 or CMT2S, as determined by genotype. Patients will receive a one-time gene transfer dose of ssAAV per patient. ssAAV will be formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, and 0.001% poloxamer 188, and delivered via intrathecal injection. Safety will be assessed based on clinical evidence and by review of the safety label. There will be a minimum of 3-4 weeks between enrollment of each subject to allow for review of safety data 30 days after gene transfer. Disease progression will be measured, and the impact of treatment on quality of life and the likelihood of long-term survival will be assessed. In certain embodiments, for example, the following are provided: (Item 1) A polynucleotide comprising: (a) one or more regulatory control elements; (b) a polynucleotide comprising the cDNA sequence of immunoglobulin-μ binding protein 2 (IGHMBP2). (Item 2) 2. The polynucleotide of item 1, wherein the regulatory control element is a CBA promoter or a P546 promoter, or a fragment thereof. (Item 3) 3. The polynucleotide of item 1 or 2, wherein the IGHMBP2 cDNA comprises a polynucleotide sequence comprising at least 95% sequence identity to SEQ ID NO: 1, or the polynucleotide sequence set forth in SEQ ID NO: 1. (Item 4) 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) 6. The rAAV of item 5, wherein the genome comprises a P546 promoter and an IGHMBP2 cDNA. (Item 7) 6. The rAAV of item 5, wherein the genome comprises a CBA promoter and an IGHMBP2 cDNA. (Item 8) 8. The rAAV according to any one of items 5 to 7, wherein the rAAV is of the 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 described in 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) 11. The composition of claim 10, further comprising an agent that increases the viscosity or density of the composition. (Item 12) 11. The composition of claim 10, wherein the agent is an imaging agent. (Item 13) 13. The composition of any one of items 9 to 12, wherein the composition is formulated for direct injection into cerebrospinal fluid, intraventricular delivery, intrathecal delivery, or intravenous delivery. (Item 14) 14. The composition of any one of items 10-13, wherein the composition is formulated for intrathecal delivery and comprises a dose of rAAV or rAAV particles of about 1e13vg per patient to about 1e15vg per patient. (Item 15) 14. The composition of any one of items 10-13, wherein the composition is formulated for intravenous delivery and comprises a dose of rAAV or rAAV particles of about 1e13vg / kg to about 2e14vg / kg. (Item 16) A method for treating an IGHMBP2-associated disorder in a subject in need thereof, comprising administering the rAAV described in any one of Items 5 to 8, the rAAV particle described in Item 9, or the composition described in any one of Items 10 to 15. (Item 17) 17. The method of item 16, wherein the disorder is SMARD1 or CMT2S. (Item 18) 18. The method of item 16 or 17, wherein the subject has a mutation in the IGHMBP2 gene. (Item 19) 19. The method of any one of items 16 to 18, wherein the rAAV or rAAV particles are administered by direct injection into cerebrospinal fluid, intraventricular delivery, intrathecal delivery, or intravenous delivery. (Item 20) 20. The method of any one of items 16-19, wherein a dose of rAAV or rAAV particles of about 1e13vg per patient to about 1e15vg per patient is administered to the subject by intrathecal delivery. (Item 21) 21. The method of any one of items 16 to 20, wherein a dose of rAAV or rAAV particles of about 1e13vg / kg to about 2e14vg / kg is administered to the subject by intravenous delivery. (Item 22) 22. The method according to any one of items 16 to 21, further comprising administering an immunosuppressant. (Item 23) Use of the rAAV of any one of items 5 to 8, the rAAV particle of item 9, or the composition of any one of items 10 to 15 in the preparation of a medicament for the treatment of an IGHMBP2-associated disorder. (Item 24) 24. The use according to item 23, wherein the disorder is SMARD1 or CMT2S. (Item 25) 25. The use according to item 23 or 24, wherein the medicament is formulated for direct injection into the cerebrospinal fluid, intraventricular delivery, intrathecal delivery, or intravenous delivery. (Item 26) 26. The use of any one of items 23 to 25, wherein the medicament comprises a dose of rAAV or rAAV particles of about 1e13vg per patient to about 1e15vg per patient, and the medicament is formulated for intrathecal delivery to the subject. (Item 27) 26. The use of any one of items 23 to 25, wherein the medicament comprises a dose of rAAV or rAAV particles of about 1e13vg / kg to about 2e14vg / kg, and the medicament is formulated for intravenous delivery to the subject. (Item 28) A composition comprising the rAAV of any one of items 5 to 8, the rAAV particle of item 9, or the composition of any one of items 10 to 15 for the treatment of an IGHMBP2-associated disorder. (Item 29) 29. The composition of item 28, wherein the disorder is SMARD1 or CMT2S. (Item 30) 30. The composition of claim 28 or 29, wherein the composition is formulated for direct injection into the cerebrospinal fluid, intraventricular delivery, intrathecal delivery or intravenous delivery. (Item 31) 31. The composition of any one of items 28-30, wherein the composition comprises a dose of rAAV or rAAV particles of about 1e13vg per patient to about 1e15vg per patient, and wherein the composition is formulated for intrathecal delivery. (Item 32) 31. The composition of any one of items 28-30, wherein the composition comprises a dose of rAAV or rAAV particles of about 1e13vg / kg to about 2e14vg / kg, and wherein the composition is formulated for intravenous delivery. (Item 33) 33. The composition according to any one of items 28 to 32, wherein the composition further comprises an immunosuppressant.

Claims

1. A plasmid comprising a nucleic acid sequence comprising 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.

2. The plasmid described in claim 1, wherein the plasmid lacks AAV rep and cap genes.

3. A plasmid described in claim 1 or claim 2, further comprising a selectable marker.

4. A host cell comprising a plasmid described in any one of claims 1 to 3.

5. A packaging cell comprising a plasmid described in any one of claims 1 to 4.

6. The packaging cells described in claim 5, selected from the group consisting of HeLa cells, PerC6 cells, 293 cells, low-passage 293 cells, MRC-5 cells, WI-38 cells, Vero cells, and FrhL-2 cells.

7. A method for producing recombinant adeno-associated virus (rAAV) particles, comprising transforming a cell with a plasmid, wherein the plasmid comprises a nucleotide sequence that is at least 90% identical to 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.

8. The method of claim 7, wherein the cells are selected from the group consisting of HeLa cells, 293 cells, PerC.6 cells, MRC-5 cells, WI-38 cells, Vero cells, and FRhL-2 cells.

9. The method described in claim 8, further comprising purifying rAAV particles from the supernatant of the cells.

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