WO2024081706A1 - Adeno-associated virus delivery to treat spinal muscular atrophy with respiratory distress type 1 (smard1) and charcot-marie-tooth type 2s (cmt2s) - Google Patents

Abstract

The disclosure provides gene therapy vectors, such as adeno-associated virus (AAV), designed for treatment of an immunoglobulin-μ binding protein 2 (IGHMBP2)-related disorder.

Description

ADENO-ASSOCIATED VIRUS DELIVERY TO TREAT SPINAL MUSCLE ATROPHY WITH RESPIRATAORY DISTRESS TYPE 1 (SMARD1) AND CHARCOT-MARIE-TOOTH TYPE 2S (CMT2S) [0001] This application claims priority benefit of U.S. Provisional Application No. 63/379,048, filed October 11, 2022, which is incorporated by reference herein in its entirety. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY [0002] This application contains, as a separate part of the disclosure, a Sequence Listing in computer-readable form which is incorporated by reference in its entirety and identified as follows: 58394_Seqlisting.xml; Size: 71,934 bytes; Created: October 10, 2023. FIELD OF THE INVENTION [0003] The disclosure provides gene therapy vectors, such as adeno-associated virus (AAV), designed for treatment of a disorder associated with a mutation in the immunoglobulin-μ binding protein 2 (IGHMBP2) gene, such as Spinal Muscle Atrophy with Respiratory Distress Type 1 (SMARD1) and Charcot-Marie Tooth 2S (CMT2S) . The disclosed rAAV provide a wild type IGHMBP2 cDNA to a subject suffering from SMARD1 or CMT2S, which results in expression of the wild type protein. BACKGROUND [0004] The immunoglobulin-μ binding protein 2 (IGHMBP2) gene encodes a member of the Upf1-like group within the helicase superfamily 1 (SF1). This protein is known to have a helicase domain, the R3H domain, the zinc finger domain, and the nuclear localization signal sequence. IGHMBP2 is ubiquitously expressed and comprises 15 exons encoding 993 amino acids corresponding to a 110 kDa gene product. The exact role of IGHMBP2 protein in disease development is unknown. The normal IGHMBP2 is known to play a role in ribosomal RNA maturation and translation, immunoglobulin-class switching, pre-mRNA maturation, and transcription regulation by either DNA binding activity or interaction with TATA-binding protein. The IGHMPB2 protein has been classified as a member of the Upf1- like group within the helicase superfamily1 (SF1), consisting of the helicase domain, the R3H domain, the zinc finger domain, and the 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). The majority 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 that is characterized by early distal lower limb muscle atrophy following proximal muscle weakness and respiratory failure. SMARD1 patients exhibit paralysis of the diaphragm between the ages of 6 weeks and 13 months. The 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) neuropathies are the most common hereditary neuropathies. 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, although typically less severe. Patients have slowly progressing distal muscle weakness with muscle atrophy of the upper and lower limbs. The subtypes of CMT2 are similar clinically and distinguished only by molecular genetic findings. Most subtypes of CMT2 are inherited in an autosomal dominant manner; however, some are inherited in an autosomal recessive manner. Recessive loss of function mutations in the IGHMBP2 gene are known to cause CMT2, now subclassified as CMT2S. [0007] There are no current therapies for CMT2S and management involves treating the symptoms. Thus, there is a need to develop gene replacement therapies to treat SMARD1 and CMT2S. SUMMARY [0008] The disclosure provides for method methods of treating an IGHMBP2-related disorder in a subject in need thereof comprising administering an rAAV or an rAAV particle described herein are specifically contemplated. In some embodiments, the methods further comprise administering an immunosuppressing agent prior to, after or simultaneously with the rAAV or rAAV particle. An IGHMPB2-related disorder includes a disorder or disease caused by a mutation that results in a loss of function of the IGHMPB2 protein or causes reduced expression of the IGHMPB2 protein. An IGHMPB2-related disorder may be any disease or disorder that is related to reduced expression or activity of the IGHMPB2 protein, despite the cause of the reduced expression or activity. In disclosure contemplates IGHMPB2-related disorders in subjects that are homozygotes for a mutation in the IGHMPB2 gene or heterozygotes for a mutation in the IGHMPB2 gene. For example, an IGHMBP2-related disorder is a neurological disorder that is associated with the presence of a mutation in the IGHMBP2 gene, such as SMARD1 or CMT2S. The IGHMBP2-related disorder also includes disorders wherein the patient has a mixed phenotype, such that the severity of the neurological disorder is between the severity observed in patients suffering from SMARD1 and CMT2S. [0009] In an exemplary aspect, the disclosure provides for methods of treating an IGHMBP2 -related disorder in a subject in need thereof comprising administering to the subject a dose of about 7x10¹³ vg to about 9.9 x 10¹³ vg of a rAAV or rAAV particles, wherein the rAAV or rAAV particles comprises a nucleotide sequence that is at least 90% or 95% identical to nucleotides 1 to 4375 of SEQ ID NO: 8 or that is at least 90% or 95% identical to nucleotides 1 to 4364 of SEQ ID NO: 17 or the nucleotide sequence comprises nucleotides 1 to 4375 of SEQ ID NO: 8 or the nucleotide sequence comprises nucleotides 1 to 4364 of SEQ ID NO: 17. For example. the dose is based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vector. [0010] In addition, in any of the disclosed methods, the rAAV or rAAV particles are of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13 or Anc80, AAV7m8 and their derivatives. [0011] In one aspect, in any of the disclosed methods, the dose of rAAV or rAAV particles is about 9.0 x 10¹³ vg, for example the dose of 9.0 x 10¹³ vg is based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vector. In any of the disclosed methods, the dose of rAAV or rAAV particles is administered as a single injection having a volume of 5 mL. [0012] In any of the disclosed methods, the dose of rAAV or rAAV particles is administered in an aqueous composition comprising an agent that increases the viscosity or density of the formulation. For example, the agent that increases the viscosity or density of the formulation is about 20 to 40% of the composition. In any of the disclosed methods, the agent that increases the viscosity or density of the composition is a non-ionic, low-osmolar compound or contrast agent. [0013] In any of the disclosed methods, the composition comprises about 10 mM to about 30 mM Tris, 0.5 mM to about 5 mM MgCl2, about 100 mM to about 500 mM NaCl and about 0.0001% to about 0.01% poloxamer 188. For example, the composition comprises about 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, and 0.001% poloxamer 188. [0014] In any of the disclosed methods, the dose of rAAV or rAAV particles is administered by for direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery or intravenous delivery. In addition, any of the disclosed methods may further comprise a step of administering an immunosuppressing agent. In some embodiments, the dose of rAAV or rAAV particles is administered simultaneously, prior to or after administration of an immunosuppressing agent. [0015] In another aspect, described herein is the use of a rAAV or an rAAV particle described herein in the preparation of a medicament for the treatment of an IGHMBP2- related disorder, such as SMARD1 or CMT2S. For example, any of the disclosed medicaments are formulated for direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery or intravenous delivery. In some embodiments, the medicament is administered simultaneously, prior to or after administration of an immunosuppressing agent. [0016] For example, the disclosure provides for use of a dose of a rAAV or rAAV particles for the preparation of a medicament for treating an IGHMBP2 -related disorder in a subject in need thereof, wherein the a dose is about 7x10¹³ vg to about 9. x 10¹³ vg of rAAV or rAAV particles, and wherein the rAAV or rAAV particles comprises a nucleotide sequence that is at least 90% or 95% identical to nucleotides 1 to 4375 of SEQ ID NO: 8 or that is at least 90% or 95% identical to nucleotides 1 to 4364 of SEQ ID NO: 17, or comprises nucleotides 1 to 4375 of SEQ ID NO: 8 or comprises nucleotides 1 to 4364 of SEQ ID NO: 17. For example. the dose is based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vector. [0017] In another aspect, described herein is a composition comprising an rAAV or an rAAV particle described herein suitable for treating an IGHMBP2-related disorder, such as SMARD1 or CMT2S. For example, any of the disclosed compositions are formulated for direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery or intravenous delivery. In some embodiments, the composition is administered simultaneously, prior to or after administration of an immuno-suppressing agent. In another embodiment, the composition further comprises an immuno-suppressing agent. [0018] For example, the disclosure provides for compositions suitable for treating an IGHMBP2-related disorder in a subject in need thereof, wherein the composition comprises a dose is about 7x10¹³ vg to about 9. x 10¹³ vg of rAAV or rAAV particles, and wherein the rAAV or rAAV particles comprises a nucleotide sequence that is at least 90% or 95% identical to nucleotides 1 to 4375 of SEQ ID NO: 8 or that is at least 90% or 95% identical to nucleotides 1 to 4364 of SEQ ID NO: 17, or comprises nucleotides 1 to 4375 of SEQ ID NO: 8 or comprises nucleotides 1 to 4364 of SEQ ID NO: 17. For example. the dose is based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vector. [0019] In addition, in any of the disclosed uses or compositions, the rAAV or rAAV particles are of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13 or Anc80, AAV7m8 and their derivatives. [0020] In one aspect, in any of the disclosed uses or compositions, the dose of rAAV or rAAV particles is about 9.0 x 10¹³ vg, for example the dose of 9.0 x 10¹³ vg is based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vector. In any of the disclosed uses or compositions, the dose of rAAV or rAAV particles is administered as a single injection having a volume of 5 mL. [0021] In any of the disclosed uses or compositions, the dose of rAAV or rAAV particles is administered in an aqueous composition comprising an agent that increases the viscosity or density of the formulation. For example, the agent that increases the viscosity or density of the aqueous composition is about 20 to 40% of the composition. In any of the disclosed methods, the agent that increases the viscosity or density of the aqueous composition is a non-ionic, low-osmolar compound or contrast agent. [0022] In any of the disclosed uses or compositions, the aqueous composition comprises about 10 mM to about 30 mM Tris, 0.5 mM to about 5 mM MgCl2, about 100 mM to about 500 mM NaCl and about 0.0001% to about 0.01% poloxamer 188. For example, the composition comprises about 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, and 0.001% poloxamer 188. [0023] In any of the disclosed uses or compositions, the dose of rAAV or rAAV particles is administered by for direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery or intravenous delivery. In addition, any of the disclosed uses or compositions, the dose of rAAV or rAAV particles is administered with an immunosuppressing agent. In some embodiments, the dose of rAAV or rAAV particles is administered simultaneously, prior to or after administration of an immunosuppressing agent. [0024] In another embodiment, the disclosure provides methods, uses and compositions wherein the rAAV comprises comprising a nucleotide sequence that encodes a functional IGHMBP2 protein, wherein the nucleotide has, e.g., 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, wherein the protein retains IGHMBP2 activity. For example, the nucleotide sequence that encodes a functional IGHMBP2 protein may comprise one or more base pair substitutions, deletions or insertions which do affect the function of the IGHMBP2. Furthermore, the nucleotide sequence that encodes a functional IGHMBP2 protein may comprise one or more base pair substitutions, deletions or insertions may increase or reduce expression of the IGHMBP2 protein, and this change in expression pattern may be desired for treatment of an IGHMBP2-related disorder, such as SMARD1 or CMT2S. [0025] In another embodiment, the disclosure provides methods, uses and compositions wherein the rAAV compria nucleotide sequence that encodes a functional IGHMBP2 protein, wherein the protein comprises an amino acid sequence that has, e.g., 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 that encodes a functional IGHMBP2 protein may comprise one or more amino acid substitutions, deletions or insertions which do affect the function of the IGHMBP2 protein. [0026] The terms “sequence identity”, “percent sequence identity”, or “percent identical” in the context of nucleic acid or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired. The percentage identity of the sequences can be determined by techniques known in the art. For example, homology can be determined by a 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: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. [0027] In another aspect, the disclosure provides for an rAAV construct contained in the plasmid comprising the nucleotide sequence of SEQ ID NO: 7. For example, the ssAAV9.CB.IGHMBP2 vector comprises the nucleotide sequence within and inclusive of the ITR’s of SEQ ID NO: 7 and shown in Figure 5. The rAAV vector comprises the 5’ ITR, CMV enhancer, CB promoter, a modified SV40 intron sequence, the coding sequence for the human IGHMBP2 gene, bGH polyA, and 3’ ITR. In one embodiment, the vector comprises nucleotides 1-4397 of SEQ ID NO: 7. The nucleotides within the ITRs may be in forward or reverse orientation. For example, the CMV enhancer sequence, CB promoter sequence, the SV40 sequence, human IGHMBP2 gene sequence, and bGH polyA sequence and may be in forward or reverse orientation. In another embodiment, the vector comprises a nucleotide sequence that has about at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotides of 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. [0028] In an exemplary embodiment, the disclosure provides for an rAAV construct contained in the plasmid comprising the nucleotide sequence of SEQ ID NO: 18. For example, the ssAAV9.CB.IGHMBP2-clinical vector comprises the nucleotide sequence within and inclusive of the ITR’s of SEQ ID NO: 18 and shown in Figure 10. The rAAV vector comprises the 5’ ITR as set out in SEQ ID NO: 19. In addition, the rAAV vector comprises the CMV enhancer, CB promoter, a modified SV40 intron sequence, the coding sequence for the human IGHMBP2 gene, bGH polyA, each in reverse orientation, and 3’ ITR as set out in SEQ ID NO: 12. In one embodiment, the vector comprises nucleotides 1-4386 of SEQ ID NO: 18. The nucleotides within the ITRs may be in forward or reverse orientation. In another embodiment, the vector comprises a nucleotide sequence that has about at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotides of 1-4386 of SEQ ID NO: 18. The plasmid set forth in SEQ ID NO 18 further comprises kanamycin resistance gene and a pUC origin of replication. The kanamycin resistance gene may be in forward or reverse orientation. [0029] In a further aspect, the disclosure provides for an rAAV construct contained in the plasmid comprising the nucleotide sequence of SEQ ID NO: 8. For example, the ssAAV9.P546.IGHMBP2 vector comprises the nucleotide sequence within and inclusive of the ITR’s of SEQ ID NO: 8 and shown in Figure 6. The rAAV vector comprises the 5’ ITR, P546 promoter, a modified SV40 intron sequence, the coding sequence for the human IGHMBP2 gene, bGH polyA, and 3’ ITR. In one embodiment, the vector comprises nucleotides 1-4375 of SEQ ID NO: 8. The nucleotides within the ITRs may be in forward or reverse orientation. For example, the P546 promoter sequence, the SV40 sequence, the human IGHMBP2 gene, and bGH polyA sequence, and may be in forward or reverse orientation. In another embodiment, the vector comprises a nucleotide sequence that has about at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotides of 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. [0030] In an exemplary embodiment, the disclosure provides for an rAAV construct contained in the plasmid comprising the nucleotide sequence of SEQ ID NO: 17. For example, the ssAAV9.P546.IGHMBP2-clinical vector comprises the nucleotide sequence within and inclusive of the ITR’s of SEQ ID NO: 17 and shown in Figure 8. The rAAV vector comprises the 5’ ITR as set out in SEQ ID NO: 19. In addition, the rAAV vector comprises theP546 promoter sequence, a modified SV40 intron sequence, the coding sequence for the human IGHMBP2 gene, and bGH polyA sequence, each in reverse orientation, and 3’ ITR as set out in SEQ ID NO: 12. In one embodiment, the vector comprises nucleotides 1-4364 of SEQ ID NO: 17. The nucleotides within the ITRs may be in forward or reverse orientation. In another embodiment, the vector comprises a nucleotide sequence that has about at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotides of 1-4364 of SEQ ID NO: 17. The plasmid set forth in SEQ ID NO 17 further comprises kanamycin resistance gene and a pUC origin of replication. The kanamycin resistance gene may be in forward or reverse orientation. [0031] In another aspect, described herein is a recombinant adeno-associated virus (rAAV) having a genome comprising a polynucleotide sequence described herein. In some embodiments, the rAAV is of the 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 the rAAV comprises promoter fragment and an IGHMBP2 cDNA. [0032] In some embodiments, the genome of the rAAV comprises a CBA promoter and an IGHMBP2 cDNA. An exemplary genome comprises the CBA promoter, and the IGHMBP2 cDNA such as the ssAAV9.CB.IGHMBP2, the rAAV set out as nucleotides 1-4397 of SEQ ID NO: 7 or the rAAV set out as nucleotides 1-4386 of SEQ ID NO: 18. . [0033] In some embodiments, the genome of the rAAV comprises a P546 promoter and an IGHMBP2 cDNA. An exemplary genome comprises the P546 promoter, the IGHMBP2 cDNA such as the ssAAV9.P546.IGHMBP2, the rAAV set out as nucleotides 1-4375 of SEQ ID NO: 8 or the rAAV set out as nucleotides 1-4364 of SEQ ID NO: 17. [0034] 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 fragment of the promoter retains promoter activity. [0035] In another aspect, described herein is an rAAV particle comprising an rAAV described herein. [0036] In any of the methods, uses or compositions disclosed herein, the rAAV or any of the viral particles are administered in a composition. In some embodiments, the compositions further comprise an agent that increases the viscosity and/or density of the composition. For example, in some embodiments that agent is a contrast agent. The contrast agent may be 20 to 40% non-ionic, low-osmolar compound or contrast agent or about 25% to about 35% non-ionic, low-osmolar compound, such as iohexol. The disclosed composition may be formulated for any means of delivery, such as direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery or intravenous delivery. [0037] In some embodiments, the composition comprises an agent that increase the viscosity of the composition by about 0.05%, or by about 1% or by 1.5% or about 2% or by about 2.5% or by about 3% or by about 4% or by about 5% or by about 6% or by about 7% or by about 8% or by about 9% or by about 10%. In some embodiments, an agent increases the viscosity of the composition by about 1% to about 5%, or by about 2% to 12%, or by about 5% to about 10%, or by about 1% to about 20% or by about 10% to about 20%, or by about 10% to about 30%, or by about 20% to about 40%, or by about 20% to about 50%, or by about 10% to about 50%, or by about 1% to about 50%. [0038] In some embodiments, the composition comprises an agent that increases the density of the composition by about 0.05%, or by about 1% or by 1.5% or about 2% or by about 2.5% or by about 3% or by about 4% or by about 5% or by about 6% or by about 7% or by about 8% or by about 9% or by about 10%. In some embodiments, an agent increases the density of the composition by about 1% to about 5%, or by about 2% to 12%, or by about 5% to about 10%, or by about 1% to about 20%, or by about 10% to about 20%, or by about 10% to about 30%, or by about 20% to about 40% or by about 20% to about 50%, or by about 10% to about 50%, or by about 1% to about 50%. [0039] For example, the disclosed composition is formulated for intrathecal delivery and comprises of a dose of rAAV or rAAV particles of about 1e13 vg per patient to about 1e15 vg per patient. For example, the does is 9e13 vg. [0040] In addition, the disclosed composition is formulated for intravenous delivery and comprises of a dose of rAAV or rAAV particles of about 1e13 vg/kg to about 2e14 vg/kg. [0041] In any of the methods, uses and compositions disclosed herein, the subject has a mutation in the IGHMBP2 gene. These mutations include those currently known, such as those set out in Table 1 or 2 herein, or a mutation(s) in the IGHMBP2 gene identified in the future that is associated with a neurological disorder. [0042] A "subject," as used herein, can be any animal, and may also be referred to as the patient. Preferably the subject is a vertebrate animal, and more preferably the subject is a mammal, such as a domesticated farm animal (e.g., cow, horse, pig) or pet (e.g., dog, 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 a subject ranging in age from 1 to 10 years. In some embodiments, the subject is 4 to 15 years of age. The subject, in on embodiment, is an adolescent subject, such as 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, the rAAV or the viral particle is 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 of about 1e13 vg per patient to about 1e15 vg per patient is administered by intrathecal delivery to the subject. In addition, in any of the disclosed methods, a dose of rAAV or rAAV particles of a dose of about 1e13 vg/kg to about 2e14 vg/kg is administered by intravenous delivery to the subject. BRIEF DESCRIPTION OF THE FIGURES [0043] Figure 1 provides schematics for AAV9.CB.IGHMBP2 (promoter 1 = CB promoter) and AAV9.P546.IGHMBP2 (promoter 2 = P546 promoter). [0044] Figure 2 provides a plasmid map of ssAAV.CB.IGHMBP2.Kan-Fw (SEQ ID NO: 7). [0045] Figure 3 provides a plasmid map of ssAAV.P546.IGHMBP2.Kan-Fw (SEQ ID NO: 8). This vector is identical to the ssAAV.P546.IGHMBP2.Kan described herein. The P5465 promoter (SEQ ID NO: 4) is also referred to as MeCp2 or 546 herein and these terms may be used interchangeably. [0046] Figure 4 demonstrates efficient targeting of brain and spinal cord in non-human primates (NHPs) dosed with 3.54 x 10¹³ vg/animal via lumbar intrathecal injection regardless of size or age. [0047] Figure 5 provide the annotated sequence of the plasmid ssAAV.CB.IGHMBP2.Kan-Fw (SEQ ID NO: 7). [0048] Figure 6 provides an annotated sequence of the plasmid ss.AAV.P546.IGHMBP2.Kan-Fw (SEQ ID NO: 8). [0049] Figure 7 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 as MeCp2 or 546 herein and these terms may be used interchangeably. In this plasmid, the P546 promoter sequence, SV40 intron sequence and the IGHMBP2 cDNA sequence are in reverse orientation. [0050] Figure 8 provides the annotated sequence of the plasmid ssAAV.P546.IGHMBP2.Kan-Clinical (SEQ ID NO: 17). [0051] Figure 9 provides a plasmid map of ssAAV.CB.IGHMBP2.Kan-Clinical (SEQ ID NO: 18). In this plasmid, the CMV enhancer sequence, the CB promoter sequence, SV40 intron sequence and the IGHMBP2 cDNA sequence are in reverse orientation. [0052] Figure 10 provides the annotated sequence of the plasmid ssAAV.CB.IGHMBP2.Kan-Clinical (SEQ ID NO: 18). DETAILED DESCRIPTION [0053] The immunoglobulin-μ binding protein 2 (IGHMBP2) gene encodes protein that is a member of the Upf1-like group within the helicase superfamily1 (SF1), consisting of the helicase domain, the R3H domain, the zinc finger domain, and the 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 the patient mutations in the IGHMPB2 gene cluster within the helicase domain and are missense mutations. IGHMPB2 Mutations [0054] 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 that has seven putative helicase motifs and a DEAD box-like motif typical for RNA helicases. The mutations may lead to dysfunction of helicase activity. The IGHMPB2 gene is known to have 15 exons. Mutations in the IGHMPB2 gene were found to be associated with SMARD1 and CMT2S. [0055] At this time, almost 40 disease-causing mutations in the IGHMBP2 gene have been described. Although these mutations do not follow a clear genotype-phenotype relationship, mutations that reduce or abrogate protein function or production lead to a more severe phenotype compared to mutations that retain some function or protein production. The most severe phenotype is found in individuals with mutant IGHMBP2 that leads to the development of spinal muscular atrophy with respiratory distress type 1 (SMARD1). SMARD1 is a devastating disease, which initially manifests as inspiratory stridor and weak cry, leading to diaphragmatic paralysis 2,4. SMARD1 patients also develop distal lower-limb muscle weakness that progresses proximally and to the upper limbs, concomitant with central and autonomic nervous system abnormalities. Death generally occurs from respiratory failure or complications from mechanical ventilation in early childhood. The estimated incidence of SMARD1 is 1 in 100,0004. [0056] There are about 26 known IGHMPB2 mutation that cause SMARD1. The mutations include recessive missense mutations, nonsense mutations, frameshifts, a in- frame deletion, a frameshift insertion and a splice donor site mutation and span the 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 methods of treatment are not limited to disorders caused by the mutations provided in Table 1 or those that are known at the time of filing as other mutations of the IGHMPB2 may be identified in the future that cause SMARD1. Table 1

[0057] Charcot-Marie-Tooth (CMT) disease type 2S (CMT2S) falls on the less severe side of the spectrum in patients harboring mutant IGHMBP25,6. CMT2S usually begins to manifest in the first decade of life by more slowly progressive distal muscle weakness, atrophy in the lower and upper limbs, sensorimotor neuropathy, decreased reflexes, and eventual loss of movement in the arms and legs. One study estimated the prevalence of mutant IGHMBP2 CMT2S to be ~1.6% of CMT disease, which itself has a prevalence of approximately 1 in 10,000 to 1 in 1,1250 in various populations globally. Importantly, even CMT2S patients can develop respiratory complications and diaphragmatic weakness requiring respiratory support at later time points in the disease course. [0058] The known IGHMPB2 mutation causing CMT2S are an autosomal recessive mutation that causes 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 in Table 2 below. The disclosed gene therapy vectors and methods of treatment are not limited to disorders caused by the mutations provided in Table 2 or those that are known at the time of filing as other mutations of the IGHMPB2 may be identified in the future that cause CMT2S. Table 2

Diagnosis and Progression of SMARD1 [0059] SMARD1 is also known as autosomal recessive distal spinal muscular atrophy 1 distal hereditary motor neuronopathy type VI (dHMN6 or HMN6) or distal muscular dystrophy type 1 (DSMA-1). This disorder is a variant of infantile SMA. The most prominent symptoms of SMARD1 are severe respiratory distress resulting from diaphragmatic paralysis with eventration shown on chest x-ray, low birth weight below the 3^rd centile, inability to wean and progressive muscle weakness in the upper limbs and distal muscles are also affected. Additional symptoms include low motor nerve conduction velocities, and a reduction in the size of myelinated fibers on sural nerve biopsy. Sensory and autonomic nerves were also affected in some patients, as demonstrated by decreased pain perception, excessive sweating, constipation, and bladder incontinence. Clinical features include: intrauterine growth retardation, prematurity, weak cry, and foot deformities. The symptoms usually present at age 1 month to 6 months. [0060] SMARD1 presents as distal symmetric muscle weakness, particularly of the lower limbs, resulting in foot deformities, areflexia, fatty finger pads, joint contractures, and respiratory distress or failure due to diaphragmatic paralysis (unilateral or bilateral) without thorax deformity.. The respiratory failure often has a sudden onset, preceding the muscle weakness and, without instant mechanical ventilation, leads to early death1. Most patients require mechanical ventilation within the first 12 months and are unable to wean from it. Patients often have a history of intrauterine growth restriction, low birth weight, and premature birth. In some cases, they can also present signs of autonomic neuropathy, as heart blocks or arrhythmias, constipation, gastroparesis, bladder incontinence, hyperhidrosis, and high blood pressure. In the past years, considerable variability in the presentation of SMARD1 and its phenotype in childhood has been reported, including cases with severe early infantile onset sensory-motor neuropathy and patients with a later, juvenile onset of respiratory failure. Diagnosis and Progression of Charcot-Marie-Tooth Hereditary Neuropathy 2 (CMT2) [0061] CMT2 is a progressive peripheral motor and sensory neuropathy and it is generally diagnoses as measuring one or more of i) nerve conduction velocities (NCVs) that are with the normal range (>40-50 m/s) although occasionally in mildly abnormal range (30-40 m/s), ii) EMG testing that shows evidence of axonal neuropathy with such findings as positive waves, polyphasic potentials, or fibrillations and reduced amplitudes of evoked motor and sensory responses, iii) greatly reduced compound motor action potentials (CMAP) and/or family history that is typically (but not always) consistent with recessive manner. [0062] CMT2S is characterized by a more slowly progressive sensorimotor polyneuropathy. However, it can be quite variable in its age of onset and severity. On the milder end of the spectrum, individuals have onset in childhood, do not require respiratory support, and maintain ambulation into adulthood. On the more severe end, symptoms develop in infancy (foot deformities), diaphragmatic weakness develops in childhood, and loss of ambulation occurs in childhood/adolescence. CMT2S is reserved for those who do not present with respiratory symptoms; however, there are reports in literature of patients presenting with late onset respiratory failure leading to sudden death without previous diaphragmatic involvement. [0063] A nerve biopsy is not required for diagnosis but it may be used as a method of monitoring progression or confirming diagnosis. Nerve biopsies show loss of myelinated fibers with signs of regeneration, axonal sprouting, and atrophic axons with neurofilaments, and large nodal gaps and shorter internodal lengths than controls, suggesting a developmental abnormality of internode formation. [0064] CMT2S more prominently involves the nerves of the motor system rather than the sensory system, although both are involved. The affected individual typically has slowly progressive weakness and atrophy of distal muscles in the feet and/or hands usually associated with depressed tendon reflexes and mild or no sensory loss. Affected individuals usually become symptomatic between ages five and 25 years, though onset ranges from infancy with delayed walking to after the third decade. The typical presenting symptom is weakness of the feet and ankles. The initial physical findings are depressed or absent tendon reflexes with weakness of foot dorsiflexion at the ankle. [0065] The 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 extremities. Brisk tendon reflexes and extensor plantar responses have also been reported as well as asymmetric muscle atrophy in up to 15% of affected individuals. Vocal cord or phrenic nerve involvement resulting in difficulty with phonation or breathing has been observed. In addition, restless leg syndrome and sleep apnea have also been observed. AAV Gene Therapy [0066] The present disclosure provides for gene therapy vectors, e.g. rAAV vectors, expressing the IGHMPB2 cDNA and methods of treating an IGHMPB2-related disorder. The IGHMPB2-related disorder includes disorders caused by a mutation that causes a loss of function of the IGHMPB2 protein or causes reduced expression of the IGHMPB2 protein. Furthermore, any disease or disorder that is related to reduced expression or activity of the IGHMPB2 protein, despite the cause of the reduced expression or activity. [0067] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are currently thirteen serotypes of AAV that have been characterized General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol.1, pp.169- 228, and Berns, 1990, Virology, pp.1743-1764, Raven Press, (New York). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (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 apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear 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 analogous self-annealing segments at the termini that correspond to "inverted terminal repeat sequences" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. [0068] An "AAV vector" as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. [0069] An "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a 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, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle. [0070] Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length including an inverted terminal repeat (ITRs). Exemplary ITR sequences may be 130 base pairs in length or 141 base pairs in length, such as the ITR sequence set out in SEQ ID NOS: 11, 12 and 19. There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the 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 other examples, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos.7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); 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). Cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45 (2007). Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start 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). [0071] AAV possesses 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 of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non- dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the 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 replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56^oC to 65^oC for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection. [0072] Multiple studies have demonstrated long-term (>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). Moreover, because muscle is highly vascularized, recombinant AAV transduction has resulted in 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). Moreover, Lewis et al., J Virol, 76: 8769-8775 (2002) demonstrated that skeletal myofibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics. [0073] Recombinant AAV genomes of the disclosure comprise nucleic acid molecule of the disclosure and one or more AAV ITRs flanking a nucleic acid molecule. AAV DNA in the rAAV genomes may be from any AAV serotype for 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). Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. [0074] The provided recombinant AAV (i.e., infectious encapsidated rAAV particles) comprise a rAAV genome. The term “rAAV genome” refers to a polynucleotide sequence that is 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 the endogenous 5’ and 3’ inverted terminal repeats (ITRs). In some embodiments, the rAAV genome comprises ITRs from an AAV serotype that is 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 repeat (ITR). In some embodiments, the rAAV genome comprises a “gene cassette.” In exemplary embodiments, the genomes of both rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genomes. [0075] The rAAV genomes provided herein, in some embodiments, comprise one or more AAV ITRs flanking the transgene polynucleotide sequence. The transgene polynucleotide sequence is operatively 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 are the pIRF promoter, 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, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as 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. [0076] Additionally provided herein are a CB promoter sequence, a P546 promoter sequence, and promoter sequences 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 CBA (SEQ ID NO: 3) or P546 (SEQ ID NO: 4) sequence which exhibit transcription promoting activity. [0077] Other examples of transcription control elements are tissue specific control elements, for example, promoters that allow expression specifically within neurons or specifically within astrocytes. Examples include 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 a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter. The gene cassette may also include intron sequences to facilitate processing of a transgene RNA transcript when expressed in mammalian cells. One example of such an intron is the SV40 intron. [0078] rAAV genomes provided herein comprises a polynucleotide (SEQ ID NO: 1) encoding IGHMPB2 protein. In some embodiments, the rAAV genomes provided herein comprises a polynucleotide that encodes 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). [0079] rAAV genomes provided herein comprises a 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 comprises a polynucleotide that at least: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequences of nucleotides 1- 4397 of SEQ ID NO: 7 or nucleotides 1- 4375 of SEQ ID NO: 7 SEQ ID NO: 7 or 8. [0080] rAAV genomes provided herein, in some embodiments, a polynucleotide sequence that encodes an IGHMPB2 protein and that hybridizes under stringent conditions to the polynucleotide sequence set forth in SEQ ID NO: 1 or the complement thereof. [0081] DNA plasmids of the disclosure comprise rAAV genomes of the disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1-deleted adenovirus or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome 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. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety. [0082] A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells. [0083] 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 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; WO 95/13365 and corresponding U.S. Patent No. 5,658.776 ; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; 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 herein, with particular emphasis on those sections of the documents relating to rAAV production. [0084] The disclosure thus provides packaging cells that produce infectious rAAV. In one embodiment packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), 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). [0085] The rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example, 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 WO 98/09657. [0086] Compositions provided herein comprise rAAV and a pharmaceutically acceptable excipient or excipients. Acceptable excipients are nontoxic to recipients and are 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; 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 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). Compositions provided herein can comprise a pharmaceutically acceptable aqueous excipient containing a non-ionic, low-osmolar compound such as iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, or ioxilan, where the aqueous excipient containing the non-ionic, low-osmolar compound can have one or more of the following characteristics: about 180 mgI/mL, an osmolality by vapor-pressure osmometry of about 322mOsm/kg water, an osmolarity of about 273mOsm/L, an absolute viscosity of about 2.3cp at 20°C and about 1.5cp at 37°C, and a specific gravity of about 1.164 at 37°C. [0087] Exemplary compositions comprise an agent to increase the viscosity and/or density of the composition. For example, the composition comprises a contrast agent to increase the viscosity and/or density of the composition. Exemplary compositions comprise about 20 to 40% non-ionic, low-osmolar compound or contrast agent or about 25% to about 35% non-ionic, low-osmolar compound. An exemplary composition comprises scAAV or rAAV viral particles formulated in 20mM Tris (pH8.0), 1mM MgCl₂, 200mM NaCl, 0.001% poloxamer 188 and about 25% to about 35% non-ionic, low-osmolar compound. Another exemplary composition comprises scAAV formulated in and 1X PBS and 0.001% Pluronic F68. [0088] Dosages of rAAV to be administered in methods of the disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the time of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Dosages may be expressed in units of viral genomes (vg). Dosages contemplated herein include about 7x10¹³ vg, about 7.5x10¹³ vg, about 8x10¹³ vg, about 8.5x10¹³ vg, about 8.6 x10¹³ vg, about 8.7 x10¹³ vg, about 8.8x10¹³ vg, about 8.9x10¹³ vg, about 9.0x10¹³ vg, about 9.1x10¹³ vg, about 9.2x10¹³ vg, about 9.3x10¹³ vg, about 9.4x10¹³ vg, about 9.5x10¹³ vg, about 9.6x10¹³ vg, about 9.7x10¹³ vg, about 9.8x10¹³ vg, about 9.9x10¹³ vg, or more total viral genomes. For example. the dose is based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vec _[0089] Dosages of about 7x10¹³ vg to about 9.9 x 10¹³ vg, about 7.5 x10¹³ vg to about 9.5 x 10¹³ vg, 7.5 x10¹³ vg to about 9.0 x 10¹³ vg, about 8.0 x10¹³ vg to about 9.9 x 10¹³ vg, about 8.0 x10¹³ vg to about 9.5 x 10¹³ vg, about 8.0 x10¹³ vg to about 9.0 x 10¹³ vg, about 8.5 x10¹³ vg to about 9.9 x 10¹³ vg, about 8.5 x10¹³ vg to about 9.5 x 10¹³ vg, about 8.5 x10¹³ vg to about 9.3 x 10¹³ vg, about 8.5 x10¹³ vg to about 9.0 x 10¹³ vg, about 8.8 x10¹³ vg to about 9.9 x 10¹³ vg, about 8.8 x10¹³ vg to about 9.5 x 10¹³ vg, about 8.8 x10¹³ vg to about 9.0 x 10¹³ vg, about 8.7 x10¹³ vg to about 9.5 x 10¹³ vg, about 8.7 x10¹³ vg to about 9.2 x 10¹³ vg, about 8.7 x10¹³ vg to about 9.0 x 10¹³ vg, about 9.0 x10¹³ vg to about 9.9 x 10¹³ vg are also contemplated. One dose exemplified herein is 9.0 x10¹³ vg administered via intrathecal delivery. For example. the dose is based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vectors. [0090] Dosages are also may be expressed in units of vg/kg. Dosages of about 1x 10¹³ vg/kg to about 1x10¹⁴ vg/kg, 7x10¹³ vg/kg to about 9.9 x 10¹³ vg/kg, about 7.5 x10¹³ vg/kg to about 9.5 x 10¹³ vg/kg, 7.5 x10¹³ vg/kg to about 9.0 x 10¹³ vg/kg, about 8.0 x10¹³ vg/kg to about 9.9 x 10¹³ vg/kg, about 8.0 x10¹³ vg/kg to about 9.5 x 10¹³ vg/kg, about 8.0 x10¹³ vg/kg to about 9.0 x 10¹³ vg/kg, about 8.5 x10¹³ vg/kg to about 9.9 x 10¹³ vg/kg, about 8.5 x10¹³ vg/kg to about 9.5 x 10¹³ vg/kg, about 8.5 x10¹³ vg/kg to about 9.3 x 10¹³ vg/kg, about 8.5 x10¹³ vg/kg to about 9.0 x 10¹³ vg/kg, about 8.8 x10¹³ vg/kg to about 9.9 x 10¹³ vg/kg, about 8.8 x10¹³ vg/kg to about 9.5 x 10¹³ vg/kg, about 8.8 x10¹³ vg/kg to about 9.0 x 10¹³ vg/kg, about 8.7 x10¹³ vg/kg to about 9.5 x 10¹³ vg/kg, about 8.7 x10¹³ vg/kg to about 9.2 x 10¹³ vg/kg, about 8.7 x10¹³ vg/kg to about 9.0 x 10¹³ vg/kg, about 9.0 x10¹³ vg/kg to about 9.9 x 10¹³ vg/kg are also contemplated. One dose exemplified herein is about 1x10¹³ vg/kg, about 2x10¹³ vg/kg, about 3x10¹³ vg/kg, about 4x10¹³ vg/kg, about 5,x10¹³ vg/kg, 6 about x10¹³ vg/kg, about 7x10¹³ vg/kg, about 8x10¹³ vg/kg, about 8.5x10¹³ vg/kg, about 9.0 x10¹³ vg/kg, about 9.5 x10¹³ vg/kg, or about 9.9 x10¹³ vg/kg administered via intrathecal delivery. For example. the dose is based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vectors [0091] Methods of transducing a target cell with rAAV, in vivo or in vitro, are contemplated by the disclosure. The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a rAAV of the disclosure to an animal (including a human being) in need thereof. If the dose is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development of a disorder/disease, the administration is therapeutic. In embodiments of the disclosure, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival. Example of a disease contemplated for prevention or treatment with methods of the disclosure is SMARD1 and CMT2S. [0092] Combination therapies are also contemplated by the disclosure. Combination as used herein includes both simultaneous treatment and sequential treatments. Combinations of methods of the disclosure with standard medical treatments are specifically contemplated, as are combinations with novel therapies. In some embodiments, the combination therapy comprises administering an immunosuppressing agent in combination with the gene therapy disclosed herein. [0093] Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. Route(s) of administration and serotype(s) of AAV components of the rAAV (in particular, the AAV ITRs and capsid protein) of the disclosure may be chosen and/or matched by those skilled in the art taking into account the infection and/or disease state being treated and the target cells/tissue(s) that are to express the wild type IGHMPB2 protein. [0094] The disclosure provides for local administration and systemic administration of an effective dose of rAAV and compositions of the disclosure. For example, systemic administration is administration into the circulatory system so 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 implantation. [0095] Transduction of cells with rAAV of the disclosure results in sustained expression of the IGHMPB2 protein. The present disclosure thus provides methods of administering/delivering rAAV which express IGHMPB2 protein to an animal, preferably a human being. These methods include transducing cells with one or more rAAV of the present disclosure. [0096] The term “transduction” is used to refer to the administration/delivery of the coding region of the IGHMPB2 to a recipient cell either in vivo or in vitro, via a replication-deficient rAAV of the disclosure resulting in expression of IGHMPB2 the recipient cell. Immunosuppressing Agents [0097] The immunosuppressing agent may be administered before or after the onset of an immune response to the rAAV in the subject after administration of the gene therapy. In addition, the immunosuppressing agent may be administered simultaneously with the gene therapy or the protein replacement therapy. The immune response in a subject includes an adverse immune response or an inflammatory response following or caused by the administration of rAAV to the subject. The immune response may be the production of antibodies in the subject in response to the administered rAAV. [0098] Exemplary immunosuppressing agents include glucocorticosteroids, janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, cyctostatic agents such as purine analogs, methotrexate and cyclophosphamide, inosine monophosphate dehydrogenase (IMDH) inhibitors, biologics such as monoclonal antibodies or fusion proteins and polypeptides, and di peptide boronic acid molecules, such as Bortezomib. [0099] The immunosuppressing agent may be an anti-inflammatory steroid, which is a steroid that decreases inflammation and suppresses or modulates the immune system of the subject. Exemplary anti-inflammatory steroid are glucocorticoids such as prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, budesonide or prednisone. [00100] Janus kinase inhibitors are inhibitors of the JAK/STAT signaling pathway by targeting one or more of the Janus kinase family of enzymes. Exemplary janus kinase inhibitors include tofacitinib, baricitinib, upadacitinib, peficitinib, and oclacitinib. [00101] Calcineurin inhibitors bind to cyclophilin and inhibits the activity of calcineurin Exemplary calcineurine inhibitors includes cyclosporine, tacrolimus and picecrolimus. [00102] mTOR inhibitors reduce or inhibit the serine/threonine-specific protein kinase mTOR. Exemplary mTOR inhibitors include sirolimus, everolimus, and temsirolimus. [00103] The immunosuppressing agents include immune suppressing macrolides. The term “immune suppressing macrolides” refer to macrolide agents that suppresses or modulates the immune system of the subject. A macrolide is a class of agents that comprise a large macrocyclic lactone ring to which one or more deoxy sugars, such as cladinose or desoamine, are attached. The lactone rings are usually 14-, 15-, or 16-membered. Macrolides belong to the polyketide class of agents and may be natural products. Examples of immunosuppressing macrolides include tacrolimus, pimecrolimus, and sirolimus. [00104] Purine analogs block nucleotide synthesis and include IMDH inhibitors. Exemplary purine analogs include azathioprine, mycophenolate and lefunomide. [00105] Exemplary immunosuppressing biologics include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinenumab, vedolizumab, basiliximab, belatacep, and daclizumab. [00106] In particular, the immunosuppressing agent is an anti-CD20 antibody. The term anti-CD20 specific antibody refers to an antibody that specifically binds to or inhibits or reduces the expression or activity of CD20. Exemplary anti-CD20 antibodies include rituximab, ocrelizumab or ofatumumab. [00107] Additional examples of immuosuppressing antibodies include anti-CD25 antibodies (or anti-IL2 antibodies or anti-TAC antibodies) such as basiliximab and daclizumab, and anti-CD3 antibodies such as muromonab-CD3, otelixizumab, teplizumab and visilizumab, anti-CD52 antibodies such as alemtuzumab. [00108] The following EXAMPLES are provided by way of illustration and not limitation. Described numerical ranges are inclusive of each integer value within each range and inclusive of the lowest and highest stated integer. EXAMPLES [00109] Experiments using mouse models of SMARD1 and CMT2S to compare the effect of CSF delivery of AAV expressing IGHMBP2 are described in the International Patent Application WO 2021/102435; which is incorporated by reference herein in its entirety. Example 1 – Gene Therapy Constructs Encoding IGHMBP2 [00110] AAV genome constructs encoding IGHMBP2 were generated as set forth in Figure 1, which depicts the AAV9 vector design with the full-length transcript of IGHMBP2 cDNA under the control of ubiquitous promoters. The promoters contemplated for inclusion in these constructs are either i) the cmv-enhancer chicken beta actin promoter (CBA; SEQ ID NO: 3) or a synthetic truncated methyl CpG binding protein 2 (MeCp2) promoter referred to as P546 (SEQ ID NO: 4) or 546. [00111] 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 either i) the P546 promoter or v) the hybrid chicken β- Actin promoter (CB). The plasmid construct also included an intron such as the simian virus 40 (SV40) chimeric intron, and a Bovine Growth Hormone (BGH) polyadenylation signal (BGH PolyA). The constructs were packaged into either AAV9 genome. [00112] The map for plasmid ssAAV.CB.IGHMBP2.Kan.-Fw (the kanamycin resistance gene is in the forward orientation) is set out in Figure 2 and the sequence of the entire plasmid is provided in SEQ ID NO: 7. The ssAAV.CB.IGHMBP2 vector comprises the nucleotide sequence within and inclusive of the ITR’s of SEQ ID NO: 7 and as shown in Figure 5. The rAAV vector comprises the 5’ AAV2 ITR, CMV enhancer, CBA promoter, a modified SV40 intron sequence, the coding sequence for the IGHMBP2 gene, bGH polyA, and 3’ AAV2 ITR. The plasmid set forth in SEQ ID NO: 7 further comprises kanamycin resistance with pUC origin of replication. Plasmid ssAAV.CB.IGHMBP2.Kan.-Rv (in which the Kanamycin resistance gene is in reverse orientation) is provided as SEQ ID NO: 9. [00113] Table 3 shows the molecular features of the plasmid ssAAV.CB.IGHMBP2.Kan.- Fw (SEQ ID NO: 7), in which range refers to the nucleotides in SEQ ID NO: 7 and ► indicates the kanamycin gene is in the forward orientation. Table 3

[00114] The map for plasmid ssAAV.P546.IGHMBP2.Kan.-Fw (kanamycin resistance gene is in the forward orientation) is set out in Figure 3 and the sequence of the entire plasmid is provided in SEQ ID NO: 8. The ssAAV.P546.IGHMBP2 vector comprises the nucleotide sequence within and inclusive of the ITR’s of SEQ ID NO: 8 and as shown in Figure 6. The rAAV vector comprises the 5’ AAV2 ITR, P546 promoter (also denoted herein as MeCp2 promoter or P546 promoter), a modified SV40 intron sequence, the coding sequence for the IGHMBP2 gene, bGH polyA, and 3’AAV2 ITR. The plasmid set forth in SEQ ID NO:8 further comprises kanamycin resistance with pUC origin of replication. Plasmid ssAAV.P546.IGHMBP2.Kan.-Rv (in which the kanamycin gene is in reverse orientation) is provided as SEQ ID NO: 10. [00115] The AAV2 ITR sequences are identical on both ends, but in opposite orientation. The 5’ ITR with the terminal resolution site (trs) allows replication from this ITR and facilitates generation of the monomeric single-stranded vector packaging. The AAV2 ITR sequences function as both the origin of vector DNA replication and the packaging signal of the vector genome in the vector producer cells when AAV and adenovirus helper functions are provided in trans. As such, the ITR sequences represent the only cis element that is required for the vector genome to be replicated and packaged into AAV particles. While the ITR sequences are derived from AAV2, these constructs were into AAV9 viral particles. The P546 promoter allows widespread expression of the transgene in neurons and astrocytes. The promoter allows expression at a more moderate level compared to the high expressing promoters used in previous studies, further improving the safety profile of this vector. The full-length consensus IGHMBP2 human cDNA is used for expression followed by the bGH polyA signal. The plasmid backbone also contains a kanamycin resistance gene for antibiotic selection during plasmid amplification [00116] Table 4 shows the molecular features of the plasmid ssAAV.P546.IGHMBP2.Kan.-Fw (SEQ ID NO: 8), in which range refers to the nucleotides in SEQ ID NO: 8 and the ► indicates the kanamycin resistance gene in the forward orientation. Table 3

[00117] The map for plasmid ssAAV.P546.IGHMBP2.Kan-clinical (the P546 promoter sequence, IGHMBP2 cDNA sequence, the SV40 intron and the bGH polyadenylation sequence are in reverse orientation) is set out in Figure 7 and the sequence of the entire plasmid is provided in 17. The ssAAV.P546.IGHMBP2-clinical vector comprises the nucleotide sequence within and inclusive of the ITR’s, and as shown in Figure 8. The rAAV vector comprises the 5’ AAV2 ITR (SEQ ID NO: 19), P546 promoter, a modified SV40 intron sequence, the 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 comprises kanamycin resistance with pUC origin of replication. The kanamycin resistance gene is in the forward orientation in Figure 7, but plasmids with the kanamycin resistance gene in the reverse orientation are also contemplated. [00118] Table 5 shows the molecular features of the plasmid ssAAV.P546.IGHMBP2.Kan- clinical (SEQ ID NO: 17), in which range refers to the nucleotides in SEQ ID NO: 17 and ► indicates the element is in the forward orientation and the ◄ indicates the element is in the reverse orientation.

Table 5 C

[00119] The map for plasmid ssAAV.CB.IGHMBP2.Kan-clinical (the CMV enhancer sequence, CB promoter sequence, IGHMBP2 cDNA sequence, the SV40 intron and the bGH polyadenylation sequence are in reverse orientation) is set out in Figure 9 and the sequence of the entire plasmid is provided in SEQ ID NO: 18. The ssAAV.CB.IGHMBP2- clinical vector comprises the nucleotide sequence within and inclusive of the ITR’s, and as shown in Figure 10. The rAAV vector comprises the 5’ AAV2 ITR (SEQ ID NO: 19), CMV enhancer, CB promoter, a modified SV40 intron sequence, the 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 comprises kanamycin resistance with pUC origin of replication. The kanamycin resistance gene is in the forward orientation in Figure 9, but plasmids with the kanamycin resistance gene in the reverse orientation are also contemplated. [00120] Table 6 shows the molecular features of the plasmid ssAAV.CB.IGHMBP2.Kan- clinical (SEQ ID NO: 18), in which range refers to the nucleotides in SEQ ID NO: 18 and ► indicates the element is in the forward orientation and the ◄ indicates the element is in the reverse orientation. Table 6

Example 2 –Toxicology and Biodistribution Study in Non Human Primate [00121] The purpose of this study was to assess safety of ssAAV9.P546.IGHMBP2 delivered via a single intrathecal (IT) injection at a dose of 6.30 x 10¹³ vg/animal followed by Trendelenburg tilting in cynomolgus The safety and efficacy studies were conducted while qualification of the final titration assay (a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vector) was still ongoing. During that time, viral vector concentrations were determined using a non-qualified ddPCR assay directed against the AAV2 ITR sequences. The doses used in the pre-clinical studies were calculated based on the AAV2 ITR assay. Following qualification of the bGH polyA ddPCR assay, a bridging study was executed to determine a conversion factor of 1.70-fold ± 0.3 between the two titration assays. The clinical dose for the proposed clinical trial was selected based on the pre-clinical efficacy and safety data with the established conversion factor applied. [00122] Effects of this treatment were evaluated at 2, 4, 8, 12, and 24 weeks post- injection via histopathology (4, 12, 24 weeks only), blood and serum chemistry analysis (4, 8, 12, and 24 weeks only), and humoral and cellular immune response analysis. Transgene expression was also confirmed at 4 and 24 weeks post-injection in all three levels of the spinal cord, dorsal root ganglia, as well as various regions of the brain. The study also included control animals injected with an equal volume of control article (0.9% saline). [00123] To test vector safety, the effects of treatment at 1 day and at 4, 8, 12, and 24 weeks were analyzed by hematology, serum chemistry, AAV9 antibodies, and T-cell reactivity against AAV9 and human IGHMBP2 (Table 7). T-cell reactivity was additionally assessed at 2 weeks post-injection. Safety was also evaluated by histopathology; at terminal necroscopy at 4, 12, and 24 weeks, major organs were harvested, fixed, and processed for histopathology analysis. Samples were sent to a third party board-certified pathologist for independent assessment. Brain and spinal cord transgene expression of vector derived IGHMBP2 was assessed at 4, 12, and 24 weeks by ddPCR. Additional parameters assessed during the study included weekly body weights and clinical observations. Table 7. Study Design of Safety and Biodistribution Studies of Research Grade ssAAV9.P546.IGHMBP2 in NHPs.

Rationale for Dose [00124] A one-time lumbar IT injection was performed for delivery of the ssAAV9.P546.IGHMBP2 vector at 6.30 x 10¹³ vg/animal according to according to an AAV2 ITR ddPCR assay. Table 8 provides the administered dose based on the ITR titer assay and the conversion of the dose to the qualified bGH polyA titration assay used for dosing of patients. The numerical relationship between the two assays was determined by a bridging study. [00125] Table 8. CSF-AAV9-IGHMBP2-NHP-001 Dose Based on Different Titration Methods. l^)*

*The dose calculation (vg/animal) for the bGH poly A ddPCR titer is based on the conversion factor between the two assays, which is 1.70. This fold difference was determined in a bridging study. [00126] Previous studies conducted in NHPs to compare the targeting efficiency of viral vector demonstrate that with the injection protocol used, there is no observable difference in distribution or expression when looking across age and size of animals. This likely has to do with the development of the nervous system as the number of cells mostly increases in prenatal phases while the main post-natal growth focuses on increasing connectivity (dendrites and synapses) and myelination. Thus, the number of cells that need to be targeted does not change very significantly at different ages of the animals. The NHP study conducted with ssAAV9.P546.IGHMBP2 also supports that age and size do not impact targeting distribution and expression since there was a broad range of animal age and weight enrolled in the clinical trial described below. To remain consistent with the planned dosing regimen in humans, all NHPs in this study, regardless of age and weight, received the same dose of 6.30 x 10¹³ vg/animal by AAV2 ITR ddPCR titer (equivalent to 3.54 x 10¹³ vg/animal application of conversion factor to the bGH polyA ddPCR titer obtained in the bridging study). Importantly, this dose reflects a higher viral vector burden per number of cells as planned for the phase I/IIa clinical trial considering that the number of cells in the nervous system between cynomolgus macaques and humans differ by approximately 13- fold. This relatively higher dose was chosen for the NHP safety study to generate an additional safety margin to better assess over-expression toxicity. Results [00127] The presented safety study was designed to evaluate the safety of ssAAV9.P546.IGHMBP2 treatment in a broad age range of 16- to 55-month old cynomolgus macaques after a single IT injection. Results show that all cynomolgus macaques survived the injection procedure and initial 24-hour observation period without any major signs of distress. The most notable observations were transient tail and limb stiffness in one test article-treated and one saline-treated animal. Transient tail stiffness after injection was observed in previous studies and is most likely due to the presence of tail nerve roots in the lumbar site of injection. All animals displaying tail stiffness recover within a few minutes post- injection. All animals maintained a healthy weight during the course of this study with minimal to no fluctuation compared to baseline. [00128] All subjects generally had normal red and white blood cell counts, hematocrit, platelet counts, and white blood cell sub-populations (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), with a few minor and transient excursions outside of reference ranges. Most subjects were within or close to reference range in total protein, globulin, aspartate aminotransferase, alanine aminotransferase, total bilirubin, creatinine, calcium, sodium, potassium, and chloride. Levels were generally within reference range for albumin, albumin/globulin, alkaline phosphatase, gamma-glutamyl transferase, blood urea nitrogen (BUN), BUN/creatinine ratio, with some transient excursions outside reference range. Overall, serum chemistry indicated very little changes from baseline, no stress to the liver during the study course and no alterations were seen to only occur in test article-treated animals. The low values found, especially for the liver transaminases and other parameters that could indicate liver or muscle injury (ALP, ALT, AST, GGT, bilirubin, CK), indicate that the test article was well-tolerated and did not cause excessive liver stress. [00129] Transgene expression was analyzed in NHPs 4 and 24 weeks post-injection in all regions of the spinal cord, as well as dorsal root ganglia and multiple brain regions including motor cortex, temporal lobe cortex, cerebellum, striatum, thalamus, and amygdala. Transgene expression was similar in all animals analyzed with fluctuations between individual brain regions and animals without correlation to age or weight of the animals. The differences are likely due to slightly varying side of biopsy between animals. There was no difference in level of transgene expression between 4 weeks and 24 weeks post-injection. [00130] Binding ELISAs were performed to detect antibodies against the AAV9 capsid. All animals that received the test article developed antibodies against the AAV9 capsid, which was evident at 2 weeks post-injection except for animal 16C66, which was still negative at 2 weeks post-injection and antibodies were only detected starting from 4 weeks post-injection. One of the saline-injected control animals was positive for AAV9 antibodies at baseline and remained positive throughout the study, the other two control animals remained negative at all time points. [00131] ELISpot analysis for T-Cell reactivity against AAV9 and the human IGHMBP2 protein for all groups at all time points remained negative. Pathology [00132] Pathology was analyzed by a board-certified third party veterinary pathologist who has extensive experience with AAV gene therapy programs. At 4 weeks post-injection, the test article ssAAV9.P546.IGHMBP2 did not induce lesions in any protocol-specified tissue. In particular, no evidence was seen of IGHMBP2-related tissue injury, such as neuron degeneration, necrosis, or reactive response (e.g., gliosis or inflammation in central nervous system tissue). [00133] At 12 weeks, AAV class-related changes, such as mononuclear cell inflammation associated with neuron necrosis or mononuclear cell infiltration, were found in the sacral dorsal root ganglia and lumbar spinal cord dorsal funiculi of test article-treated animals. In the brain, mononuclear cell infiltration was observed in the choroid plexus and meninges of one animal. All changes were localized and interpreted to be non-adverse due to their minimal severity. [00134] At 24 weeks, the test article ssAAV9.P546.IGHMBP2 was associated with several AAV class-related changes. In dorsal root ganglia, this included mononuclear cell inflammation associated with neuron necrosis, mononuclear cell infiltration, and increased satellite glial cellularity. These findings were more common in lumbar and sacral dorsal root ganglia but were occasionally seen in cervical and thoracic dorsal root ganglia. Minimal degeneration of nerve fibers in the spinal cord in areas harboring dorsal root ganglia axons was evident in one animal. These changes were interpreted to be non-adverse due to their minimal severity. Human IGHMBP2 Transgene Expression [00135] Expression levels in brain and spinal cord were available from 4 NHPs sacrificed at 4 and 24 weeks post-injection, respectively are shown in Figure 4. All animal showed good distribution of the viral vector with individual variability that might be due to differences in biopsy location or individual parameters such as breathing and heart rate. Importantly, the treatment was safe and well-tolerated in all animals. [00136] While vector derived IGHMBP2 expression levels between individual NHPs were variable, there was no correlation between weight or age of the animal and expression levels. For example, very similar levels of expression were found in the spinal cord of the 1.94 kg heavy 16-month old animal vs. the 55-month old 6 kg heavy animal on all levels of the spinal cord while another animal (H17C121, 3.45 kg) showed higher levels of transduction on all levels. Similar observations were made in the brain regions, while both the 1.94 kg and 6 kg heavy animals showed lower expression levels in the motor cortex compared to the other two animals, levels were similar in the temporal lobe cortex and higher in amygdala for the 6 kg heavy animal. Overall, this data indicates that the spread and targeting efficiency of ssAAV9.P546.IGHMBP2 was similar regardless of age and size of the animals. Differences in expression levels could be due to slight variation in biopsy location or individual expression differences between animals. [00137] All test article-treated animals developed an antibody response against the AAV9 capsid. However, none of the treated animals showed T-cell reactivity at a test article dose of 3.54 x 10¹³ vg/animal (6.30 x 10¹³ vg/animal based on ITR ddPCR). [00138] Overall, there were no observed safety signals at a dose of 3.54 x 10¹³ vg/animal (6.30 x 10¹³ vg/animal based on ITR ddPCR) at any time point and all findings were interpreted to be non-adverse. Conclusion [00139] In conclusion, hematology, serum chemistry, immune response analysis, and histopathology results indicate that ssAAV9.P546.IGHMBP2 treatment was well-tolerated and safe in six 16-to 55-month old cynomolgus macaques up to 6 months post-injection. As mentioned previously, the selected NHP study dose of 3.54 x 10¹³ vg/animal (6.30 x 10¹³ vg/animal based on ITR ddPCR) represents a higher viral vector burden per cell compared to participants in the proposed phase I/II clinical trial (9.00 x 10¹³ vg per participant based on bGH polyA ddPCR titer), which underlines the strong safety profile of the vector. [00140] The intrathecal route of administration including a single lumbar intrathecal injection followed by Trendelenburg tilting used in the NHP toxicology study is the same route and procedure used in the human clinical trial described in Example X. There were no treatment- or procedure-related adverse reactions observed across the studies. [00141] In the NHP safety study, a dose of 3.54 x 10¹³ vg/animal (calculated using the qualified bGH polyA based ddPCR assay used for the clinical trial) was used to ensure a sufficient safety margin. Based on the fold change for number of neurons, the dose used in the NHP study would be equivalent to 4.83 x 10¹⁴ vg/patient (see Table 8). In terms of safety margins, the proposed clinical dose of 9.0 x 10¹³ vg/patient is still 5.4 times lower than the scaled dose that would be equivalent to what was tested in the NHPs. Importantly, no safety signals were observed in the NHP safety study at any time point tested including hematology, serum chemistry, immunology, and histopathology Table 9. Dose Comparison per Number of Neurons Between NHPs and Planned Dose for Human Patients. in l se P

*The dose calculation (vg/animal) for NHP efficacious dose is based on the conversion factor of 1.70, which was determined in the bridging study. [00142] Although AAV9 is capable of efficiently crossing the blood-brain-barrier (BBB), our recent studies indicate that direct CSF delivery via IT administration reduces the amount of viral vector needed to equally target cells throughout the CNS vs. systemic intravenous injection (Patel et al., Mol Ther.22(3):498-510, 2014, McCarty et al. Gene Ther. 2001;8(16):1248-1254, 2001). Thus, CSF delivery allows for optimal targeting of the most affected cell type in SMARD1 and CMT2S disease - the motoneurons in the brain and spinal cord - while reducing the exposure of peripheral organs to the virus, making the treatment safer for the patients. Example 3 – Human Phase I/IIa Gene Therapy Study [00143] Overexpression of IGHMBP2 has been shown to be well-tolerated in wild-type mice and expression of IGHMBP2 was highly effective in three disease mouse models spanning the entire severity spectrum of SMARD1 and CMT2S. In the following experiments, a truncated version of the MeCP2 (P546) promoter that allows for widespread expression of the transgene in neurons and astrocytes at more moderate levels compared to the chicken beta-actin promoter used in other gene therapy programs (Foust et al., Mol. Ther.21(12):2148-2159, 2013; Meyer et al., Mol. Ther.23(3): 477-487, 2015). Lumbar intrathecal delivery of AAV9 has been used in several other clinical trials also using the same promoter (P546)). Lumbar intrathecal delivery of AAV9 is known to be better tolerated than systemic delivery and the treatment was known to be safe and well-tolerated in other human clinical trials. [00144] This is an intrathecal phase I/IIa gene therapy study for treatment of patients with loss-of-function mutations in IGHMBP2. Individuals harboring mutations in IGHMBP2 display a spectrum of disease severity ranging from spinal muscular atrophy with respiratory distress type 1 (SMARD1) to Charcot-Marie-Tooth disease type 2S (CMT2S). The primary objective of this study is to determine the safety and tolerability of intrathecal (IT) administration of ssAAV9.P546.IGHMBP2, a single-stranded (ss) adeno-associated virus serotype 9 (AAV9) carrying the IGHMBP2 cDNA under control of a truncated mouse methyl CpG binding protein 2 promoter (called P546). SMARD1 patients are affected in infancy and show progressive muscle weakness, diaphragm paralysis, and respiratory distress. CMT2S patients usually display a slowly progressive weakness, which first involves the distal muscles with disease onset usually within the first decade of life. [00145] In this study, 6-10 pediatric subjects (2 months to less than 14 years of age) with IGHMBP2-related disease receive a single dose of 9.0 x 10¹³ vg/subject administered directly to the CSF via lumbar puncture. Subjects having two confirmed pathogenic variants in the IGHMBP2 gene will be eligible, and 6-10 subjects (approximately 2-3 per cohort) matching the inclusion/exclusion criteria will be enrolled in the study. [00146] Direct administration of the viral vector to the CSF does not require adjusting the dose to body weight as the size of the brain and spinal cord differ much less amongst individuals compared to body weight. In addition, the brain and spinal cord display a unique development and growth pattern that differs from other organs such that the neuronal growth component mainly happens prenatally. Thus, the brain nuclei, cortical structures, and major connections and organization are already established at birth. Cortical neurogenesis and migration are also completed within the first week post-birth (Herculano-Houzel, Proc Natl Acad Sci U S A.109(SUPPL.1):10661-10668, 2012). Post-natally, the brain mainly matures and grows in size by fine tuning and establishing long-lasting connections rather than by adding a significant amount of neurons. This maturation process includes neuronal death, elimination of existing synapses, growth of new synapses, glial expansion, as well as myelination. Hence, the number of targetable cells (especially neurons) is already established at, or shortly after, birth and the main growth of the brain results from myelination and increase in connectivity. The extrapolation of the clinical dose was achieved by comparing the number of neurons between species and scaling up the dose accordingly. The rationale for this approach is that the efficacy of treatment is dependent on targeting a sufficient number of cells. This rationale is supported by the observation that even in milder disease models, a similar dose-response was seen compared to the more severe mouse models. Neurons were used as a surrogate for number of cells in the brain and spinal cord as the ratio of neurons and glial cells are similar between species (Herculano-Houzel, Glia 62(9): 1377-1391, 2014). The most efficacious dose in mice was 1.30 x 10¹¹ vg/animal (by ITR ddPCR; equivalent to 7.29 x 10¹⁰ vg/animal by bGH polyA ddPCR following application of conversion factor obtained in the bridging study). The number of neurons between mice and humans differ by approximate 1,211-fold (Herculano-Houzel, Proc Natl Acad Sci U S A. 109(SUPPL.1):10661-10668, 2012). Based on these calculations, a dose of 8.82 x 10¹³ vg or 9.0 x 10¹³ vg per patient was identified as a dose expected to deliver a durable and highly efficacious dose to SMARD1 and CMT2S patients. This dose is supported by the safety studies in mice and NHPs. [00147] All patients received 9.0 x 10¹³ viral genomes (vg) of ssAAV9.P546.IGHMBP2 delivered intrathecally via radiologic-guided lumbar puncture. The 9.0 x 10¹³ vg total viral vector dose will be mixed with OMNIPAQUE (stock conc. of 180 mgI/mL) and diluent to a total injection volume of 5 mL. (The titer was based on WuXi AppTec’s qualified bGH polyA ddPCR assay) in a total volume of 5 mL and containing 54 mgI/mL of OMNIPAQUE (30% of the total volume). The ssAAV9.P546.IGHMBP2 vector is formulated in a clear and sterile aqueous solution comprised of 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, and 0.001% (w/w) poloxamer 188. HCl is used to adjust the pH of the solution (20 mM Tris (pH 8.0), 1mM MgCl2, 200 mM NaCl) prior to its use in final formulation of the vector. [00148] The vector was delivered by a lumbar puncture into the L3-L4 or L4-L5 interspinous space into the subarachnoid space to the CSF of patients. The injection will follow the removal of CSF and immediately after the injection, subjects will be tilted into the Trendelenburg position, with the patient at 15-degree angle for 15 min. Since the vector is delivered directly into the CSF, the dose does not need to be adjusted by kg body weight. The aim is to increase the level of functional IGHMBP2 protein in the brain and spinal cord in various cell types, but especially neurons which are most affected by the disease. This should lead to modification of the disease course and prolonged survival of the patients. [00149] The primary objective of the clinical trial is to assess safety and tolerability. Patient dosing in the trial is staggered to allow sufficient review of safety data. Short-term safety is evaluated over an active study period of 3 years, followed by transfer to an annual monitoring program where data will be collected from annual standard of care visits for an additional 2 years. Safety endpoints are assessed by noting any changes in complete blood counts (CBC) with differential, serum clotting factors, serum enzyme levels, serum chemistries, CSF cell counts, glucose and protein, urinalysis, ECG, ECHO, immunologic responses to AAV9 (ELISA and ELISpots) and to IGHMBP2 (ELISpots) and monitoring for the development of any new clinical signs and/or symptoms. [00150] Stopping criteria are based on the development of unacceptable toxicity, defined as the occurrence of two or more Grade 3 or higher adverse events (based upon CTCAE v5.0 criteria), that are unexpected and possibly, probably, or definitely related to the study drug. [00151] There will be at least 6 weeks between dosing of the first and second subjects within this cohort. The allowance of 6 weeks between dosing of the first and second subjects in the cohort provides time for a Data Safety Monitoring Board (DSMB) review of the safety analysis from five time points (Days 1, 2, 7, 14, 21 and 30) prior to dosing of the next subject. The study will also require 30 days between all subsequent subjects to allow for further safety monitoring [00152] The secondary and exploratory objectives evaluate efficacy via various outcome measures. Comparison of functional outcomes between baseline and 90 and 180 days, and 12, 18, 24, and 36 months post-gene transfer are the basis of the secondary efficacy analysis. For functional outcomes, the subjects are separated into three different cohorts based on ambulatory status. Cohort 1: pre-ambulant subjects (< 18 months of age) will be evaluated using the Neuromuscular Gross Motor Outcome (GRO). Cohort 2: ambulant subjects are evaluated using the 100-meter timed test. Cohort 3: non-ambulant subjects (18 months to 6 years of age) are evaluated using the Neuromuscular GRO, and non-ambulant subjects (> 6 years of age to 14 years of age) are evaluated using the revised upper limb module for SMA (RULM). Efficacy analysis is assessed after all patients have completed the three-year active study period [00153] There are several exploratory objectives in this trial. For functional assessments: Cohort 1 (pre-ambulant): acquisition of milestones, CHOP Intend, and Peabody Fine Motor; Cohort 2 (ambulant): Neuromuscular GRO, North Star Ambulatory Assessment for limb girdle type muscular dystrophies (NSAD), reachable workspace by ACTIVE testing, nine- hole peg test (9HPT), Box and Blocks; Cohort 3 (non-ambulant): acquisition of Milestones and Peabody Fine Motor if 18 months to 6 years of age, and Neuromuscular GRO, North Star Ambulatory Assessment for limb girdle type muscular dystrophies (NSAD), reachable workspace by ACTIVE testing, and nine-hole peg test (9HPT) if > 6 years of age to 14 years of age. We will also assess patient reported outcomes using the PROMIS upper extremity (non-ambulant subjects) and PROMIS mobility (ambulant subjects). Other exploratory outcomes include survival, time off ventilatory support, diaphragm thickness and maximal excursion, pulmonary function testing for age 5 or older if subject able (change in FVC % predicted), change in compound motor action potential (CMAP) amplitude of the median nerve, and change in sensory nerve action potential (SNAP) amplitude of the radial and sural nerves. [00154] In our pre-ambulant subgroup, we will use the Neuromuscular GRO. This is a functional outcome measure that was developed here at Nationwide Children’s Hospital and validated in 5q SMA, a similar disorder that results in significant hypotonia, weakness, and limited or no motor milestone achievement33. Although the Neuromuscular GRO was designed as a tool to quantify motor function across the age range and spectrum of abilities, it is of note that this measure was found to mitigate the ceiling and floor effects seen while using traditional outcome measures in younger, pre-ambulant patients. Further efforts in additional cohorts, including but not limited to congenital muscular dystrophies, Menkes disease, and central core myopathies, have found that the Neuromuscular GRO is highly and significantly correlated to performance on other functional tests across diagnosis groups34. In our 1 subject with consecutive data, the GRO was stable over a 6-month period. [00155] In the ambulant subgroup, the 100-meter timed test will be used. Normative reference values and regression modeling is available to compare patient performance to performance of healthy individuals of the same age and body type and accounting for expected performance changes based on development and maturity. [00156] In the non-ambulant subgroup (ages 18 months – 6 years) Neuromuscular GRO is used. In the natural history study, there are 2 subjects with consecutive data, and a 2-point decline over 6 months in 1 subject was observed and a 4-point decline over 12 months was observed in the second subject. [00157] In our non-ambulant subgroup (age > 6 years – 14 years) the revised upper limb module for SMA (RULM) is used. In the 1 subject with consecutive visits, there was a 2-point decline in 6 months and 5-point decline over 12 months. [00158] No biopharmaceutic studies have been conducted for ssAAV9.P546.IGHMBP2. T-cell responses to AAV9 and IGHMBP2 by ELISpot is measured. The development of antibodies to AAV9 by ELISA is also assessed, but this will not be carried out for IGHMBP2 as there is no suitable antigen/antibodies that could be used in such assays. CSF and serum is collected at multiple time points from each patient and will be stored for future research on biomarkers. [00159] If ssAAV9.P546.IGHMBP2 is shown to be safe in this population and demonstrates early signs of efficacy, a second study with a more focused treatment cohort and a larger number of subjects with an efficacy measure as a primary objective is carried out. Subject number will be determined based upon effect measures in this current trial. Example 4 – Preliminary Safety and Outcomes from Phase I/IIa Trial of IGHMBP2 gene Replacement in SMARD1/CMT2S [00160] Preclinical studies in three IGHMBP2 disease mouse models resulted in the development of the ssAAV9.p546.IGHMBP2 vector. With no evidence of toxicity in mice and non-human primates, the first-in-human IGHMBP2 gene replacement trial is described above in Example 3. Herein the post-infusion data of the first four participants (2.5 – 10 months of follow up) is provided. [00161] The key selection criteria were: two pathogenic variants in IGHMBP2, age 2 months or greater and less than 14 years, and ability to cooperate with functional outcome assessments. The key exclusion criteria were: abnormal liver function, AAV9 antibody titer >1:50, a positive JCV antibody test, contraindication for intrathecal injection, concomitant illness/active infection/any additional medical condition that increases the risk of complications during gene transfer. All participants were administered 9.0 x 10¹³ vg well of ssAAV9.p546.IGHMBP2 via IR guided lumbar puncture infused over 5 minutes. The participants were also administered prednisone at day -3 and through at least 30 days at a dose of 1 mg/kg. Proton pump inhibitor initiated at day -3 as well through end of prednisone dosing. [00162] The primary outcome was safety and tolerability. The secondary outcome were stratified by functional status and compared to baseline. For pre-ambulant participants (<18 months of age, the secondary outcome was neuromuscular GRO (time frame days 90 and 180, months 12, 18, 24 and 36). For ambulant participants, the secondary outcome was 100 m timed test (time frame days 90 and 180, months 12, 18, 24 and 36). For non-ambulant participants (18 months – 6 years), the secondary outcome was neuromuscular GRO (time frame days 90 and 180, months 12, 18, 24 and 36). For non-ambulant participants (> 6 years – 14 years, the secondary outcome was revised upper limb module for SMA (RULM) (time frame days 90 and 180, months 12, 18, 24 and 36). The active study phase will be 3 years, then annual monitoring for 2 years. [00163] All participants tolerated the intrathecal infusion of 9.0 x 10¹³ vg well of ssAAV9.p546.IGHMBP2. Only one participant experienced post lumbar puncture pain and nausea. One participant was hospitalized for supportive care during a viral gastrointestinal illness that was unrelated to gene transfer. There were no other SAEs and no hepatic toxicity. Functional outcome measures were limited due to the short length of follow up, but function remains stable. [00164] Five participants have been screened. Participant 2 was a screen failure. Four participants have been enrolled as of September 15, 2022. [00165] Table 9A: Demographic and Additional Information n

Table 9B: Demographic and Additional Information n s

Table 10: CSF Analyses e )

Table 11: Outcomes

Table 12: Functional and Respiratory Outcomes

Revised upper limb module for SMA (RULM): higher score = better function; FVC = forced vital capacity; MIP = maximum inspiratory; MEP = maximum expiratory pressure

Table 13: Diaphragm Ultrasound Assessment al ion e ) to re

[00166] Cardiac function remains normal as assessed via electrocardiogram and exchocardiogram. Respiratory function remains stable. [00167] This is the first-in-human study and intrathecal delivery of ssAAV9.p456.IGHMBP2 at a dose of 9.0x10¹³ has been well tolerated.

Claims

What is claimed is: 1. A method of treating an IGHMBP2 -related disorder in a subject in need thereof comprising administering to the subject a dose of about 7x10¹³ vg to about 9.9 x 10¹³ vg of a rAAV or rAAV particles, wherein the rAAV or rAAV particles comprises a nucleotide sequence that is at least 90% identical to nucleotides 1 to 4375 of SEQ ID NO: 8 or that is at least 90% identical to nucleotides 1 to 4364 of SEQ ID NO: 17.

2. The method of claim 1 wherein the nucleotide sequence is at least 95% identical to nucleotides 1 to 4375 of SEQ ID NO: 8 or that is at least 90% identical to nucleotides 1 to 4364 of SEQ ID NO: 17.

3. The method of claim 1 or 2 wherein the nucleotide sequence comprises nucleotides 1 to 4375 of SEQ ID NO: 8 or nucleotides 1 to 4364 of SEQ ID NO: 17.

4. The method of any one of claims 1-3, wherein the dose of rAAV or rAAV particles is about 9.0 x 10¹³ vg based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vector.

5. The method of any one of claims 1-4 wherein the rAAV or rAAV particles are of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13 or Anc80, AAV7m8 and their derivatives.

6. The method of any one of claims 1-4 wherein the rAAV or rAAV particles are of the serotype AAV9.

7. The method of any one of claims 1-6 wherein the dose of rAAV or rAAV particles is administered as a single injection having a volume of 5 mL.

8. The method of any one of claims 1-7 wherein the dose of rAAV or rAAV particles is administered in an aqueous composition comprising an agent that increases the viscosity or density of the formulation.

9. The method of claim 8 wherein the agent that increases the viscosity or density of the formulation is about 20 to 40% of the composition .

10. The method of claim 8 or 9 wherein the agent that increases the viscosity or density of the composition is a non-ionic, low-osmolar compound or contrast agent.

11. The method of any one of claims 8-10 wherein the composition comprises about 10 mM to about 30 mM Tris, 0.5 mM to about 5 mM MgCl_2, about 100 mM to about 500 mM NaCl and about 0.0001% to about 0.01% poloxamer 188.

12. The method of any one of claims 8-11 wherein the composition comprises about 20 mM Tris, 1 mM MgCl₂, 200 mM NaCl, and 0.001% poloxamer 188.

13. The method of any one of claims 1-12 wherein the IGHMBP2-related disorder is SMARD1 or CMT2S.

14. The method of any one of claims 1-13 wherein the subject has a mutation in the IGHMBP2 gene.

15. The method of any one of claims 1-14 wherein the dose of rAAV or rAAV particles is administered by for direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery or intravenous delivery.

16. The method of any one of claims 1-15, further comprising a step of administering an immunosuppressing agent.

17. Use of a dose of a rAAV or rAAV particles for the preparation of a medicament for treating an IGHMBP2 -related disorder in a subject in need thereof, wherein the a dose is about 7x10¹³ vg to about 9. x 10¹³ vg of rAAV or rAAV particles, and wherein the rAAV or rAAV particles comprises a nucleotide sequence that is at least 90% identical to nucleotides 1 to 4375 of SEQ ID NO: 8 or that is at least 90% identical to nucleotides 1 to 4364 of SEQ ID NO: 17.

18. The use of claim 17 wherein the nucleotide sequence is at least 95% identical to nucleotides 1 to 4375 of SEQ ID NO: 8 or that is at least 95% identical to nucleotides 1 to 4364 of SEQ ID NO: 17.

19. The use of claim 17 or 18 wherein the nucleotide sequence comprises nucleotides 1 to 4375 of SEQ ID NO: 8 or nucleotides 1 to 4364 of SEQ ID NO: 17.

20. The use of any one of claims 17-18, wherein the dose of rAAV or rAAV particles is about 9.0 x 10¹³ vg based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vector

21. The use of any one of claims 17-20, wherein the rAAV or rAAV particles are of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13 or Anc80, AAV7m8 and their derivatives.

22. The use of any one of claims 17-20 wherein the rAAV or rAAV particles are of the serotype AAV9.

23. The use of any one of claims 17-22 wherein the dose of rAAV or rAAV particles is formulated for administration as a single injection having a volume of 5 mL.

24. The use of any one of claims 17-23 wherein the dose of rAAV or rAAV particles is in an aqueous composition comprising an agent that increases the viscosity or density of the formulation.

25. The use of claim 24 wherein the agent that increases the viscosity or density of the formulation is about 20 to 40% of the composition.

26. The use of claim 24 or 25 wherein the agent that increases the viscosity or density of the composition is a non-ionic, low-osmolar compound or contrast agent.

27. The use of any one of claims 24-26 wherein the composition comprises about 10 mM to about 30 mM Tris, 0.5 mM to about 5 mM MgCl_2, about 100 mM to about 500 mM NaCl and about 0.0001% to about 0.01% poloxamer 188.

28. The use of any one of claims 24-27 wherein the composition comprises about 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, and 0.001% poloxamer 188.

29. The use of any one of claims 17-28 wherein the IGHMBP2-related disorder is SMARD1 or CMT2S.

30. The use of any one of claims 17-28 wherein the subject has a mutation in the IGHMBP2 gene.

31. The use of any one of claims 17-30 wherein the dose of rAAV or rAAV particles is formulated for administration by for direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery or intravenous delivery.

32. The use of any one of claims 17-31, wherein the medicament further comprises an immunosuppressing agent.

33. A composition suitable for treating for treating an IGHMBP2 -related disorder in a subject in need thereof, wherein the composition comprises a dose is about 7x10¹³ vg to about 9. x 10¹³ vg of rAAV or rAAV particles, and wherein the rAAV or rAAV particles comprises a nucleotide sequence that is at least 90% identical to nucleotides 1 to 4375 of SEQ ID NO: 8 or that is at least 90% identical to nucleotides 1 to 4364 of SEQ ID NO: 17.

34. The composition of claim 33 wherein the nucleotide sequence is at 95% identical to nucleotides 1 to 4375 of SEQ ID NO: 8 or that is at least 95% identical to nucleotides 1 to 4364 of SEQ ID NO: 17.

35. The composition of claim 33 or 34 wherein the nucleotide sequence comprises nucleotides 1 to 4375 of SEQ ID NO: 8 or nucleotides 1 to 4364 of SEQ ID NO: 17.

36. The composition of any one of claims 33-35, wherein the dose of rAAV or rAAV particles is about 9.0 x 10¹³ vg based on a ddPCR assay targeting the bovine growth hormone (bGH) polyadenylation (polyA) sequence in the vector.

37. The use of any one of claims 33-36, wherein the rAAV or rAAV particles are of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13 or Anc80, AAV7m8 and their derivatives.

38. The composition of any one of claims 33-37 wherein the rAAV or rAAV particles are of the serotype AAV9.

39. The composition of any one of claims 33-38 wherein the dose of rAAV or rAAV particles is formulated for administration as a single injection having a volume of 5 mL.

40. The composition of any one of claims 33-39 wherein the dose of rAAV or rAAV particles is in an aqueous composition comprising an agent that increases the viscosity or density of the formulation.

41. The composition of claim 40 wherein the agent that increases the viscosity or density of the formulation is about 20 to 40% of the aqueous composition.

42. The composition of claim 40 or 41 wherein the agent that increases the viscosity or density of the aqueous composition is a non-ionic, low-osmolar compound or contrast agent.

43. The composition of any one of claims 40-42 wherein the composition comprises about 10 mM to about 30 mM Tris, 0.5 mM to about 5 mM MgCl2, about 100 mM to about 500 mM NaCl and about 0.0001% to about 0.01% poloxamer 188.

44. The composition of any one of claims 40-43 wherein the composition comprises about 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, and 0.001% poloxamer 188.

45. The composition of any one of claims 33-44 wherein the IGHMBP2-related disorder is SMARD1 or CMT2S.

46. The composition of any one of claims 33-45 wherein the subject has a mutation in the IGHMBP2 gene.

47. The composition of any one of claims 33-46 wherein the dose of rAAV or rAAV particles is formulated for administration by for direct injection into the cerebrospinal fluid, intracerebroventricular delivery, intrathecal delivery or intravenous delivery.

48. The composition of any one of claims 33-47, wherein the composition further comprises an immunosuppressing agent.