Products and methods for treating myelin protein null silencing and CMT1B disease

Abstract

Disclosed are RNA interference-based products and methods for inhibiting expression of mutant myelin protein zero (MPZ) gene in a cell or cells of a subject. The disclosure includes microRNAs that specifically target various regions of the MPZ gene to knock down expression of the abnormal protein. Additionally, the disclosure includes delivery of nucleic acids encoding normal, wild-type, or functionally active MPZ protein. Additionally, the disclosure includes recombinant adeno-associated viruses to deliver nucleic acids encoding microRNAs that knock down expression of the abnormal MPZ protein and / or to deliver nucleic acids encoding normal, wild-type, or functionally active MPZ protein. The disclosure includes methods of using these nucleic acids in the treatment of diseases associated with MPZ gene mutations, including but not limited to Charcot-Marie-Tooth type 1B (CMT1B) disease.

Description

[Technical field]

[0001] Incorporation by reference of sequence listing This application includes, as a separate part of the disclosure, a sequence listing in computer-readable form (Filename: 57017_Seqlisting.XML, Size: 111,501 bytes, Created: October 3, 2022), which is incorporated by reference in its entirety herein.

[0002] Products and methods are provided for silencing or inhibiting expression of the Myelin Protein Zero (MPZ) gene (1q22), where mutations in the gene are associated with underexpression of wild-type MPZ protein and severe, early-onset congenital hypomyelination, including various types of Charcot-Marie-Tooth (CMT) disease. MPZ is a well-known CMT causative gene with a wide phenotypic spectrum. Several mutations in the MPZ gene cause many different types of hypomyelination and CMT disease, including, but not limited to, dominant intermediate CMT (DI-CMT), CMT type 1B (CMT1B), CMT type 2I (CMT2I), and CMT type 2J (CMT2J), as well as Degerines-Sottas syndrome (DSS or CMT type 3) disease. CMT1B disease presents with symptoms of peripheral neuropathy, including, but not limited to, distal muscle weakness and atrophy, foot deformities, and sensory loss. The present disclosure provides a gene therapy approach for treating CMT1B using a nucleic acid encoding an artificial microRNA (miRNA) that specifically hybridizes to a target nucleic acid sequence encoding the MPZ gene, where the binding of the complex with the target nucleic acid sequence results in knockdown of MPZ gene expression, and involves using a nucleic acid encoding a codon-optimized MPZ gene (coMPZ or resMPZ) that is resistant to the artificial miRNA designed to knockdown the MPZ gene. A delivery vehicle such as a recombinant adeno-associated virus delivers one or more MPZ microRNAs as well as the nucleic acid encoding the coMPZ gene. These products and methods have application in the treatment of CMT1B disease and other disorders that exhibit abnormal MPZ expression. [Background technology]

[0003] Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous collection of inherited peripheral neuropathies with a prevalence of up to 1 in 2,500. CMT neuropathy type 1B (CMT1B) is the third most common subtype of CMT1, accounting for 10% of cases and affecting approximately 1 in 30,000 individuals. CMT1B is characterized by slowly progressive distal muscle weakness and atrophy, foot drop and deformity, loss of sensation, and very severe lack of reflexes with two typical onsets: early childhood and adolescent onset. CMT1B is caused by over 200 different mutations in the myelin protein zero (MPZ or P0) gene. The MPZ gene encodes the MPZ protein, the major protein in the myelin sheath. MPZ is an essential protein for maintaining a healthy and efficient peripheral nervous system. Accumulation of the defective protein in Schwann cells leads to demyelination and cell death over time. The pathological mechanisms of CMT1B disease can be mostly divided into two major groups: (1) toxic gain-of-function mutations that directly affect normal myelination, and (2) defective unfolded protein response (UPR) or endoplasmic reticulum (ER) stress response. Both mechanisms of disease ultimately lead to the accumulation of mutant myelin proteins in Schwann cells (SCs), reduced myelination, muscle weakness and atrophy, and loss of sensation in the lower limbs and feet. For example, the R98C mutation causes early-onset severe disease due to retaining mutated MPZ protein in the ER and defective UPR. Patients with this mutation have very low or almost no myelin. These patients are classified as CMT1B, but also as very severe CMT1B or Degerines-Sottas syndrome (DSS), another definition of congenital hypomyelination. There is no cure for this disease yet.

[0004] There remains a need in the art for products and methods of treatment for MPZ genetic disorders, including but not limited to CMT1B disease. Summary of the Invention

[0005] Provided herein are products, methods and uses for treating mutations in the Myelin Protein Zero (MPZ) gene, as well as for treating, ameliorating, slowing the progression of, and / or preventing diseases resulting from mutations in the MPZ gene.

[0006] The present disclosure relates to a) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; b) a polynucleotide sequence comprising an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19-30, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19-30; c) a polynucleotide sequence comprising or encoding an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42; d) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, the target nucleotide sequence being set forth in any one of SEQ ID NOs: 43 to 54; or e) providing a nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 3 or 6.

[0007] The present disclosure relates to a) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; or ii) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, the target nucleotide sequence being represented by any one of SEQ ID NOs: 43 to 54; b) a polynucleotide sequence encoding a human MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in i) SEQ ID NO:1; ii) a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:3; or iii) a polynucleotide sequence encoding an MPZ polypeptide sequence that comprises at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:4.

[0008] In some embodiments, the nucleic acid further comprises a promoter or multiple promoters. In some embodiments, the promoter is a U6 promoter, a U7 promoter, a H19 promoter, a neuron specific promoter, a H1 promoter, an EF1-alpha promoter, a minimal EF1-alpha promoter, an unc45b promoter, a CK1 promoter, a CK6 promoter, a CK7 promoter, a mini CMV promoter, a CMV promoter, a muscle creatine kinase (MCK) promoter, an alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), a tMCK promoter, a minimal MCK promoter, a desmin promoter, a chicken beta actin promoter, The promoter may be a promoter selected from the group consisting of a promoter for the gene encoding the miMPZ promoter, ... In some more specific embodiments, the promoter is a U6 promoter, a U7 promoter, an H19 promoter, a neuron-specific promoter, or a Schwann cell-specific promoter. In some embodiments, the Schwann cell-specific promoter is an MPZ promoter.In some embodiments, the MPZ promoter comprises at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:5.

[0009] The present disclosure provides nanoparticles, extracellular vesicles, exosomes, or vectors comprising any of the nucleic acids of the present disclosure, or any one or more combinations thereof. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, or a synthetic virus. In some embodiments, the viral vector is an AAV. In some embodiments, the AAV lacks the rep and cap genes. In some embodiments, the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV). In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAVv66, or AAV-F. In some specific embodiments, the AAV is AAV9, AAVrh.10, AAV-PHP.eB, AAVv66, or AAV-F.

[0010] The present disclosure provides a composition comprising any of the nucleic acids of the present disclosure, or any one or more combinations thereof.The present disclosure provides a composition comprising any of the nanoparticles, extracellular vesicles, exosomes, vectors, or viral vectors of the present disclosure, or any one or more combinations thereof.In some embodiments, the composition also comprises a pharma- ceutically acceptable carrier.

[0011] The present disclosure provides a method of reducing expression of a mutant myelin protein zero (MPZ) gene in a cell, the method comprising contacting the cell with any of the nucleic acids of the present disclosure, or a combination of any one or more thereof. In some embodiments, the method comprises contacting the cell with: a) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; b) a polynucleotide sequence comprising an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19-30, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19-30; c) a polynucleotide sequence comprising or encoding an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42; d) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, the target nucleotide sequence being represented by any one of SEQ ID NOs: 43 to 54; and / or e) contacting with a nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 3 or 6.

[0012] In some embodiments, the method comprises: a) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; or ii) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, the target nucleotide sequence being represented by any one of SEQ ID NOs: 43 to 54; b) a polynucleotide sequence encoding a human MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in i) SEQ ID NO:1; ii) a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:3; and / or iii) contacting the polynucleotide sequence encoding an MPZ polypeptide sequence comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:4.

[0013] In some embodiments, the nucleic acid further comprises a promoter or promoters. In some embodiments, the promoter is a U6 promoter, a U7 promoter, a H19 promoter, a neuron specific promoter, a H1 promoter, an EF1-alpha promoter, a minimal EF1-alpha promoter, an unc45b promoter, a CK1 promoter, a CK6 promoter, a CK7 promoter, a mini CMV promoter, a CMV promoter, a muscle creatine kinase (MCK) promoter, an alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), a tMCK promoter, a minimal MCK promoter, a desmin promoter, a chicken beta actin promoter, The promoter may be a promoter selected from the group consisting of a promoter for the expression vector (CBA), a P546 promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukosis virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, an elongation factor-1a promoter, a hemoglobin promoter, a creatine kinase promoter, a Schwann cell specific promoter, a myelin specific promoter, or a native promoter. In some embodiments, the promoter may be a MPZ promoter, a non-compact myelin associated protein (NCMPA or MP11) promoter, a PMP22 promoter, a MBP promoter, a SOX10 promoter, or a GAP43 promoter. In some embodiments, the promoter used with miMPZ is a U6 promoter. In some embodiments, the promoter used with MPZ replacement gene is a MPZ promoter or a miniMPZ promoter. In some more specific embodiments, the promoter is a U6 promoter, a U7 promoter, an H19 promoter, a neuron-specific promoter, or a Schwann cell-specific promoter. In some embodiments, the Schwann cell-specific promoter is an MPZ promoter or a mini-MPZ promoter.In some embodiments, the MPZ promoter comprises at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleic acid is in a nanoparticle, extracellular vesicle, exosome, or vector. In some embodiments, the nucleic acid is in a viral vector. In some embodiments, the nucleic acid, nanoparticle, extracellular vesicle, exosome, or vector is in a composition. In some embodiments, the cell is a neuronal cell. In some embodiments, the neuronal cell is a Schwann cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is in a human subject.

[0014] The present disclosure provides methods of treating a subject comprising a mutant myelin protein zero (MPZ) gene, the methods comprising administering to the subject an effective amount of any of the nucleic acids of the present disclosure, or any one or more combinations thereof. In some embodiments, the methods comprise administering to the subject: a) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; b) a polynucleotide sequence comprising an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19-30, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19-30; c) a polynucleotide sequence comprising or encoding an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42; d) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, the target nucleotide sequence being represented by any one of SEQ ID NOs: 43 to 54; and / or e) administering an effective amount of a nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 3 or 6.

[0015] In some embodiments, the method comprises administering to a subject: a) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; or ii) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, the target nucleotide sequence being represented by any one of SEQ ID NOs: 43 to 54; b) a polynucleotide sequence encoding a human MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in i) SEQ ID NO:1; ii) a polynucleotide sequence encoding a codon-optimized MPZ DNA that contains at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO: 3 or 6; or iii) administering an effective amount of a nucleic acid comprising a polynucleotide sequence encoding an MPZ polypeptide sequence comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:4.

[0016] In some embodiments, the nucleic acid further comprises a promoter or promoters. In some embodiments, the promoter is a U6 promoter, a U7 promoter, a H19 promoter, a neuron specific promoter, a H1 promoter, an EF1-alpha promoter, a minimal EF1-alpha promoter, an unc45b promoter, a CK1 promoter, a CK6 promoter, a CK7 promoter, a mini CMV promoter, a CMV promoter, a muscle creatine kinase (MCK) promoter, an alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), a tMCK promoter, a minimal MCK promoter, a desmin promoter, a chicken beta actin promoter, The promoter may be a promoter selected from the group consisting of a promoter for the expression vector (CBA), a P546 promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukosis virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, an elongation factor-1a promoter, a hemoglobin promoter, a creatine kinase promoter, a Schwann cell specific promoter, a myelin specific promoter, or a native promoter. In some embodiments, the promoter may be a MPZ promoter, a non-compact myelin associated protein (NCMPA or MP11) promoter, a PMP22 promoter, a MBP promoter, a SOX10 promoter, or a GAP43 promoter. In some embodiments, the promoter used with miMPZ is a U6 promoter. In some embodiments, the promoter used with MPZ replacement gene is a MPZ promoter or a miniMPZ promoter. In some more specific embodiments, the promoter is a U6 promoter, a U7 promoter, an H19 promoter, a neuron-specific promoter, or a Schwann cell-specific promoter. In some embodiments, the Schwann cell-specific promoter is an MPZ promoter or a mini-MPZ promoter.In some embodiments, the MPZ promoter comprises at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the nucleic acid is present in a nanoparticle, extracellular vesicle, exosome, or vector. In some embodiments, the nucleic acid is in a viral vector. In some embodiments, the viral vector is an AAV that is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAVv66, or AAV-F. In some embodiments, the AAV is AAV9, AAVrh.10, AAV-PHP.eB, AAVv66, or AAV-F. In some embodiments, the nucleic acid, nanoparticle, extracellular vesicle, exosome, or vector is in a composition. In some embodiments, the subject is a human subject. In some embodiments, the subject is afflicted with hypomyelination disease or Charcot-Marie-Tooth disease. In some embodiments, the disease is CMT (DI-CMT), CMT type 1B (CMT1B), CMT type 2I (CMT2I), CMT type 2J (CMT2J), or Degerine-Sottas syndrome (DSS or CMT type 3) disease.

[0017] The present disclosure provides a method of reducing expression of a mutant myelin protein zero (MPZ) gene in a cell and expressing a functional MPZ protein in the cell, the method comprising administering to the cell an effective amount of (a) (i) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; (ii) a polynucleotide sequence comprising an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19-30, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19-30; (iii) a polynucleotide sequence comprising or encoding an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42; and / or (iv) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, the target nucleotide sequence being one or more of the polynucleotide sequences shown in any one of SEQ ID NOs: 43 to 54; and (b) delivering a nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 3 or 6.

[0018] In some embodiments, any one or more of the nucleic acids further comprises a promoter or promoters. In some embodiments, the promoter is a U6 promoter, a U7 promoter, an H19 promoter, a neuron-specific promoter, or a Schwann cell-specific promoter. In some embodiments, the Schwann cell-specific promoter is an MPZ promoter or a mini-MPZ promoter. In some embodiments, the MPZ promoter comprises at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the cell is a neuronal cell. In some embodiments, the neuronal cell is a Schwann cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is in a human subject. In some embodiments, the subject is afflicted with a hypomyelination disease or Charcot-Marie-Tooth disease. In some embodiments, the disease is CMT (DI-CMT), CMT type 1B (CMT1B), CMT type 2I (CMT2I), CMT type 2J (CMT2J), or Degerines-Sottas syndrome (DSS or CMT type 3) disease. In some embodiments, the nucleic acid is delivered to the cell in a nanoparticle, an extracellular vesicle, an exosome, or a vector, or a combination of any one or more thereof. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, or a synthetic virus. In some embodiments, the viral vector is an AAV. In some embodiments, the AAV lacks the rep and cap genes. In some embodiments, the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV).In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, or AAVv66, or AAV-F. In some specific embodiments, the AAV is AAV9, AAVrh.10, AAV-PHP.eB, AAVv66, or AAV-F. In some embodiments, a nucleic acid comprising a polynucleotide sequence comprising or encoding an MPZ miRNA and / or a polynucleotide that specifically hybridizes to a target nucleotide sequence in the MPZ gene, and a nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA are delivered simultaneously to a cell. In some embodiments, the nucleic acids are delivered to the cell in the same vector. In some embodiments, the nucleic acid comprising a polynucleotide sequence comprising or encoding an MPZ miRNA and / or a polynucleotide that specifically hybridizes to a target nucleotide sequence in the MPZ gene, and the nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA are delivered to the cell at different times.

[0019] The present disclosure provides a method of treating a subject suffering from aberrant expression of a mutant myelin protein zero (MPZ) gene, the method comprising reducing expression of the mutant MPZ gene in the subject and expressing functional MPZ, the method comprising administering to the subject an effective amount of (a) (i) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; (ii) a polynucleotide sequence comprising an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19-30, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19-30; (iii) a polynucleotide sequence comprising or encoding an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42, or a variant thereof comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 31 to 42; and / or (iv) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, the target nucleotide sequence being one or more of the polynucleotide sequences shown in any one of SEQ ID NOs: 43 to 54; and (b) delivering a nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 3 or 6.

[0020] The present disclosure also provides a method of treating a subject suffering from abnormal expression of a mutant myelin protein zero (MPZ) gene, the method comprising expressing a functional MPZ protein in the subject by delivering to the subject an effective amount of a nucleic acid comprising a polynucleotide sequence encoding MPZ DNA or codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs:1, 3, and 6.

[0021] In some embodiments, any one or more of the nucleic acids further comprises a promoter or promoters. In some embodiments, the promoter is a U6 promoter, a U7 promoter, an H19 promoter, a neuron-specific promoter, or a Schwann cell-specific promoter. In some embodiments, the Schwann cell-specific promoter is an MPZ promoter or a mini-MPZ promoter. In some embodiments, the MPZ promoter comprises at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the subject is a human. In some embodiments, the subject is afflicted with hypomyelination disease or Charcot-Marie-Tooth disease. In some embodiments, the disease is CMT (DI-CMT), CMT type 1B (CMT1B), CMT type 2I (CMT2I), CMT type 2J (CMT2J), or Degerines-Sottas syndrome (DSS or CMT type 3) disease. In some embodiments, the nucleic acid is delivered to the subject in a nanoparticle, an extracellular vesicle, an exosome, or a vector, or a combination of any one or more thereof. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, or a synthetic virus. In some embodiments, the viral vector is an AAV. In some embodiments, the AAV lacks the rep and cap genes. In some embodiments, the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV).In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, or AAVv66, or AAV-F. In some specific embodiments, the AAV is AAV9, AAVrh.10, AAV-PHP.eB, AAVv66, or AAV-F. In some embodiments, a nucleic acid comprising a polynucleotide sequence comprising or encoding an MPZ miRNA and / or a polynucleotide that specifically hybridizes to a target nucleotide sequence in the MPZ gene, and a nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA are delivered simultaneously to a subject. In some embodiments, the nucleic acids are delivered to a subject in the same vector. In some embodiments, the nucleic acid comprising a polynucleotide sequence that comprises or encodes an MPZ miRNA and / or that specifically hybridizes to a target nucleotide sequence in an MPZ gene, and the nucleic acid comprising a polynucleotide sequence that encodes a codon-optimized MPZ DNA are delivered to a cell at different times.

[0022] The present disclosure provides for the use of any of the nucleic acids of the present disclosure, or combinations thereof. The present disclosure provides for the use of any of the nanoparticles, extracellular vesicles, exosomes, or vectors of the present disclosure. The present disclosure provides for the use of any of the viral vectors of the present disclosure. The present disclosure provides for the use of any of the compositions of the present disclosure. In some embodiments, the use is for the preparation of a medicament for reducing expression of a mutant myelin protein zero (MPZ) gene in a cell. In some embodiments, the cell is in a human subject. Thus, in some other embodiments, the cell is ex vivo or in vitro and external to the subject. In some embodiments, the use is for treating a subject comprising a mutant myelin protein zero (MPZ) gene. In some embodiments, the subject is a human subject. In some embodiments, the subject is afflicted with a hypomyelination disease or Charcot-Marie-Tooth disease. In some embodiments, the disease is CMT (DI-CMT), CMT type 1B (CMT1B), CMT type 2I (CMT2I), CMT type 2J (CMT2J), or Degerine-Sottas syndrome (DSS or CMT type 3) disease.

[0023] The present disclosure provides that any of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, compositions, or pharmaceutical agents described herein are in some embodiments formulated for intramuscular injection, oral administration, subcutaneous, intradermal, or transdermal delivery, injection into the bloodstream, or aerosol administration.

[0024] Other features and advantages of the present disclosure will become apparent from the following description of the drawings and detailed description, although the drawings, detailed description, and examples illustrate embodiments of the disclosed subject matter and are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent from the drawings, detailed description, and examples. [Brief description of the drawings]

[0025] [Figure 1]A schematic diagram of human and mouse MPZ cDNA is provided. The most common dominant mutations or small deletions in MPZ cDNA that cause CMT1B are indicated. Stars represent conserved regions on mouse and human MPZ sequences. Thick horizontal lines indicate target positions with miRNAs designed in MPZ cDNA. miRNAs were designed to target highly conserved sequences and targeted both human and mouse MPZ sequences in preclinical trials in mice using one single vector. [Figure 2A]Each of the MPZ microRNA (miMPZ) nucleotide sequences of the present disclosure are shown, i.e., FIG. 2A shows miMPZ_225, FIG. 2B shows miMPZ_226, FIG. 2C shows miMPZ_315, FIG. 2D shows miMPZ_316, FIG. 2E shows miMPZ_317, FIG. 2F shows miMPZ_718, FIG. 2G shows miMPZ_719, FIG. 2H shows miMPZ_720, FIG. 2I shows miMPZ_721, FIG. 2J shows miMPZ_722, FIG. 2K shows miMPZ_723, and FIG. 2L shows miMPZ_1852. In each of FIG. 2A-L, the top sequence shows DNA template synthesis by PCR using the primer pairs described in the context. Each DNA template transcribes the respective miMPZ. The folded miRNA transcript is shown as a hairpin structure. The mature miMPZ sequence is generated after processing in the target cell by the host miRNA processing machinery (including Drosha, DGCR8, Dicer, and exportin-5). The grey-tinted sequences indicate the restriction enzyme cleavage sites used to clone each miRNA into the U6T6 vector. In the RNA, CTCGAG (SEQ ID NO: 79) is the XhoI site, and ACTAGT (SEQ ID NO: 80) is the SpeI site (CUCGAG (SEQ ID NO: 81) and ACUAGU (SEQ ID NO: 82) in the RNA, where U is a uracil base). The underlined italicized sequence indicates the mature miRNA antisense guide strand that ultimately serves to catalyze the cleavage of the DUX4 target mRNA. This sequence is also underlined in the miRNA hairpin portion of this figure. The arrows indicate the Drosha and Dicer catalytic cleavage sites, respectively. The numbers 13, 35, 53, and 75 are provided for positional explanation. The sequence between (and including) positions 35-53 is derived from the native human mir-30a sequence, except for the A at position 39, where G is the normal mir-30a sequence. This nucleotide was changed to an A to facilitate folding of the miRNA loop, based on computational RNA folding models. The bases of the stem (5' at position 13 and 3' at position 75) were derived from the mir-30a structure and sequences with some modifications depending on the primary sequence of the guide strand.Specifically, the nucleotide at position 13 can be altered to help promote the necessary mismatch between nucleotides 13 and 75. It is hypothesized that this bulged structure promotes proper Drosha cleavage. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 2H] Same as above. [Figure 2I] Same as above. [Figure 2J] Same as above. [Figure 2K] Same as above. [Figure 2L] Same as above. [Diagram 3]The results of the preliminary screening of the miMPZ of the present disclosure for the effectiveness of knocking down MPZ expression are shown. Figure 3A shows a schematic diagram of the dual luciferase plasmid used in various tests described in this disclosure. To generate the dual luciferase reporter plasmid, human MPZ cDNA was cloned downstream of Renilla luciferase gene as 3' untranslated region (3' UTR) in psiCheck2 plasmid (Promega). This structure does not produce luciferase-MPZ fusion protein because the MPZ sequence is placed after Renilla luciferase stop codon. Instead, a fusion mRNA is produced in which the MPZ sequence acts as the 3' UTR of Renilla luciferase. As a result, any effective MPZ targeting miRNA reduces Renilla luciferase-MPZ fusion mRNA, which subsequently reduces Renilla luciferase protein expression in transfected cells. There is a separate firefly luciferase gene expressed under a different promoter in the same plasmid, which does not contain the MPZ sequence and is therefore not affected by miMPZ. Figure 3B shows the luciferase assay results of the initial miMPZ efficacy screen. All samples in this assay were normalized to cells co-transfected with the reporter vector and U6T6 plasmid. All miMPZs, except miMPZ_720, efficiently reduced Renilla luciferase expression. miMPZ 225, 226, 721, and 723 demonstrated the greatest efficiency in this experiment. Figure 3C shows a Western blot showing that miMPZ 225, 226, 317, 721, and 723 significantly reduced MPZ expression in co-transfected HEK293 cells. Figure 3D shows the relative expression of hMPZ relative to untreated controls using qRT-PCR, demonstrating that miMPZ reduces MPZ gene expression. Based on these results, miMPZ_225, 226, 317, 721, and 723 most significantly reduced MPZ mRNA levels in co-transfected HEK293 cells.These miMPZs, in various embodiments, show that they are efficient in targeting and reducing MPZ R98C expression levels in co-transfected HEK293 cells. [Figure 4] Figure 4 shows the reduction of MPZ R98C expression achieved by the artificial miMPZ described herein. Figure 4A shows the luciferase assay results of the miMPZ efficacy screen targeting mutant MPZ R98C in HEK293 cells. All samples in this assay were normalized to cells co-transfected with reporter vector and U6T6 plasmid. All miMPZs efficiently reduced Renilla luciferase expression. Figure 4B shows the Western blot results of MPZ R98C knockdown by various miMPZs in co-transfected HEK293 cells. miMPZs 225, 226, 317, 719, 721, and 723 more significantly reduced R98C expression in co-transfected HEK293 cells. Figure 4C shows the qRT-PCR results of MPZ R98C expression levels in HEK293 cells co-transfected with each miMPZ and R98C expression plasmid. All MPZ miRNAs significantly reduced R98C mRNA levels in co-transfected cells compared to untreated R98C. [Diagram 5]Shown is SEQ ID NO:6, which is the sequence of MPZ expression cassette made from 1200bp human MPZ promoter (ENSG00000158887), 63bp human MPZ 5'UTR, and 747bp partially codon-optimized MPZ cDNA. It also has SV40 polyA signal and CMV amplicon sequence. CMV amplicon is used for AAV titration using qRT-PCR or ddPCR assay. miMPZ is cloned into NsiI or NotI site located after CMV amplicon. In some exemplary embodiments, expression cassette is cloned into AAV pre-plasmid using two XbaI restriction enzyme sites at each end. The sequence order in the coMPZ expression cassette is: XBAI-stui-SalI-kasi-human MPZ promoter (1200bp)-NheI-human MPZ 5'UTR-partially codon-optimized human mpz cds-SPEI-ndei-SV40 PA-paci-CMV amplicon for DRP-NSII-noti-XBAI. Various MPZ promoters including human, rat, mouse, or combinations with universal enhancers such as CMV or Schwann-specific enhancers such as PMP22 can also be used in this construct. [Figure 6] Base pairing between mi225 and human and mouse MPZ, and resistance of coMPZ to miMPZ knockdown. Figure 6A shows perfect base pairing of miMPZ-225 with wild-type human and mouse MPZ. Figure 6B shows mismatches between miMPZ225 and coMPZ. The mutated nucleotide (bold) is at the wobble position and maintains the wild-type MPZ amino acid. G·U is the wobble base pair. Figure 6C shows qRT-PCR results. miMPZ-225 reduced wild-type human MPZ by >75% in cotransfected HEK293 cells, whereas coMPZ was relatively resistant to high levels of silencing. The slight reduction in coMPZ expression in this experiment may reflect differences in transcription or qRT-PCR detection of the two MPZ transcripts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present disclosure provides a novel strategy to achieve inhibition of mutant myelin protein zero (MPZ or P0) gene expression by post-transcriptionally suppressing or inhibiting mutant MPZ gene expression and protein production, since expression of mutant MPZ protein is known to cause congenital neurological disorders associated with hypomyelination, including but not limited to Charcot-Marie-Tooth disease (CMT). Thus, in some embodiments, the products, methods, and uses described herein find use in treating, ameliorating, slowing progression, and / or preventing neurological disorders associated with hypomyelination, including but not limited to CMT.

[0027] CMT is a clinically and genetically heterogeneous collection of inherited peripheral neuropathies with a prevalence of up to 1 in 2,500. Charcot-Marie-Tooth type 1B (CMT1B) is the third most common form of inherited demyelinating neuropathies, accounting for 10% of cases. CMT1B is caused by a DNA mutation, an autosomal dominant gain-of-function mutation in the MPZ gene. MPZ is an essential protein and is the major protein in the myelin sheath required to maintain a healthy and efficient peripheral nervous system. Accumulation of defective MPZ protein in Schwann cells, supporting cells in peripheral nerves, causes progressive nerve damage, leading to CMT1B symptoms. CMT1B features include slowly progressive distal muscle weakness and atrophy, foot drop and deformity, loss of sensation, and very severe lack of reflexes with two typical onsets: early childhood and adolescent onset. Treatment of very severe CMT1B or Degerines-Sottas Syndrome (DSS), another definition of congenital hypomyelination, is included in the methods and uses of the present disclosure.

[0028] MPZ is a 27 kDa single membrane glycoprotein expressed by myelinating Schwann cells (Magnaghi et al., Brain Research Reviews, 2011, 37(1-3):360-371). It accounts for more than 50% of all proteins in the peripheral nervous system, making it the most common protein expressed in the PNS (Shy, Journal of the Neurological Sciences, 2006, 242(1-2):55-66). Most MPZ mutations causing CMT1B are dominantly inherited. Various pathological mechanisms have been suggested for each MPZ gene mutation. However, the main pathological mechanisms of dominant mutations in the MPZ gene can be divided into two major groups: (1) toxic gain-of-function mutations that directly affect normal myelination, and (2) defective unfolded protein response (UPR) or endoplasmic reticulum (ER) stress response (Bai et al., Ann Clin Transl Neurol, 2018, 5:445-455; Bai et al., Rare Dis, 2013, 1:e24049; Wrabetz et al., J Neurosci, 2006, 26:2358-2368; Fratta et al., Hum Mol Genet, 2019, 1;28(1):124-132). Intracellular accumulation of mutant proteins is common to both mechanisms. For example, the R98C mutation causes early-onset severe disease due to retention of mutant MPZ protein in the ER and defective UPR. Patients with this mutation have very low or almost no myelin. Some patients may never be able to walk or may use a wheelchair independently by the end of the first decade. The median motor nerve conduction velocity (MNCV) is generally less than 6 m / sec, whereas normal is approximately 40 m / sec. These patients are classified as CMT1B.

[0029] Additional diseases associated with abnormal MPZ expression include, but are not limited to, severe early-onset congenital hypomyelination, Degerines-Sottas syndrome or disease (DSS or CMT type 3). Some mutations in the MPZ gene also cause dominant intermediate Charcot-Marie-Tooth disease (DI-CMT), CMT type 2I (CMT2I), and CMT type 2J (CMT2J).

[0030] MPZ levels must lie within a narrowly defined range. Lack of Mpz expression in homozygous Mpz null mice leads to poorly compacted myelin sheaths (Grandis et al., Hum Mol Genet, 2008, 1;17(13):1877-89), dysregulation of myelin-specific gene expression, and abnormal myelin protein localization in Schwann cells (Bai et al., Ann Clin Transl Neurol, 2018, 5:445-455; Wrabetz et al., J Neurosci, 2006, 26(8):2358-2368). Heterozygous Mpz null mice display a very mild phenotype similar to the late, adult-onset form of CMT1B. Interestingly, overexpression of Mpz by more than 80% (>1.8-fold) also inhibits Schwann cell association with axons and disrupts myelination (Bai et al., Rare Dis, 2013, 1:e24049).

[0031] Several transgenic CMT1B mouse models have been generated, and it has been confirmed that dominant mutations in the MPZ gene cause peripheral neuropathy (Saporta et al., Brain, 2012, 135(7):2032-47; Wrabetz et al., ibid.; Giese et al., Cell, 1992, 71:565-576). One of the CMT1B mouse models is the MPZR98C transgenic line. The R98C "knock-in" mouse model of CMT1B (provided by Prof. Michael Shy, University of Iowa, Saporta et al., ibid.) was generated by site-directed mutagenesis using a homologous recombination method. MPZR98C blocks Schwann cell development in a mouse model of early-onset CMT1B. These mice display histological and behavioral phenotypes. Both heterozygous (R98C / +) and homozygous (R98C / R98C) mice develop weakness, abnormal nerve conduction velocity, and morphologically abnormal myelin, with R98C / R98C mice being more severely affected: these mice show accumulation of MpzR98C in the endoplasmic reticulum and developmental delay in myelination, similar to patients with the same mutation.

[0032] The present disclosure includes the use of homozygous R98C / R98C and heterozygous R98C / + mice in gene therapy studies. Homozygous mice have a slow unsteady gait, evident by persistent tremors during walking by the time of weaning at about postnatal day 21. Heterozygous R98C / + mice do not show consistent observable clinical abnormalities until at least one year of age. However, both heterozygous and homozygous mice show abnormalities in tests of motor skills. R98C / R98C mice were unable to maintain their balance on a rotating rod, with R98C / + mice performing significantly worse than wild-type mice. Homozygous and heterozygous mice have delayed motor nerve conduction velocity (MNCV) of about 4 m / s and about 15 m / s, respectively, compared to a MNCV of about 40 m / s in wild-type mice at 6-8 weeks of age (MpzR98C arrests Schwann cell development in a mouse model of early-onset Charcot-Marie-Tooth disease type 1B) (Saporta et al., supra). Thus, homozygous mice are more associated with severe early-onset CMT1B (DSS) and heterozygous mice develop a late-onset form of CMT1B. Thus, in various embodiments, MPZR98C mice are used as a model. Thus, MpzR98C is used in various embodiments of the present disclosure as a therapeutic model (http-colon-forwardslash-forwardslash-www.medlink.com-forwardslash-article-forwardslash-charcot-marie-tooth-disease-type-1b). Once the therapeutic vectors and doses are optimized in these mice, additional mouse models are included for use in this disclosure to explore the applicability of the proposed therapeutic strategies in various MPZ mutations.

[0033] Mouse models for milder forms of CMT1B, such as S63del (Wrabetz et al., supra), two other common gain-of-function variants, and H39P, represent childhood-onset and adult-onset CMT1B, respectively, and these models are included in various aspects of the present disclosure. S63del mice develop obvious neuromuscular disorders at 4-8 weeks of age, characterized by tremor, ataxia, weakness, and muscle atrophy in the hind limbs. S63del mice also develop marked hypomyelination at 6 months of age, with occasional naked axons and onion-like structures (concentrically layered Schwann cell processes surrounding nerve fibers) increasing in number after 1 year of age. MNCV movement is reduced by 50% in S63del / / + / - mice (Wrabetz et al., supra).

[0034] The present disclosure provides a novel strategy to achieve defective or mutant myelin protein zero (MPZ or P0) gene silencing at the mRNA level using RNA inhibition (RNAi). MPZ inhibitory RNA includes, but is not limited to, antisense RNA, small inhibitory RNA (siRNA), short hairpin RNA (shRNA), or artificial miRNA (MPZ miRNA) that inhibits the expression of wild-type and mutant MPZ genes.

[0035] MPZ miRNAs can specifically bind to a segment of messenger RNA (mRNA) encoded by the human MPZ gene (represented by SEQ ID NO:1, the human MPZ cDNA), which segment is conserved compared to the mRNA encoded by the wild-type mouse MPZ gene (represented by SEQ ID NO:82, the mouse MPZ cDNA). For example, MPZ miRNAs can specifically bind to an mRNA segment that is complementary to a sequence within nucleotides 225-247, 315-318, or 718-744 of SEQ ID NO:1.

[0036] The present disclosure focuses on inhibiting mutated MPZ expression in Schwann cells while maintaining healthy levels of normal MPZ protein. To achieve this, artificial miRNAs specifically designed to silence endogenous mutant and wild-type MPZ expression are provided by simultaneously replacing wild-type MPZ expression with miRNA-resistant (rMPZ) genes. As used herein, the term "miRNA-resistant (rMPZ)" is used interchangeably with "resistant MPZ (resMPZ)" or "codon-optimized resistant MPZ (coMPZ)". Thus, the present disclosure provides miRNAs (called miMPZ) designed to specifically and equally target both human MPZ and mouse Mpz genes, without predicted non-specific binding to other transcripts. This strategy allows for the translation of results from murine models to human models, which is particularly important in clinical trials.

[0037] The present disclosure provides products, methods, and uses for directly targeting the underlying genetic causes of CMT disease, including CMT1B disease, and the MPZ gene itself. The present disclosure provides a universal therapy applicable to all early and late onset forms of CMT1B caused by dominant mutations in the MPZ gene. Using miRNA-mediated silencing of the MPZ gene is a novel approach to CMT1B treatment. miRNAs are highly conserved, small (approximately 22 nucleotides long), non-coding RNA molecules that negatively regulate the expression of many genes at the post-transcriptional level (He et al., Nat Rev Genet, 2004, 5:522-531; Carrington et al., Science, 2003, 301:336-338). Because natural miRNAs are involved in several disease phenotypes and regulate more than one endogenous gene transcript, novel approaches utilize artificial miRNAs for therapeutic purposes (Hammond, Trends Mol Med, 2006, 12:99-101; McBride et al., Proc Natl Acad Sci USA, 2008, 105:5868-5873).

[0038] The present disclosure provides engineered artificial miRNAs based on natural human miR-30 by maintaining the essential structural and sequence elements required for normal miRNA biogenesis, but replacing the mature mir-30 sequence with one that targets the MPZ gene of interest. These novel artificial miRNAs contain 22-nt of perfect complementarity with the MPZ gene. The miRNAs of the present disclosure (miMPZ) are designed to target highly conserved sequences between mouse and human MPZ cDNAs. miMPZ-6-11 are designed in regions of the MPZ gene that do not have any reported mutations associated with CMT1B (Figure 1). Figure 1 provides a schematic diagram of human and mouse MPZ cDNA, showing the positions where the most common dominant mutations or small deletions that cause CMT1B are represented, and where the miRNAs of the present disclosure were designed so that a single miRNA can target both human and mouse MPZ sequences.

[0039] In some embodiments, the present disclosure includes the treatment or amelioration of CMT1B by reducing defective MPZ protein and replacing it with healthy MPZ protein. This strategy "knockdown and replacement" is achieved by using gene therapy to deliver MPZ reducing molecules called microRNAs (miRNAs) to Schwann cells along with healthy copies of the MPZ gene. Thus, in some embodiments, the products and methods described herein are used to treat MPZ genetic disorders, including but not limited to CMT diseases such as CMT1B.

[0040] This disclosure provides data showing that several miMPZs significantly reduced human mutant and wild-type MPZ expression by more than 80% in MPZ-overexpressing HEK293 cells. This disclosure also provides data demonstrating that rMPZ expression is resistant to miMPZ silencing in transiently transfected HEK293 cells.

[0041] The disclosure provides various nucleic acids and polypeptides. The disclosure includes various nucleic acids comprising, consisting essentially of, or consisting of various nucleotide sequences described herein. In some embodiments, the nucleic acid comprises a nucleotide sequence. In some embodiments, the nucleic acid consists essentially of a nucleotide sequence. In some embodiments, the nucleic acid consists of a nucleotide sequence. The disclosure includes various peptides comprising, consisting essentially of, or consisting of various amino acid sequences described herein. In some embodiments, the polypeptide comprises an amino acid sequence. In some embodiments, the polypeptide consists essentially of an amino acid sequence. In some embodiments, the polypeptide consists of an amino acid sequence.

[0042] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding human MPZ as set forth in the nucleotide sequence set forth in SEQ ID NO: 1. In various embodiments, the nucleic acid is an isoform or variant of a nucleotide sequence encoding human MPZ comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to the nucleotide sequence set forth in SEQ ID NO: 1.

[0043] In some embodiments, the polypeptide is a human MPZ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4. In various embodiments, the polypeptide is an isoform or variant of a human MPZ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0044] Thus, in some embodiments, the nucleic acid comprises a nucleotide sequence encoding human MPZ as set forth in the amino acid sequence set forth in SEQ ID NO: 4. In various embodiments, the nucleic acid is an isoform or variant of the nucleotide sequence encoding human MPZ as set forth in the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4.

[0045] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the human MPZ 3'UTR as set forth in SEQ ID NO:2. The target site in the 3'UTR is optimized to render it resistant to silencing by miRNA. In various embodiments, the nucleic acid is an isoform or variant of the nucleotide sequence encoding the human MPZ 3'UTR comprising the nucleotide sequence as set forth in SEQ ID NO:2. In some embodiments, the isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to the nucleotide sequence as set forth in SEQ ID NO:2.

[0046] RNA inhibition (RNAi) is described herein as an effective long-term treatment for dominantly inherited disorders. By way of example, products and methods are provided for treating subjects with MPZ gene mutations by knocking down both the wild type and mutant of the gene involved, while also delivering an RNAi-resistant replacement MPZ gene. By way of example, products and methods are described herein for knocking down the expression of mutant and wild type MPZ genes in a subject. The method utilizes MPZ inhibitory RNA to knock down mutant MPZ gene expression. The method also provides an RNAi-resistant replacement MPZ gene. The use of the methods and products is adapted, for example, to prevent, treat, or ameliorate diseases associated with mutations in the MPZ gene, such as Charcot-Marie-Tooth type 1B (CMT1B) disease.

[0047] All miRNAs described herein were designed to target both wild-type and mutant MPZ genes. CMT1B is caused by an autosomal dominant mutation, so only one mutant allele is sufficient to cause CMT1B symptoms. Patients suffering from MPZ mutations may have one wild-type and one mutant MPZ allele, or two mutant MPZ alleles. Patients with only one mutant MPZ allele have a milder phenotype compared to patients with two mutant MPZ alleles. The miRNAs described herein were designed to target and silence both alleles. Additionally, because expression of the wild-type MPZ allele is necessary to maintain healthy myelin sheath, we designed a miRNA-resistant MPZ gene by codon-optimizing the MPZ cDNA sequence and mutating the miRNA target site in the gene (as described herein and shown in Figure 6A-B).

[0048] An exemplary miRNA-resistant MPZ gene is the codon-optimized MPZ cDNA (alternatively referred to as resistant MPZ (resMPZ or rMPZ) or coMPZ) sequence of the present disclosure, set forth in SEQ ID NO: 3, or found within the MPZ expression cassette set forth in SEQ ID NO: 6. The terms "miRNA-resMPZ", "resMPZ", "rMPZ", and "coMPZ" are interchangeable as used herein.

[0049] Provided is an RNAi-resistant replacement MPZ gene. An "RNAi-resistant replacement MPZ gene" has a nucleotide sequence that encodes an MPZ protein whose expression is not knocked down by the MPZ miRNA described herein, and the nucleotide sequence still has MPZ protein activity. An exemplary RNAi-resistant replacement MPZ gene is found in the MPZ expression cassette shown in SEQ ID NO:3 or SEQ ID NO:6.

[0050] Thus, the present disclosure provides nucleic acids for replacing mutant MPZ or wild-type MPZ that are downregulated by MPZ microRNA (miRNA). In some embodiments, the nucleic acid is codon-optimized to be resistant to exposure to MPZ miRNA. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a partially codon-optimized human MPZ (human coMPZ) as set forth in SEQ ID NO: 3. In various embodiments, the nucleic acid is an isoform or variant of the nucleotide sequence encoding a partially codon-optimized human MPZ, comprising the nucleotide sequence as set forth in SEQ ID NO: 3. In some embodiments, an isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to the nucleotide sequence set forth in SEQ ID NO:3.

[0051] In some embodiments, the nucleic acid for replacing mutant MPZ or wild type MPZ comprises a nucleotide sequence encoding a human MPZ promoter comprising the nucleotide sequence set forth in SEQ ID NO: 5. In various embodiments, the nucleic acid is an isoform or variant of the nucleotide sequence encoding a human MPZ promoter comprising the nucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to the nucleotide sequence set forth in SEQ ID NO: 5.

[0052] In some embodiments, the nucleic acid comprises a nucleotide sequence comprising a human MPZ expression cassette comprising a human MPZ promoter, a human MPZ 5'UTR, and a partially codon-optimized human MPZ cDNA. In some embodiments, such a nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:6. In various embodiments, the nucleic acid is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to the nucleotide sequence set forth in SEQ ID NO:6.

[0053] Exemplary RNAi-resistant replacement MPZ genes are set forth in any one of SEQ ID NOs: 3 and 6, or variants thereof comprising at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 3 and 6. Exemplary RNAi-resistant replacement MPZ genes further include an MPZ promoter comprising the nucleotide sequence set forth in SEQ ID NO:5, or a variant thereof comprising at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5.

[0054] Table 1, set forth below, provides various nucleotide and amino acid sequences of the present disclosure. Specifically, Table 1 provides the sequences of the human MPZ cDNA, 3'UTR DNA, partially codon-optimized human cDNA, human MPZ polypeptide, human MPZ promoter, and human MPZ expression cassette of the present disclosure.

[0055] [Table 1-1]

[0056] [Table 1-2]

[0057] [Table 1-3]

[0058] [Table 1-4]

[0059] The present disclosure includes the use of RNA interference to inhibit or interfere with expressed mutant MPZ to improve and / or treat subjects with diseases or disorders caused by mutated MPZ genes and the resulting altered forms of mRNA. RNA interference (RNAi) is a mechanism of gene regulation in eukaryotic cells that has been considered for the treatment of various diseases. RNAi refers to the post-transcriptional control of gene expression mediated by inhibitory RNA. miRNAs are small (21-25 nucleotides), non-coding RNAs that share sequence homology and base-pair with the 3' untranslated region of cognate messenger RNA (mRNA). The interaction between miRNA and mRNA supports cellular gene silencing mechanisms that prevent translation of mRNA.

[0060] As the understanding of natural RNAi pathways has improved, researchers have designed artificial shRNAs and snRNAs for use in regulating the expression of target genes to treat diseases. Several classes of small RNAs are known to trigger the RNAi process in mammalian cells, including short (or small) interfering RNAs (siRNAs), as well as short (or small) hairpin RNAs (shRNAs) and microRNAs (miRNAs), which constitute a similar class of vector expression triggers [Davidson et al., Nat. Rev. Genet. 12: 329-40, 2011; Harper, Arch. Neurol. 66: 933-8, 2009]. shRNAs and miRNAs are expressed in vitro from plasmid- or virus-based vectors, so that a single dose can achieve long-term gene silencing as long as the vector is present in the target cell nucleus and the driving promoter is active (Davidson et al., Methods Enzymol. 392: 145-73, 2005). Importantly, this vector expression approach builds on decades of progress already made in the muscle gene therapy field, but instead of expressing a protein-coding gene, the vector cargo in the RNAi therapy strategy is an artificial shRNA or miRNA cassette that targets the disease gene of interest. This strategy is used to express a natural miRNA. Each shRNA / miRNA is based on the hsa-miR-30a sequence and structure. The natural mir-30a mature sequence is replaced by unique sense and antisense sequences derived from the target gene.

[0061] The present disclosure provides an artificial miRNA specifically designed to silence endogenous mutant MPZ expression and wild-type MPZ expression. The present disclosure also provides an artificial miRNA specifically designed to silence endogenous mutant MPZ expression and wild-type MPZ expression, while simultaneously replacing wild-type MPZ expression with a miRNA-resistant (rMPZ) gene. This "knockdown and replacement" strategy reduces defective MPZ protein and replaces the defective protein with healthy MPZ protein. In some embodiments, the artificial MPZ-reducing miRNA is delivered alone. In some embodiments, the artificial MPZ-reducing miRNA is delivered together with the rMPZ gene to neural cells, including but not limited to Schwann cells. The artificial miRNA (herein referred to as miMPZ) is designed to specifically and equally target both human MPZ and mouse Mpz genes, and has no predicted non-specific binding with other transcripts. This strategy allows for the translation of results from murine models to human clinical trials. Therefore, the miMPZ described and disclosed herein is useful as a therapeutic agent for treating MPZ mutations and diseases associated with these MPZ mutations, including but not limited to CMT diseases.

[0062] The present disclosure provides nucleic acids comprising polynucleotides encoding inhibitory RNAs (microRNAs (miRNAs)) that target MPZ to knockdown, inhibit, or prevent expression of MPZ genes and proteins, including mutant MPZ genes and proteins. The inhibitory RNA comprises an antisense sequence that inhibits expression of the MPZ gene. In some embodiments, the present disclosure provides nucleic acids comprising polynucleotides encoding MPZ miRNAs and RNAi-resistant MPZ genes. The present disclosure provides full-length unprocessed MPZ miRNAs, and mature or processed miRNAs. The present disclosure also provides MPZ sequences that miRNA sequences are designed to target.

[0063] In some embodiments, the disclosure provides a nucleic acid comprising a polynucleotide encoding an MPZ miRNA that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs:7-18.

[0064] In some embodiments, the disclosure provides a nucleic acid comprising a polynucleotide encoding an MPZ miRNA that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs:7-18.

[0065] In some embodiments, the disclosure provides nucleic acids comprising a polynucleotide comprising an MPZ miRNA comprising a full length miRNA antisense guide strand that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 19-30. Thus, in some embodiments, the disclosure provides a nucleic acid comprising a polynucleotide that comprises or encodes a MPZ final processed guide strand miRNA that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs:31-42. Thus, in some more particular aspects, the disclosure provides a nucleic acid comprising a polynucleotide sequence encoding a MPZ final processed guide strand miRNA that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs:31-42.

[0066] In some embodiments, the present disclosure provides nucleic acids comprising a polynucleotide targeting a DNA sequence of the MPZ gene that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 43-54.

[0067] Table 2, shown below, provides exemplary miRNA coding sequences, RNA sequences, and MPZ target sequences. Table 3, shown below, provides PCR primers (SEQ ID NOs: 55-78) used in synthesizing DNA encoding microRNAs (miRNAs).

[0068] [Table 2-1]

[0069] [Table 2-2]

[0070] [Table 3-1]

[0071] [Table 3-2]

[0072] In some embodiments, the nucleic acids of the present disclosure comprise a polynucleotide sequence that is operably linked to a transcriptional control element (including, but not limited to, a promoter, an enhancer, and / or a polyadenylation signal) that is functional in a target cell. In some embodiments, the polynucleotide (or nucleotide) sequence is linked to a promoter.

[0073] Inducible promoters are also contemplated. Non-limiting examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline-regulated promoters. The gene cassette containing the MPZ replacement gene may also include an intron sequence to facilitate processing of the MPZ RNA transcript when expressed in mammalian cells.

[0074] In some embodiments, the polynucleotide sequence encoding the MPZ miRNA and / or the polynucleotide sequence encoding the replacement MPZ gene are expressed under the same promoter, or each polynucleotide sequence is expressed under its own promoter, such as U6 promoter, U7 promoter, H19 promoter, neuron-specific promoter, H1 promoter, EF1-alpha promoter, minimal EF1-alpha promoter, unc45b promoter, CK1 promoter, CK6 promoter, CK7 promoter, mini-CMV promoter, CMV promoter, muscle creatine kinase (MCK) promoter, alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), tMCK promoter, minimal MCK promoter, desmin promoter, chicken β-actin promoter (CBA), P546 promoter, simia promoter, or the like. Examples of promoters that may be used include, but are not limited to, SV40 (simian virus 40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, elongation factor-1a promoter, hemoglobin promoter, creatine kinase promoter, neuron-specific promoter, myelin-specific promoter, or native promoter. In some embodiments, the promoter may be a Schwann cell-specific promoter or a myelin-specific promoter, including, but not limited to, those promoters described in, for example, WO2020 / 245169. In some embodiments, the promoter may be a mouse, rat, or human promoter. In some embodiments, the nucleotide sequence encoding the MPZ miRNA and / or replacement MPZ gene is expressed under a U6 promoter, a U7 promoter, a H19 promoter, or a neuron-specific promoter.In some embodiments, such neuron-specific promoters are MPZ promoters, non-compacted myelin associated protein (NCMPA or MP11) promoters, PMP22 promoters, MBP promoters, SOX10 promoters, or GAP43 promoters. In some embodiments, the promoters used with miMPZ are U6 promoters. In some embodiments, the promoters used with MPZ replacement genes are MPZ promoters or miniMPZ promoters. In some embodiments, the MPZ promoters or miniMPZ promoters are mouse, rat, or human promoters. In some embodiments, the MPZ promoters or miniMPZ promoters are human MPZ promoters or human miniMPZ promoters. In some embodiments, the human MPZ promoter comprises a nucleotide sequence set forth in SEQ ID NO:5, or a variant thereof comprising at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5.

[0075] In some embodiments, the products, methods, and uses of the present disclosure also include short hairpin RNA or small hairpin RNA (shRNA) for affecting (e.g., knocking down or inhibiting) MPZ expression. Short hairpin RNA (shRNA / hairpin vector) is an artificial RNA molecule with a tight hairpin turn that can be used to silence expression of a target gene via RNA interference (RNAi). shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover, but it requires the use of an expression vector. Once the vector has transduced the host genome, the shRNA is then transcribed in the nucleus by polymerase II or polymerase III depending on the promoter selection. The product mimics a pri-microRNA (pri-miRNA) and is processed by Drosha. The resulting pre-shRNA is exported from the nucleus by exportin 5. The product is then processed by Dicer and loaded into the RNA-induced silencing complex (RISC). The sense (passenger) strand is degraded. The antisense (guide) strand directs RISC to the mRNA with a complementary sequence. In the case of perfect complementarity, RISC cleaves the mRNA. In the case of imperfect complementarity, RISC suppresses the translation of the mRNA. In both of these cases, the shRNA results in targeted gene silencing. In some embodiments, the present disclosure includes the production and administration of AAV vectors that express MPZ antisense sequences via shRNA. The expression of shRNA is regulated by the use of various promoters. Promoter selection is essential to achieve robust shRNA expression. In various embodiments, polymerase II promoters such as U6 and H1 are used, as well as polymerase III promoters. In some embodiments, U6 shRNA is used.

[0076] Thus, in some embodiments, the present disclosure uses U6 shRNA molecules to inhibit, knock down, or interfere with MPZ gene expression. Conventional small / short hairpin RNA (shRNA) sequences are usually transcribed into cell nuclei from vectors containing PolIII promoters such as U6. The endogenous U6 promoter normally controls the expression of U6 RNA, a small nuclear RNA (snRNA) involved in splicing, and has been well characterized [Kunkel et al., Nature. 322(6074): 73-7 (1986); Kunkel et al., Genes Dev. 2(2): 196-204 (1988); Paule et al., Nucleic Acids Res. 28(6): 1283-98 (2000)]. In some embodiments, the U6 promoter is used to control vector-based expression of shRNA molecules in mammalian cells [Paddison et al., Proc. Natl. Acad. Sci. USA 99(3):1443-8(2002); Paul et al., Nat. Biotechnol. 20(5):505-8(2002)] because (1) the promoter is recognized by RNA polymerase III (polyIII) and controls high-level constitutive expression of shRNAs, and (2) the promoter is active in most mammalian cell types. In some embodiments, the promoter is a type III PolIII promoter in which all elements required to control expression of the shRNA are located upstream of the transcription start site (Paule et al., Nucleic Acids Res. 28(6):1283-98(2000)). The present disclosure includes both murine and human U6 promoters. The shRNA, which contains the sense and antisense sequences from a target gene connected by a loop, is transported from the nucleus to the cytoplasm, where Dicer processes it into small / short interfering RNAs (siRNAs).

[0077] In some embodiments, the products, methods, and uses of the present disclosure include small nuclear ribonucleic acids (snRNAs), also commonly referred to as U-RNAs, to knock down or even inhibit MPZ gene expression. snRNAs are a class of small RNA molecules found inside splicing speckles and Cajal bodies of the cell nucleus in eukaryotic cells. Small nuclear RNAs are associated with a set of specific proteins, and the complexes are called small nuclear ribonucleoproteins (snRNPs, often pronounced "snoop"). Each snRNP particle consists of an snRNA component and several snRNP-specific proteins, including Sm proteins, a family of nuclear proteins. snRNAs, together with their associated proteins, form ribonucleoprotein complexes (snRNPs) that bind to specific sequences on pre-mRNA substrates. They are transcribed by either RNA polymerase II or RNA polymerase III. snRNAs are often divided into two classes based on both common sequence features and associated protein factors, such as RNA-binding LSm proteins. The first class, known as the Sm class of snRNAs, is composed of U1, U2, U4, U4atac, U5, U7, U11, and U12. The Sm class of snRNAs are transcribed by RNA polymerase II. The second class, known as the Lsm class of snRNAs, is composed of U6 and U6atac. The Lsm class of snRNAs are transcribed by RNA polymerase III and, in contrast to the Sm class of snRNAs, never leave the nucleus. In some embodiments, the present disclosure includes the production and administration of AAV vectors containing U7 snRNA for delivery of MPZ antisense sequences.

[0078] In some embodiments, the present disclosure further inhibits, knocks down, or interferes with MPZ gene expression using U7 snRNA molecules. U7 snRNA is normally involved in histone pre-mRNA 3' end processing, but in some embodiments, it is converted into a versatile tool for splicing regulation or as an antisense RNA continuously expressed in cells [Goyenvalle et al., Science 306(5702):1796-9(2004)]. By replacing the wild-type U7 Sm binding site with a consensus sequence derived from spliceosomal snRNA, the resulting RNA assembles with the seven Sm proteins found in the spliceosomal snRNA. As a result, this U7 Sm OPT RNA accumulates more efficiently in the nucleoplasm and no longer mediates histone pre-mRNA cleavage, but can still bind to histone pre-mRNA and act as a competitive inhibitor of wild-type U7 snRNP. By further replacing the sequence that binds to the histone downstream element with one that is complementary to a specific target in the splicing substrate, it is possible to generate U7 snRNAs that can regulate specific splicing events. The advantage of using U7 derivatives is that the antisense sequence is embedded in the small nuclear ribonucleoprotein (snRNP) complex. Furthermore, when embedded in gene therapy vectors, these small RNAs can be permanently expressed in target cells after a single injection [Levy et al., Eur. J. Hum. Genet. 18(9): 969-70 (2010); Wein et al., Hum. Mutat. 31(2): 136-42, (2010); Wein et al., Nat. Med. 20(9): 992-1000 (2014)].

[0079] U7 snRNA is usually involved in histone pre-mRNA 3' end processing, but is also used as a general tool for splicing regulation or as an antisense RNA that is continuously expressed in cells. One advantage of using U7 derivatives is that the antisense sequence is embedded in the small nuclear ribonucleoprotein (snRNP) complex. Furthermore, when embedded in gene therapy vectors, these small RNAs can be permanently expressed in target cells after a single injection.

[0080] In some embodiments, the present disclosure includes nanoparticles, extracellular vesicles, exosomes, or vectors that include any of the nucleic acids of the present disclosure, or any one or more combinations thereof. In some embodiments, one or more copies of these sequences are combined into a single nanoparticle, extracellular vesicle, exosome, or vector.

[0081] Thus, the present disclosure includes vectors comprising a nucleic acid of the present disclosure or a combination of nucleic acids of the present disclosure.Embodiments of the present disclosure utilize vectors (e.g., viral vectors such as adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus, poxvirus, herpes virus, herpes simplex virus, poliovirus, Sindbis virus, vaccinia virus, or synthetic viruses, e.g., chimeric viruses, mosaic viruses, or pseudotyped viruses, and / or viruses containing foreign proteins, synthetic polymers, nanoparticles, or small molecules) to deliver the nucleic acids disclosed herein.

[0082] In some embodiments, the present disclosure utilizes AAV to deliver inhibitory RNA, such as DNA encoding MPZ miRNA, to target MPZ mRNA and inhibit mutant MPZ expression. AAV is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length, including an inverted terminal repeat (ITR) of 145 nucleotides. There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077, and the complete genome of AAV-2 is provided under GenBank Accession No. NC_001401 and Srivastava et al. al., J. Virol., 45:555-564 {1983), the complete genome of AAV-3 is provided under GenBank accession number NC_1829, the complete genome of AAV-4 is provided under GenBank accession number NC_001829, the AAV-5 genome is provided under GenBank accession number AF085716, the complete genome of AAV-6 is provided under GenBank accession number NC_001862, at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 regarding AAV-8), and the AAV-9 genome is provided by Gao et al. The AAV genome is provided in, for example, J. Virol., 78:6381-6388 (2004), the AAV-10 genome is provided in, for example, Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is provided in, for example, Virology, 330(2):375-383 (2004). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 by their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (nucleotide positions 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are 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, Curr Topics in Microbiol and Immunol, 158:97-129 (1992).

[0083] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV can infect many mammalian cells, offering the possibility to target many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the lifetime of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Moreover, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb genome (encoding the replication and structural capsid protein rep-cap) can be replaced with foreign DNA. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. This makes AAV more easily resistant to the conditions used to inactivate adenovirus, making cryopreservation of AAV less important. AAV can be lyophilized, and AAV-infected cells are not resistant to superinfection. In some embodiments, AAV is used to deliver a nucleic acid encoding an inhibitory RNA under the control of a U6 promoter, a U7 promoter, or a neuron-specific promoter. In some embodiments, AAV is used to deliver a nucleic acid encoding an inhibitory RNA under the control of a MPZ promoter or a mini-MPZ promoter. In some embodiments, the MPZ promoter or the mini-MPZ promoter is mouse, rat, or human. In some embodiments, AAV is used to deliver a nucleic acid encoding an RNAi-resistant replacement MPZ gene under the control of a U6 promoter, a U7 promoter, or a neuron-specific promoter. In some embodiments, AAV is used to deliver a nucleic acid encoding an RNAi-resistant replacement MPZ gene under the control of a MPZ promoter or a mini-MPZ promoter.In some embodiments, the MPZ promoter or mini-MPZ promoter is a mouse, rat, or human promoter. In some embodiments, the MPZ promoter or mini-MPZ promoter is a human promoter.

[0084] In some embodiments, the AAV lacks the rep and cap genes, hi some embodiments, the AAV is a recombinant linear AAV (rAAV), a single-stranded AAV, or a recombinant self-complementary AAV (scAAV).

[0085] Advances in AAV vectors have led to safer and more efficient viral vehicles that deliver therapeutic transgenes with a single injection, and gene therapy is now the preferred therapeutic intervention for monogenic diseases. AAV vectors can provide long-term expression of gene products in postmitotic target tissues. Thus, current AAV-based strategies may require only a single vector administration.

[0086] The recombinant AAV genome of the present disclosure includes, for example, one or more AAV ITRs adjacent to a polynucleotide encoding one or more MPZ inhibitory RNAs or MPZ miRNAs. The genome of the rAAV provided herein may further include an RNAi-resistant replacement MPZ gene, or the RNAi-resistant replacement MPZ gene may be present in a separate rAAV. The miRNA- and replacement MPZ-encoding polynucleotides are operably linked to transcriptional regulatory DNA, such as promoter DNA, that is functional in the target cell. Commercial providers such as Ambion Inc. (Austin, TX), Darmacon Inc. (Lafayette, CO), InvivoGen (San Diego, CA), and Molecular Research Laboratories, LLC (Herndon, VA) make custom inhibitory RNA molecules. In addition, commercially available kits are available for generating custom siRNA molecules, such as the SILENCER™ siRNA Construction Kit (Ambion Inc., Austin, TX) or the psiRNA System (InvivoGen, San Diego, CA).

[0087] Provided herein is a rAAV that each encodes one or more MPZ miRNAs and / or one or more RNAi-resistant replacement MPZ genes. The rAAV that encodes one or more MPZ miRNAs and RNAi-resistant replacement MPZ genes can encode 1, 2, 3, 4, 5, 6, 7, or 8 MPZ miRNAs. In some embodiments, a separate rAAV that encodes an RNAi-resistant replacement MPZ gene is provided. In some embodiments, the rAAV encodes an RNAi-resistant replacement MPZ gene in the same rAAV vector. In some embodiments, the rAAV that encodes one or more MPZ miRNAs can encode 1, 2, 3, or 4 MPZ miRNAs.

[0088] Thus, in some embodiments, the viral vector is AAV. The present disclosure includes all types of AAV and is not limited to only the types of AAV described herein. Accordingly, such AAVs include AAV1 (i.e., an AAV comprising AAV1 inverted terminal repeats (ITRs) and AAV1 capsid protein), AAV2 (i.e., an AAV comprising AAV2 ITRs and AAV2 capsid protein), AAV3 (i.e., an AAV comprising AAV3 ITRs and AAV3 capsid protein), AAV4 (i.e., an AAV comprising AAV4 ITRs and AAV4 capsid protein), AAV5 (i.e., an AAV comprising AAV5 ITRs and AAV5 capsid protein), AAV6 (i.e., an AAV comprising AAV6 ITRs and AAV6 capsid protein), AAV7 (i.e., an AAV7 ITRs and AAV7 capsid protein), AAV8 (i.e., an AAV comprising AAV8 ITRs and AAV8 capsid protein), AAV9 (i.e., an AAV comprising AAV9 ITRs and AAV9 capsid protein), AAV10 (i.e., an AAV10 AAVrh.74 (i.e., an AAV containing AAVrh.74 ITRs and AAVrh.74 capsid protein), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid protein), AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid protein), AAV11 (i.e., an AAV containing AAV11 ITRs and AAV11 capsid protein), AAV12 (i.e., an AAV containing AAV12 ITRs and AAV12 capsid protein), AAV13 (i.e., an AAV containing AAV13 ITRs and AAV13 capsid protein), AAV-anc80 (i.e., an AAV containing AAV-anc80 ITRs and AAV-anc80 capsid protein), AAVrh.74 (i.e., an AAV containing AAVrh.74 ITRs and AAVrh.74 capsid protein), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid protein), AAVrh.10 (i.e., an AAVrh.10 ITRs and AAVrh.10 capsid protein), AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAVv66, AAV-F, or a pseudotype AAV such as AAV2 / 1, AAV2 / 8, or AAV2 / 9.

[0089] In various embodiments, the viral vector is AAV9, AAV-PHP.eB, or AAV-F. In various embodiments, the viral vector is AAV9 or AAV-F. AAV9 has become the most widely used vector for neurological indications with an established safety profile in the clinic. Intrathecal administration of AAV9 allows for dissemination of transgenes throughout the nervous system and is currently approved by the FDA for spinal muscular atrophy (SMA, NCT03381729) and in clinical trials for the treatment of neuronal ceroid lipofuscinosis 3 (CLN3, NCT03770572), CLN6 (NCT02725580), and giant axonal neuropathy (GAN, NCT02362438). AAV9 is also known to be able to target Schwann cells, which are a clear therapeutic target for CMT1B disease and other peripheral neuropathy. More importantly, AAV9 has been reported to transduce Schwann cells in large animals and non-human primates (Bradbury et al., J Clin Invest, 2020, 130(9):4906-4920; Gautier et al., Nat Commun, 2021, 12:2356), indicating that it is a desirable viral vector for clinical applications requiring delivery of therapeutic genes to human Schwann cells. AAV-F has recently been shown to be effective in the spinal cord (Beharry et al., Hum Gene Ther. 2022 Jan;33(1-2):61-75. doi:10.1089 / hum.2021.069. Epub 2021 Aug 26).

[0090] The DNA plasmid of the present disclosure comprises the rAAV genome of the present disclosure. In some embodiments, the DNA plasmid is transferred into a cell that is permissive for infection by a helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) of AAV for assembly of the rAAV genome into an infectious viral particle. Thus, in some embodiments, the present disclosure comprises an AAV vector that delivers a therapeutic agent into a cell. In some embodiments, the cell is a neuronal cell. In some embodiments, the neuronal cell is a Schwann cell.

[0091] Techniques for producing rAAV particles that provide a cell with the AAV genome to be packaged, the rep and cap genes, and helper virus functions are standard in the art. rAAV production requires the following components to be present within a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separate from (i.e., not present in) the rAAV genome, and the helper virus functions. The AAV rep gene can be from any AAV serotype from which a recombinant virus can be derived, and can be from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, or AAVv66. In some embodiments, the AAV DNA in the rAAV genome is from any AAV serotype from which the recombinant virus can be derived, including but not limited to, AAV serotypes in which the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAVv66, or AAV-F. Other types of rAAV variants are also included in the disclosure, such as rAAVs with capsid mutations. See, e.g., Marsic et al., Molecular Therapy 22(11):1900-1909(2014). As mentioned above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art.The use of homologous components is specifically contemplated.The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety.

[0092] In some embodiments, the viral vector is pseudotyped AAV and contains ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 8 (i.e., an AAV containing AAV2 ITRs and AAV8 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 1 (i.e., an AAV containing AAV2 ITRs and AAV1 capsid proteins).

[0093] In some embodiments, the AAV comprises a recombinant capsid protein, such as a capsid protein comprising one or more chimeras of capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAVv66, or AAV-F. Other types of rAAV variants are also contemplated, such as rAAVs with capsid mutations. See, e.g., Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As mentioned in the background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0094] The method for generating packaging cells is to create a cell line that stably expresses all the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separate from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the genome of the cell. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus, such as adenovirus. The advantage of this method is that the cells are selectable and are suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus, rather than a plasmid, to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0095] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbiol. and Immunol., 158:97-129). Various approaches have been proposed by Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Tratschin et al., Mo1. Cell. al., J. Virol., 62:1963 (1988), and Lebkowski et al., 1988 Mol. Cell. Biol., 7: 349 (1988), Samulski et al. al., J. Virol., 63:3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365 and corresponding U.S. Pat. No. 5,658.776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al., Vaccine, 13:1244-1250 (1995), Paul et al. al., Human Gene Therapy, 4:609-615 (1993); Clark et al., Gene Therapy, 3:1124-1132 (1996); U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; U.S. Patent No. 6,258,595; and McCarty, Mol. Ther., 16(10):1648-1656 (2008). The foregoing documents are incorporated herein by reference in their entireties, with particular emphasis on those portions of the documents that relate to rAAV production. The production and use of self-complementary (sc) rAAV is specifically contemplated and exemplified.

[0096] Thus, the present disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells are stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (synonymous 293 line). In another embodiment, the packaging cells are non-transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (fetal rhesus lung cells).

[0097] In some embodiments, the rAAV is purified by methods standard in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art and include, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO 98 / 09657.

[0098] Compositions are provided that include the nucleic acid and viral vector of the present disclosure. Compositions are provided that include the delivery vehicle (such as rAAV) described herein. In various embodiments, such compositions also include a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a diluent, excipient, or buffer. The compositions may also include other components, such as adjuvants.

[0099] Acceptable carriers, diluents, excipients, and adjuvants are nontoxic to recipients and preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparaginine, 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, Pluronic®, or polyethyl glycol (PEG).

[0100] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in a suitable solvent, together with various other ingredients as listed above as necessary, followed by filtration sterilization.In general, dispersions are prepared by incorporating sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those listed above.In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces a powder of active ingredients plus any additional desired ingredients from their previously sterile-filtered solutions.

[0101] The titer of the rAAV administered in the methods of the present disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the therapeutic goal, the individual, and the cell type being targeted, and can be determined by methods standard in the art. The titer of the rAAV is approximately 1 x 10 per ml. 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 13 ~Approx. 1×10 14Dosages may range from 1 x 10 to 100, or more than 1 x 10 DNase resistant particles (DRP). Doses may also be expressed in units of viral genomes (vg) (e.g., 1 x 10 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, and 1×10 14 vg).

[0102] Transduction of cells with the rAAV of the present disclosure results in sustained expression of MPZ miRNA and RNAi-resistant replacement MPZ gene. Thus, the present disclosure provides a method of administering / delivering a rAAV expressing MPZ miRNA and RNAi-resistant replacement MPZ gene to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. These methods include transducing cells and tissues (including, but not limited to, peripheral motor neurons, sensory motor neurons, neurons, Schwann cells, and other tissues or organs such as muscle, liver, and brain) with one or more rAAVs described herein. Transduction can be performed using gene cassettes that contain cell-specific control elements.

[0103] The term "transduction" is used, by way of example, to refer to administration / delivery of MPZ miRNA and RNAi-resistant replaced MPZ to target cells either in vivo or in vitro via a replication-deficient rAAV as described herein, resulting in expression of the MPZ miRNA and RNAi-resistant replaced MPZ by the target cells.

[0104] Methods are provided for transducing target cells with a delivery vehicle (e.g., nanoparticles, extracellular vesicles, exosomes, or vectors (rAAV)) in vivo or in vitro. In vivo methods include administering an effective dose or multiple effective doses of a composition comprising a delivery vehicle (such as rAAV) to an animal (including a human subject) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. An effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease state, reduces the extent of the disease, results in remission (partial or complete) of the disorder / disease state, and / or prolongs survival. Thus, methods are provided for administering an effective dose (or doses administered essentially simultaneously or at intervals) of a rAAV as described herein to a patient in need thereof.

[0105] Provided herein are pharmaceuticals and methods for treating, ameliorating, or preventing diseases associated with mutant MPZ genes or abnormal MPZ gene expression. The MPZ gene encodes the MPZ protein, which is the major protein in the myelin sheath. MPZ is an essential protein for maintaining a healthy and efficient peripheral nervous system. Accumulation of defective proteins in Schwann cells leads to demyelination and cell death over time. The pathological mechanisms of CMT1B disease can be mostly divided into two major groups: (1) toxic gain-of-function mutations that directly affect normal myelination, and (2) defective unfolded protein response (UPR) or endoplasmic reticulum (ER) stress response. Both mechanisms of disease ultimately lead to accumulation of mutant myelin proteins in Schwann cells (SCs), reduced myelination, muscle weakness and atrophy, and loss of sensation in the lower limbs and feet. For example, the R98C mutation causes early-onset severe disease due to retention of mutated MPZ protein in the ER and defective UPR. Patients with this mutation have very low or almost no myelin. These patients are classified as CMT1B, but also as very severe CMT1B or Degerines-Sottas syndrome (DSS), another definition of congenital hypomyelination. Thus, an example of a disease included for prevention, treatment, or amelioration by the methods of the present disclosure is CMT1B disease. In families known to have pathological MPZ mutations, the method for prevention is performed before disease onset in the subject. In other subjects, the method is performed after diagnosis.

[0106] Molecular, biochemical, histological, and functional outcome measures demonstrate the therapeutic efficacy of the method. Outcome measures are described, for example, in Dyck and Thomas, Peripheral Neuropathy, Elsevier Saunders, Philadelphia, PA, 4 thEdition, Volume 1 (2005), Chapters 32, 35, and 43, and Burgess et al., Methods Mol. Biol., 602:347-393 (2010). Outcome measures include, but are not limited to, one or more of the following: reduction or elimination of mutant MPZ mRNA or protein in affected tissues, MPZ gene knockdown, reduced demyelination, or improved myelination. Others include, but are not limited to, reduced cell death.

[0107] In various embodiments, quantitative RT-PCR (qRT-PCR) and Western blot assays are used to detect the expression of miMPZ and / or coMPZ. In some embodiments, toluidine blue staining is performed to examine demyelination / remyelination and onion-like structures in treated and untreated subjects. In some embodiments, behavioral analyses such as rotarod and footgrip tests are performed to evaluate motor balance, coordination, and muscle strength. In some embodiments, TUNEL assays are performed on the sciatic nerve to determine the amount of Schwann cell death. In some embodiments, tests are performed to examine endoplasmic reticulum (ER) changes or stress in muscle and the unfolded protein response (UPR). In some embodiments, these tests are performed using qRT-PCR assays. Skeletal muscle is a highly plastic tissue in the human body that undergoes extensive adaptations in response to environmental cues such as physical activity, metabolic perturbations, and disease states. The ER plays a key role in protein folding and calcium homeostasis in many mammalian cell types, including skeletal muscle. However, an overload of misfolded or misfolded proteins in the ER lumen causes stress, leading to the activation of a signaling network called the UPR. The UPR is initiated by three ER transmembrane sensors: protein kinase R-like endoplasmic reticulum kinase, inositol-requiring protein 1α, and activating transcription factor 6. The UPR restores ER homeostasis by regulating the rate of protein synthesis and enhancing gene expression of many ER chaperones and regulatory proteins. However, chronically elevated ER stress can also lead to many pathological outcomes, including cell death. Accumulating evidence suggests that the ER stress-induced UPR pathway plays an important role in regulating skeletal muscle mass and metabolic function in multiple conditions. In some embodiments, a nerve conduction velocity (NCV) test is used. During the test, a nerve is stimulated, usually with a surface electrode patch attached to the skin. Two electrodes are placed on the skin overlying the nerve. One electrode stimulates the nerve with a very mild electrical impulse, while the other electrode records it. The resulting electrical activity is recorded by another electrode.This is repeated for each nerve tested, providing a measurement of the conduction velocity of the electrical impulse through the nerve. Thus, in some embodiments, the NCV test can determine nerve damage and destruction, and / or improvement.

[0108] In the disclosed methods, expression of the mutant MPZ allele (or both mutant alleles) is inhibited by at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 95, at least 98 percent, at least 99 percent, or 100 percent. In the methods, expression of the wild-type MPZ allele is inhibited by at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 95, at least 98 percent, at least 99 percent, or 100 percent.

[0109] Combination therapy is also contemplated by the present disclosure. In some embodiments, a combination of two or more miRNAs is used to achieve higher silencing efficiency. In some embodiments, miRNA is used with other therapies for diseases associated with MPZ mutations. In some embodiments, miRNA described herein is used with other therapies designed to neutralize or reduce the expression of mutant MPZ genes. In some embodiments, such other therapies are described herein. In some embodiments, such other therapies are known to those skilled in the art. Combination as used herein includes both simultaneous and sequential treatment. Combination of the methods described herein with standard medical and supportive care is specifically contemplated, as is combination with therapy.

[0110] Administration of an effective dose of the nucleic acid, nanoparticle, extracellular vesicle, exosome, viral vector, or composition of the present disclosure may be by routes standard in the art, including, but not limited to, intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraventricular, intrathecal, intraosseous, intraocular, rectal, or vaginal. In various embodiments, an effective dose is delivered by a combination of routes. For example, in various embodiments, an effective dose is delivered intravenously and / or intramuscularly, or intravenously and intraventricularly, etc. In some embodiments, an effective dose is delivered sequentially or sequentially. In some embodiments, an effective dose is delivered simultaneously. The route of administration and serotype of the AAV components (specifically, AAV ITRs and capsid proteins) of the rAAV of the present disclosure can be selected and / or adapted by the skilled artisan in consideration of the infection and / or disease state to be treated, and the target cells / tissues in which the miRNA is to be expressed.

[0111] Specifically, the actual administration of the delivery vehicle (such as rAAV) can be accomplished by using any physical method that transports the delivery vehicle (such as rAAV) to the target cells of the subject. Administration includes, but is not limited to, injection into muscle, bloodstream, and / or directly into the nervous system or liver. Simply resuspending rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the usual procedures using rAAV). The capsid protein of rAAV can be modified to target rAAV to a specific target tissue of interest, such as neural cells. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or as a local formulation that is delivered to muscle by transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have already been developed and can be used in the practice of the present disclosure. The delivery vehicle (such as rAAV) can be used with any pharma- ceutically acceptable carrier to facilitate administration and handling.

[0112] Dispersions of the delivery vehicle (such as rAAV) can also be prepared in glycerol, sorbitol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, the sterile aqueous media employed are all readily obtainable by standard techniques well known to those skilled in the art.

[0113] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, sorbitol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, suMPZ or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0114] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in a suitable solvent, together with various other ingredients as listed above as necessary, followed by filtration sterilization.In general, dispersions are prepared by incorporating sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those listed above.In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces a powder of active ingredients plus any additional desired ingredients from their previously sterile-filtered solutions.

[0115] The present disclosure also provides a kit for use in the treatment of a disease or disorder described herein. Such a kit comprises at least a first sterile composition comprising any of the nucleic acids described herein above or any of the viral vectors described herein above in a pharma- ceutically acceptable carrier. Another component is a second therapeutic agent for the treatment of the disorder, optionally together with a suitable container and vehicle for administration of the therapeutic composition. The kit optionally comprises a solution or buffer for suspending, diluting, or effecting delivery of the first and second compositions.

[0116] In one embodiment, such a kit comprises the nucleic acid or vector in a diluent packaged in a container, such as a sealed bottle or vessel, with a label attached to the container or included in the package that describes the use of the nucleic acid or vector. In one embodiment, the diluent is in the container such that the amount of head space in the container (e.g., the amount of air between the liquid formulation and the top of the container) is very small. Preferably, the amount of head space is negligible (i.e., almost nonexistent).

[0117] In some embodiments, the formulation comprises a stabilizer. The term "stabilizer" refers to a substance or excipient that protects the formulation from harmful conditions, such as those that occur during heating or freezing, and / or extends the stability or shelf life of the formulation in a stable state. Examples of stabilizers include, but are not limited to, stabilizers such as sucrose, lactose, and mannose, sugars, sugar alcohols such as mannitol, amino acids such as glycine or glutamic acid, and proteins such as human serum albumin or gelatin.

[0118] In some embodiments, the formulation includes an antimicrobial preservative. The term "antimicrobial preservative" refers to any substance added to the composition that inhibits the growth of microorganisms that may be introduced upon repeated puncturing of the vial or container used. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[0119] In some embodiments, the kits include labels and / or instructions that describe the use of the reagents provided in the kit. The kits also optionally include catheters, syringes, or other delivery devices for delivering one or more of the compositions used in the methods described herein.

[0120] The entire document is intended to be related as a unified disclosure, and it is understood that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, or paragraph, or section of this document. The disclosure also includes all embodiments of the disclosure that are somewhat narrower in scope than the variations specifically mentioned above, for example. With respect to aspects of the disclosure described as genus, all individual species are considered separate aspects of the disclosure. With respect to aspects of the disclosure described or claimed as "a" or "an", these terms are understood to mean "one or more" unless the context clearly requires a more limited meaning. When aspects of the disclosure are described as "comprising" a feature, embodiments that "consist" or "consist essentially of" that feature are also contemplated.

[0121] All publications, patents, and patent applications cited in this specification are herein incorporated by reference, to the extent that each individual publication or patent application is not inconsistent with this disclosure, as if it were specifically and individually indicated to be incorporated by reference in its entirety.

[0122] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light of them will be suggested to those skilled in the art, but are within the spirit and scope of this application and the scope of the appended claims. EXAMPLES

[0123] Aspects and embodiments of the present disclosure are illustrated by the following examples, which are not intended to limit the scope of the invention in any way. Example 1 Materials and Methods MicroRNA (miRNA) design and synthesis MicroRNAs (miRNAs) specific for CMT1B disease were designed and synthesized. The sequences of human MPZ cDNA (SEQ ID NO:1, NM_000530.8) and its 3'UTR (SEQ ID NO:2, NM_000530.8) are provided in Table 1. The sequence of the partially codon-optimized MPZ cDNA (SEQ ID NO:3) is provided in Table 1. Nucleotides 225-247 of SEQ ID NO:3 (underlined in Table 1) contain the target sites for miMPZ 225 and 226. Nucleotides 315-338 of SEQ ID NO:3 (underlined in Table 1) contain the target sites for miMPZ 315, 316, and 317. Nucleotides 718-744 of SEQ ID NO:3 (underlined in Table 1) contain the target sites for miMPZ 718, 719, 720, 721, 722, and 723. A fully codon-optimized MPZ gene can be used to compare the expression level of MPZ or to create new cassettes. Therefore, other codon-optimized MPZ sequences are encompassed by the present disclosure since there are more than one codon for each amino acid. The sequence of the MPZ protein sequence (SEQ ID NO: 4, NM_000530.8) is provided in Table 1. The sequence of the human MPZ promoter (SEQ ID NO: 5, ENSG00000158887) is provided in Table 1. The sequence of the MPZ expression cassette (SEQ ID NO: 6) made from 1200 bp of the human MPZ promoter (ENSG00000158887), 63 bp of the human MPZ 5'UTR, and 747 bp of the partially codon-optimized MPZ cDNA is provided in Table 1. SEQ ID NO: 6 also includes the SV40 polyA signal and the CMV amplicon sequence. The CMV amplicon is used in the construct for AAV titration using qRT-PCR or ddPCR assays. The lead miMPZ can be cloned into the NsiI or NotI site located after the CMV amplicon. The entire sequence is cloned into the AAV pre-plasmid using two XbaI restriction enzyme sites at each end. The sequence was synthesized by Integrated DNA Technologies (IDT) (Coralville, IA).The sequence in the CoMPZ expression cassette is: XBAI-stui-SalI-kasi-human MPZ promoter (1200 bp)-NheI-human MPZ 5'UTR-partially codon-optimized human mpz cds-SPEI-ndei-SV40 PA-paci-CMV amplicon for DRP-NSII-noti-XBAI. Alternatively, the disclosure includes the use of various MPZ promoters or mini-MPZ promoters, including human, rat, or mouse promoters, in combination with a universal enhancer, such as a CMV-specific enhancer, or a Schwann-specific enhancer, such as PMP22.

[0124] Twelve synthetic miRNAs (named miMPZ) targeting the MPZ (P0) gene were designed and generated. See Table 2. These miRNAs were designed to target regions of both human and mouse MPZ genes (the MPZ gene sequence is conserved in both species). These are non-allele specific miMPZs, i.e., these miRNAs target both mutant and wild-type MPZ, silencing global MPZ expression in target cells. The miMPZ numbers indicate the target sequence in the cDNA and 3'UTR, with counting starting at the MPZ cDNA. DNA encoding miRNAs named miMPZ_225, miMPZ_226, miMPZ_315, miMPZ_316, miMPZ_317, miMPZ_718, miMPZ_719, miMPZ_720, miMPZ_721, miMPZ_722, miMPZ_723, and miMPZ_1852 were generated by PCR using 12 pairs of primers as provided in Table 3.

[0125] PCR cloning of miRNAs 1 μg of each primer was added to a 1-cycle primer extension reaction: 95° C. for 5 min, 94° C. for 2 min, 52° C. for 1 min, 68° C. for 15 min, then held at 4° C. The PCR products were cleaned up with a Qiagen QIAquick PCR purification kit and then digested overnight with XhoI and SpeI restriction enzymes. The digestion products were then run on a 1.5% TBE gel and the bands were excised and purified using a Qiagen QIAquick gel extraction kit. Sequences of each miRNA shown in Figure 1A-1L.

[0126] The two PCR products were ligated overnight into a U6T6 vector (via XhoI and XbaI) containing a mouse U6 promoter and an RNA polymerase III termination signal (six thymidine nucleotides). The miRNAs are cloned into the XhoI and XbaI restriction sites located between the 3' end of the U6 promoter and the termination signal (the SpeI site at the 3' end of the DNA template for each miRNA has a cohesive end complementary to the XbaI site). The ligation products were transformed into chemically competent E-coli cells using a heat shock at 42°C and incubated at 37°C shaking for 1 hour before being plated on kanamycin selection plates. Colonies were grown overnight at 37°C. The next day they were miniprepped and sequenced for accuracy.

[0127] Luciferase assay A luciferase assay was used to determine the expression level of MPZ in the presence of each miMPZ. 42,000 HEK293 cells were cultured in each well of a 96-well plate 16 h prior to transfection. The next day, cells were transfected with 25 ng of Renilla-Firefly plasmid containing the MPZ target sequence (Figure 3A) and 250 ng of each miMPZ-encoding plasmid using Lipofectamine 2000. After 24 h, luciferase assays using a Dual Luciferase Reporter Assay Kit (Promega, E1960) were performed according to the manufacturer's protocol. Relative expression was calculated by dividing Renilla expression by Firefly expression. Relative expression was then normalized to the expression of cells transfected with MPZ and U6T6 plasmids as negative controls. The results are shown in Figure 3B-D. MPZ-targeting miRNAs, designated miMPZ-225, 226, 721, and 723, were most effective in reducing luciferase protein expression in transfected cells in initial experiments.

[0128] Western blot HEK293 cells (250,000 cells / well) were seeded in 24-well plates 16 hours prior to transfection. The next morning, they were co-transfected with 250 ng of MPZ and 1250 ng of miMPZ expression plasmids using Lipofectamine 2000 (Thermofisher, US). 24 hours after transfection, cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% TritonX100) supplemented with a cocktail containing protease inhibitors. Protein concentrations were determined by DC protein assay kit (Bio-Rad Laboratories). 20 μg of each total protein sample was run on a 12% SDS-polyacrylamide gel. The molecular weight of protein bands was determined using GE Healthcare Rainbow molecular weight markers (Fisherscientific, USA). Proteins were transferred from SDS-PAGE gels to PVDF membranes via a semi-dry transfer method. Membranes were blocked with 5% nonfat milk and then incubated overnight at 4°C with primary rabbit anti-Flag antibody (1:10,000, Abcam, ab1162) or rabbit polyclonal anti-α-tubulin antibody (1:1,000, ab15246, Abcam, Cambridge, MA). The next day, following washing, blots were then probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:100,000, Jackson ImmunoResearch, West Grove, PA) for 1 h 30 min at room temperature. After a brief incubation in Immobilon Chemiluminescent HRP Substrate (Millipore, Billerica, MA), the relative protein bands were developed on X-ray film. The results of the Westren blots are shown in Figure 2C. miMPZ225, 226, 317, 721, and 723 were the most effective in knocking down MPZ protein expression.

[0129] qRT-PCR As described in the previous section of the Western blot assay, 250 and 1250 μg of MPZ and each miMPX plasmid, respectively, were co-transfected into HEK293 cells. After 24 h, the growth medium was discarded and the cells were washed twice with PBS. Total RNA was extracted using Trizol (Trizol from Fisher, Waltham, MA) according to the manufacturer's protocol. The quality and quantity of the isolated RNA was examined by nanodrop, then DNase treated and used in RT-PCR using random hexamers (Applied Biosystems cDNA Archive kit, Applied Biosystems, Foster City, CA). The subsequent cDNA samples were then used as templates in Taqman assays using predesigned human MPZ (Thermo Fisher, Hs01595271_g1) and human RPL13A control primer / probe sets (Thermofisher). Each sample was performed in triplicate. All data was normalized to cells transfected with MPZ alone. Figure 3D shows the qRT-PCR results. miMPZ_225, 721, and 723 were the most efficient miRNAs in reducing MPZ mRNA levels.

[0130] MPZ knockdown and replacement using rAAV vectors as gene therapy to treat CMT1B disease MPZ protein, also known as the main peripheral myelin protein or myelin protein zero (P0), is normally expressed by myelinating Schwann cells and accounts for more than 50% of the total PNS myelin protein. The miRNAs designed in this study target both mutant and wild-type MPZ genes. Thus, to restore healthy levels of normal MPZ expression, which is necessary to maintain a healthy myelin sheath, miMPZ-resistant MPZ genes are delivered to Schwann cells via AAV vectors. To do so, the miMPZ target sites in the human MPZ gene (NM_000530.8, SEQ ID NO: 1) were codon-optimized so that miMPZ cannot bind to these regions. The codon-optimized MPZ cDNA (coMPZ) is provided as SEQ ID NO: 3. Both wild-type MPZ and coMPZ produce the same healthy MPZ protein (SEQ ID NO: 4, NM_000530.8).

[0131] Finally, a gene expression cassette carrying "human MPZ promoter (hpMPZ)-coMPZ-U6-miMPZ" (SEQ ID NO: 6) was designed and created for both knockdown and replacement. To create this cassette, a cassette was designed containing the human MPZ promoter (this promoter is very suitable because MPZ is only expressed in Schwann cells), human coMPZ, and the restriction sites required for cloning miMPZ in this cassette. The complete sequence and order of each part of the cassette is shown in Figure 4. The expression of MPZ protein is tested in vitro in HEK293 cells, and then the ability of each miMPZ to target the MPZ target sequence is tested by Western blot and qRT-PCR. Then, after cloning the lead miMPZ into this plasmid, it is packaged into an AAV vector using XbaI sites at each end of the sequence for in vivo testing in CMT1B mice.

[0132] This cassette expresses two genes, one is miMPZ expressed under the U6 promoter that provides silencing of the endogenous (both mutant and wild-type) MPZ gene, and the other gene is a healthy codon-optimized MPZ (coMPZ) gene that is resistant to miMPZ-mediated gene silencing and expressed under the human Schwann-specific MPZ promoter, i.e., the 1200 bp endogenous human MPZ promoter (SEQ ID NO: 5, ENSG00000158887).

[0133] Outcome measures The expression level of Mpz is investigated by qRT-PCR and Western blot assays. Toluidine blue staining is performed to investigate demyelination / remyelination and the formation of onion-like structures. Behavioral analyses such as rotarod and foot grip tests are performed to evaluate motor balance, coordination, and muscle strength. To determine the amount of Schwann cell death, TUNEL assays are performed on the sciatic nerve. Changes in ER and UPR responses are determined using qRT-PCR assays. To examine nerve histology, peripheral sciatic nerves from 2- or 6-month-old treated or control mice are dissected, fixed, plastic embedded, sectioned at 1 μm, and stained with toluidine blue. Axon number, axon size, g-ratio, and myelin thickness, as well as onion-like structure formation, are measured by photomicrography. Grip strength is tested by placing the mouse on a wire grid and then inverting it, and the latency to fall is recorded for up to 1 min. This test is used for mice aged 3 weeks or older and is used in the longitudinal direction. The rotarod test is used to test motor balance and coordination. Motor balance and coordination are determined using an accelerating rotarod apparatus. Mice are tested at 2 and 6 months of age. Animal training consists of 3 trials per day for 3 consecutive days, with 15 minutes of rest between trials. Mice are placed on the rod and the speed is gradually increased from 4 to 40 RPM. The test ends when the mouse falls off the rod or lasts 600 seconds on the rod. The test is performed on the fourth day using two different speeds, 20 and 32 RPM. The latency to fall (in seconds) is calculated for each speed.

[0134] The present disclosure also provides nerve conduction velocity (NCV) testing. Mice are anesthetized with ketamine / xylazine, the sciatic nerve is stimulated at the hip joint (sciatic notch) and ankle, and EMG signals are recorded at the thenar muscle of the hind paw. The distance between the stimulating electrodes is divided by the difference in latency to EMG to determine the conduction velocity. The EMG amplitude is determined. Such EMG amplitude can be integrated for compound muscle action potential (iCMAP). In some embodiments, a secondary EMG signal is recorded. The EMG signal arises from the H-reflex, which is a monosynaptic reflex of activated sensory neurons to spinal motor neurons. Thus, muscle activity (EMG), sensory nerve conduction velocity, and synaptic connectivity in the spinal cord are measured.

[0135] Example 2 Efficacy of miRNAs to knockdown MPZ expression in co-transfected HEK293 cells miMPZ was generated by PCR and cloned into a plasmid under the control of the U6 promoter, which favors the expression of a small hairpin loop by RNA polymerase III. Prescreening of miMPZ was performed by co-transfecting miRNA and wild-type or mutant MPZ expression plasmids into HEK293 cells. Briefly, the target sequence was incorporated into the 3' untranslated region of Renilla luciferase in a Renilla / firefly dual reporter system (Figure 3A). Target binding and knockdown efficacy were determined by the emission ratio. Figures 3B and 4A show the luciferase assay results used to determine each miMPZ targeting wild-type and R98C mutant MPZ mRNA.

[0136] Use of miMPZ_225, 226, 317, 719, 721, and 723 resulted in approximately >80% reduction in luciferase signal for both wild-type and mutant MPZ R98C. The same results were achieved using Western blot and qRT-PCR from co-transfected HEK293 cells 24 hours after transfection (Figures 3C and 4B). The same miMPZs caused a significant reduction in wild-type and mutant MPZ.

[0137] These results indicate that miRNAs were effective in knocking down MPZ expression in co-transfected HEK293 cells. Example 3 Testing the ability of miMPZ to knock down MPZ expression in an in vitro model of dorsal root ganglion (DRG) / Schwann cell co-culture from the R98C CMT1B mouse model In an in vitro model, we use mouse dorsal root ganglion (DRG) and Schwann cell (DRG / Schwann) co-cultures to evaluate the efficacy of MPZ R98C knockdown and replacement and to investigate axonal remyelination. This in vitro CMT1B model allows for testing of CMT1B disease mechanisms and in vitro development of therapeutic constructs prior to in vivo experiments.

[0138] DRGs isolated and purified from embryonic spinal cords of R98C mice (Saporta et al., supra) were provided by Dr. Michael Shy at University of Iowa and co-cultured with primary Schwann cells as reported by Florio et al. (J Neurosci. 2018, 38(18):4275-4287). These cells are then transfected with each of the miMPZs described herein and either MPZ or coMPZ described herein, or any of the expression cassettes described herein. In some embodiments, each of the miMPZs and coMPZs are provided in the same construct. In some embodiments, each of the miMPZs and coMPZs are provided in one or more constructs. Controls are treated with empty vector, coMPZ alone, miMPZ alone, or untreated.

[0139] After a period of time, myelination is analyzed using various established markers by various experimental methods including immunostaining, Western blot, qPCR, and qRT-PCR. qRT-PCR tests and Western blot assays are used to detect the expression of miMPZ, mutant MPZ, and coMPZ. Toluidine blue staining is performed to investigate demyelination / remyelination and the formation of onion-like structures in the cells.

[0140] Improvement in myelination is obtained with treatment with miMPZ and coMPZ. Example 4 miMPZ knockdown and MPZ replacement in the R98C mouse model of CMT1B To verify and test the in vivo efficacy of knockdown (AAV9-mediated MPZ gene silencing by miMPZ) and gene replacement by AAV9-coMPZ, knockdown and gene replacement are performed in the severe R98C CMT1B mouse model provided by Dr. Michael Shy at the University of Iowa (Saporta et al., supra).

[0141] AAV vectors carrying any of the miMPZ and any of the coMPZ described herein are administered to the CSF of pre-symptomatic or post-symptomatic mice either via ICV or intrathecal injection, or via direct injection into the sciatic nerve. Controls are treated with empty vector, coMPZ alone, miMPZ alone, or are untreated.

[0142] The efficacy of mutant MPZ knockdown and coMPZ replacement in improving myelination will be investigated in mice at several time points after administration using a variety of methods, including but not limited to immunostaining, Western blotting, qPCR, and qRT-PCR, as described herein.

[0143] qRT-PCR test and Western blot assay are used to detect the expression of miMPZ, mutant MPZ, and coMPZ. Toluidine blue staining is performed to investigate demyelination / remyelination and the formation of onion-like structures in treated and untreated mice. Behavioral analyses such as rotarod and foot grip tests are performed to evaluate motor balance, coordination, and muscle strength. TUNEL assay is performed on the sciatic nerve to determine the amount of Schwann cell death. Changes in ER and UPR responses are determined using qRT-PCR assays in treated and untreated mice. NCV assay is used to investigate the improvement of motor function in treated mice compared to untreated animals.

[0144] Treatment with miMPZ knockdown and coMPZ replacement in the R98C mouse model of CMT1B improves myelination, motor balance, motor function, coordination, and / or muscle strength in mice.

[0145] Example 5 miMPZ knockdown and MPZ replacement in the S63del mouse model of CMT1B To verify and test the in vivo efficacy of knockdown (AAV9-mediated MPZ gene silencing by miMPZ) and gene replacement by AAV9-coMPZ, knockdown and gene replacement are performed in the S63del CMT1B mouse model (Wrabetz et al., supra; Sidoli et al., J Neuroscience, 2016, 36(44):11350-61). This transgenic mouse model is demonstrated by Dr. Lawrence Wrabetz and Dr. Laura Fetri at the University of Buffalo, NY.

[0146] AAV vectors carrying miMPZ and coMPZ are administered into the CSF of pre- or post-symptomatic mice either via ICV or intrathecal injection, or via direct injection into the sciatic nerve. Controls are treated with empty vector, coMPZ alone, miMPZ alone, or are untreated.

[0147] The efficacy of mutant MPZ knockdown and coMPZ replacement in improving myelination will be investigated in mice at several time points after administration using a variety of methods, including but not limited to immunostaining, Western blotting, qPCR, and qRT-PCR, as described herein.

[0148] qRT-PCR test and Western blot assay are used to detect the expression of miMPZ, mutant MPZ, and coMPZ. Toluidine blue staining is performed to investigate demyelination / remyelination and the formation of onion-like structures in treated and untreated mice. Behavioral analyses such as rotarod and foot grip tests are performed to evaluate motor balance, coordination, and muscle strength. TUNEL assay is performed on the sciatic nerve to determine the amount of Schwann cell death. Changes in ER and UPR responses are determined using qRT-PCR assays in treated and untreated mice. NCV assay is used to investigate the improvement of motor function in treated mice compared to untreated animals.

[0149] Treatment with miMPZ knockdown and coMPZ replacement in the S63del mouse model of CMT1B improves myelination, motor balance, motor function, coordination, and / or muscle strength in mice.

[0150] Example 6 miMPZ knockdown and MPZ replacement or MPZ replacement alone in the treatment of human patients with MPZ mutations Mutations in MPZ cause CMT1B, and many of the >200 mutations result in neurological damage through gain-of-function toxicities of the mutant protein, such as ER retention, activation of the unfolded protein response (UPR), or disruption of myelin compaction. Patients have been identified as heterozygous or homozygous for myelin P0 or Po gene (MPZ) mutations (Ikegami et al., Biochem. Biophys. Res. Commun. 1996;222:107-110. Ikegami et al. (ibid.) identified a novel homozygous Phe64 deletion mutation and suggested that impaired myelination was dose-dependent. Other patients have been identified with mutations in MPZ that result in haploinsufficiency of MPZ, resulting in mild neuropathy, demonstration of axonal loss, and variable degrees of disability to the lower and upper limbs (Howard et al., J. Peripher Nerv Syst. 2021;26:177-83). Five patients had heterozygous c.306del;p.Asp104fs mutations and one patient had a c.204C>A;p.Tyr68Ter mutation in MPZ (Howard et al. (supra)). Howard et al. (supra) reported that in this group, the phenotype of the patients differed from that of Mpz+ / - mice, as the mice had motor rather than sensory nerve involvement, whereas the patients showed a predominantly large fiber sensory neuropathy. Howard et al. (supra) hypothesized that these patients could serve as candidates for therapeutic approaches that have been demonstrated to be effective in Mpz+ / - mice, and also serve as a model to compare other late-onset phenotypes in patients with MPZ mutations.

[0151] To verify and test the in vivo efficacy of knockdown (AAV9-mediated MPZ gene silencing by miMPZ) and gene replacement by AAV9-coMPZ, knockdown and gene replacement therapy is contemplated for the treatment of human patients with both homozygous and heterozygous mutations of the MPZ gene. Additionally, MPZ gene replacement alone is contemplated as a means for the treatment of these patients.

[0152] Thus, subject to regulatory approval, (i) AAV vectors carrying any one or more of miMPZ and any of coMPZ, (ii) AAV vectors carrying any of coMPZ, or (iii) AAV vectors carrying MPZ genes or functional variants thereof are administered or delivered to these MPZ-deficient patients. In some embodiments, administration is into the CNS. In some embodiments, administration is via either ICV or intrathecal injection. In some embodiments, administration is by direct injection into the sciatic nerve.

[0153] Heterozygous MPZ loss of function (LOF) mutations cause a milder neuropathic phenotype, whereas homozygous MPZ LOF mutations cause severe early-onset CMT1B, also known as Degerines-Sottas syndrome (DSS). MPZ gene replacement (i.e., delivery of either a codon-optimized MPZ gene or a wild-type MPZ gene or a functional MPZ gene variant) or MPZ knockdown and gene replacement (i.e., delivery of miMPZ and coMPZ) are treatment options for both heterozygous and homozygous LOF mutation patients.

[0154] Both gene replacement alone, as well as knockdown and replacement therapy approaches, are useful in treating these patients. Similarly, therefore, both wild-type and codon-optimized MPZ can be used for treatment.

[0155] While the present disclosure has been described with respect to specific embodiments, it will be understood that variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the scope of the claims should be placed on the present disclosure.

[0156] All documents referenced in this application are incorporated herein by reference in their entirety.

Claims

1. A nucleic acid, a) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; b) a polynucleotide sequence comprising an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19-30, or a nucleotide sequence comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19-30; c) a polynucleotide sequence comprising or encoding an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 31-42, or a nucleotide sequence comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 31-42; d) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, wherein the target nucleotide sequence is set forth in any one of SEQ ID NOs: 43 to 54; or e) A nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 3 or 6.

2. A nucleic acid, a) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; or ii) a polynucleotide sequence that specifically hybridizes to a target nucleotide sequence in the MPZ gene, wherein the target nucleotide sequence is set forth in any one of SEQ ID NOs: 43 to 54; b) i) a polynucleotide sequence encoding a human MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:1; ii) a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:3; or iii) a polynucleotide sequence encoding an MPZ polypeptide sequence that is at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence set forth in SEQ ID NO:

4.

3. The nucleic acid of claim 2, further comprising a promoter or promoters.

4. The nucleic acid of claim 3 , wherein the promoter is a U6 promoter, a U7 promoter, an H19 promoter, a neuron-specific promoter, or a Schwann cell-specific promoter.

5. The nucleic acid of claim 3 , wherein the Schwann cell-specific promoter is an MPZ promoter or a mini-MPZ promoter.

6. 6. The nucleic acid of claim 5, wherein the MPZ promoter comprises at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:

5.

7. A nanoparticle, extracellular vesicle, exosome, or vector comprising the nucleic acid of claim 1.

8. A nanoparticle, extracellular vesicle, exosome, or vector containing the nucleic acid described in claim 2.

9. The vector according to claim 7 or 8, wherein the vector is a viral vector.

10. 10. The viral vector of claim 9, wherein the viral vector is an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, or a synthetic virus.

11. The viral vector of claim 9 , wherein the viral vector is an AAV.

12. The viral vector of claim 11 , wherein the AAV lacks the rep and cap genes.

13. The viral vector of claim 11, wherein the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV).

14. The AAV of claim 11, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAVv66, or AAV-F.

15. The AAV of claim 11, wherein the AAV is AAV9, AAVrh.10, AAV-PHP.eB, AAVv66, or AAV-F.

16. 1. A composition comprising: (a) a nucleic acid according to any one of claims 1 to 6, or (b) the nanoparticle, extracellular vesicle, exosome, or vector according to claim 7 or 8; and a pharmaceutically acceptable carrier.

17. 17. The composition of claim 16 for reducing the expression of a mutant myelin protein zero (MPZ) gene in a cell.

18. The composition of claim 17 , wherein the cell is a neuronal cell.

19. The composition of claim 18, wherein the neuronal cells are Schwann cells.

20. The composition of claim 18 , wherein the cell is a human cell.

21. 21. The composition of claim 20, wherein the cell is in a human subject.

22. 22. The composition of claim 21, wherein the subject is suffering from a hypomyelination disease or Charcot-Marie-Tooth disease.

23. 23. The composition of claim 22, wherein the disease is CMT (DI-CMT), CMT type 1B (CMT1B), CMT type 2I (CMT2I), CMT type 2J (CMT2J), or Dejerine-Sottas syndrome (DSS or CMT type 3) disease.

24. 17. The composition of claim 16 for treating a subject containing a mutant myelin protein zero (MPZ) gene.

25. 25. The composition of claim 24, wherein the subject is a human subject.

26. 26. The composition of claim 25, wherein the subject is suffering from a hypomyelination disease or Charcot-Marie-Tooth disease.

27. 27. The composition of claim 26, wherein the disease is CMT (DI-CMT), CMT type 1B (CMT1B), CMT type 2I (CMT2I), CMT type 2J (CMT2J), or Dejerine-Sottas syndrome (DSS or CMT type 3) disease.

28. The composition of claim 16, wherein the nucleic acid is the nucleic acid of claim 2.

29. 29. The composition of claim 28, wherein the nucleic acid is present in a nanoparticle, extracellular vesicle, exosome, or vector.

30. 30. The composition of claim 29, wherein the vector is a viral vector.

31. 31. The composition of claim 30, wherein the viral vector is an AAV that is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAVv66, or AAV-F.

32. 31. The composition of claim 30, wherein the AAV is AAV9, AAVrh.10, AAV-PHP.eB, AAVv66, or AAV-F.

33. For the preparation of a medicament for reducing the expression of mutant myelin protein zero (MPZ) in a cell, (a) a nucleic acid according to any one of claims 1 to 6, or (b) The nanoparticle, extracellular vesicle, exosome, or vector according to claim 7 or 8. Use of.

34. 34. The use of claim 33, wherein the cell is in a human subject.

35. The composition of claim 16, formulated for intramuscular injection, oral administration, subcutaneous, intradermal, or transdermal delivery, injection into the bloodstream, or aerosol administration.

36. 1. A pharmaceutical combination for use in a method for reducing expression of a mutant myelin protein zero (MPZ) gene in a cell and expressing functional MPZ protein in the cell, the pharmaceutical combination comprising: (a) (i) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; (ii) a polynucleotide sequence comprising an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19-30, or a nucleotide sequence comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19-30; (iii) a polynucleotide sequence comprising or encoding an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 31-42, or a nucleotide sequence comprising at least about 90% identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 31-42; and / or (iv) a first nucleic acid comprising one or more of the polynucleotide sequences that specifically hybridize to a target nucleotide sequence in the MPZ gene, wherein the target nucleotide sequence is set forth in any one of SEQ ID NOs: 43 to 54; (b) a second nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 3 or 6; The pharmaceutical combination, wherein the method comprises delivering to the cell effective amounts of the first nucleic acid and the second nucleic acid.

37. 37. The pharmaceutical combination of claim 36, wherein any one or more of the nucleic acids further comprises a promoter or promoters.

38. 38. The pharmaceutical combination of claim 37, wherein the promoter is a U6 promoter, a U7 promoter, an H19 promoter, a neuron-specific promoter, or a Schwann cell-specific promoter.

39. The pharmaceutical combination of claim 38, wherein the Schwann cell-specific promoter is an MPZ promoter or a mini-MPZ promoter.

40. 40. The pharmaceutical combination of claim 39, wherein the MPZ promoter comprises at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:

5.

41. The pharmaceutical combination of claim 36, wherein the cell is a neuronal cell.

42. The pharmaceutical combination of claim 41, wherein the neuronal cells are Schwann cells.

43. The pharmaceutical combination of claim 36, wherein the cell is a human cell.

44. 37. The pharmaceutical combination of claim 36, wherein the cell is in a human subject.

45. 45. The pharmaceutical combination of claim 44, wherein the subject is suffering from a hypomyelination disease or Charcot-Marie-Tooth disease.

46. 46. ​​The pharmaceutical combination of claim 45, wherein the disease is CMT (DI-CMT), CMT type 1B (CMT1B), CMT type 2I (CMT2I), CMT type 2J (CMT2J), or Dejerine-Sottas syndrome (DSS or CMT type 3) disease.

47. 37. The pharmaceutical combination of claim 36, wherein the nucleic acid is delivered to the cell in a nanoparticle, an extracellular vesicle, an exosome, or a vector, or a combination of any one or more thereof.

48. The pharmaceutical combination of claim 47, wherein the vector is a viral vector.

49. 49. The pharmaceutical combination of claim 48, wherein the viral vector is an adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, or synthetic virus.

50. The pharmaceutical combination of claim 48, wherein the viral vector is AAV.

51. 51. The pharmaceutical combination of claim 50, wherein the AAV lacks the rep and cap genes.

52. 51. The pharmaceutical combination of claim 50, wherein the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV).

53. The pharmaceutical combination of claim 50, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAVv66, or AAV-F.

54. The pharmaceutical combination of claim 53, wherein the AAV is AAV9, AAVrh.10, AAV-PHP.eB, AAVv66, or AAV-F.

55. the first nucleic acid comprising the polynucleotide sequence comprising or encoding the MPZ miRNA and / or the polynucleotide that specifically hybridizes to a target nucleotide sequence in the MPZ gene; and the second nucleic acid comprising the polynucleotide sequence encoding the codon-optimized MPZ DNA, The pharmaceutical combination according to any one of claims 47 to 54, which is delivered to said cells simultaneously.

56. 56. The pharmaceutical combination of claim 55, wherein the nucleic acids are delivered to the cells in the same vector.

57. the first nucleic acid comprising the polynucleotide sequence comprising or encoding the MPZ miRNA and / or the polynucleotide that specifically hybridizes to a target nucleotide sequence in the MPZ gene; and the second nucleic acid comprising the polynucleotide sequence encoding the codon-optimized MPZ DNA, The pharmaceutical combination according to any one of claims 47 to 54, which is delivered to the cells at different times.

58. 1. A pharmaceutical combination for treating a subject suffering from abnormal expression of a mutant myelin protein zero (MPZ) gene, said treatment comprising reducing expression of said mutant MPZ gene and expressing functional MPZ in said subject, said pharmaceutical combination comprising: (a) (i) a polynucleotide sequence encoding a myelin protein zero (MPZ) microRNA, comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 7-18; (ii) a polynucleotide sequence comprising an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 19-30, or a nucleotide sequence comprising at least about 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 19-30; (iii) a polynucleotide sequence comprising or encoding an MPZ microRNA, wherein the MPZ microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 31-42, or a nucleotide sequence comprising at least about 90% identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 31-42; and / or (iv) a first nucleic acid comprising one or more of the polynucleotide sequences that specifically hybridize to a target nucleotide sequence in the MPZ gene, wherein the target nucleotide sequence is set forth in any one of SEQ ID NOs: 43 to 54; (b) a second nucleic acid comprising a polynucleotide sequence encoding a codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO: 3 or 6; and Including, The pharmaceutical combination, wherein the treatment comprises delivering to the subject effective amounts of the first nucleic acid and the second nucleic acid.

59. 59. The pharmaceutical combination of claim 58, wherein any one or more of the nucleic acids further comprises a promoter or promoters.

60. 60. The pharmaceutical combination of claim 59, wherein the promoter is a U6 promoter, a U7 promoter, an H19 promoter, a neuron-specific promoter, or a Schwann cell-specific promoter.

61. The pharmaceutical combination of claim 60, wherein the Schwann cell-specific promoter is an MPZ promoter or a mini-MPZ promoter.

62. 62. The pharmaceutical combination of claim 61, wherein the MPZ promoter comprises at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the polynucleotide sequence set forth in SEQ ID NO:

5.

63. 59. The pharmaceutical combination of claim 58, wherein the subject is a human.

64. 64. The pharmaceutical combination of claim 63, wherein the subject is suffering from a hypomyelination disease or Charcot-Marie-Tooth disease.

65. 65. The pharmaceutical combination of claim 64, wherein the disease is CMT (DI-CMT), CMT type 1B (CMT1B), CMT type 2I (CMT2I), CMT type 2J (CMT2J), or Dejerine-Sottas syndrome (DSS or CMT type 3) disease.

66. 59. The pharmaceutical combination of claim 58, wherein the nucleic acid is delivered to the subject in a nanoparticle, an extracellular vesicle, an exosome, or a vector, or a combination of any one or more thereof.

67. The pharmaceutical combination of claim 66, wherein the vector is a viral vector.

68. 68. The pharmaceutical combination of claim 67, wherein the viral vector is an adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, or synthetic virus.

69. 68. The pharmaceutical combination of claim 67, wherein the viral vector is AAV.

70. 70. The pharmaceutical combination of claim 69, wherein the AAV lacks the rep and cap genes.

71. 70. The pharmaceutical combination of claim 69, wherein the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV).

72. The pharmaceutical combination of claim 69, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAVv66, or AAV-F.

73. The pharmaceutical combination of claim 72, wherein the AAV is AAV9, AAVrh.10, AAV-PHP.eB, AAVv66, or AAV-F.

74. the first nucleic acid comprising the polynucleotide sequence comprising or encoding the MPZ miRNA and / or the polynucleotide that specifically hybridizes to a target nucleotide sequence in the MPZ gene; and the second nucleic acid comprising the polynucleotide sequence encoding the codon-optimized MPZ DNA, The pharmaceutical combination of any one of claims 58 to 73, which is delivered to the subject simultaneously.

75. 75. The pharmaceutical combination of claim 74, wherein the nucleic acids are delivered to the subject in the same vector.

76. the first nucleic acid comprising the polynucleotide sequence comprising or encoding the MPZ miRNA and / or the polynucleotide that specifically hybridizes to a target nucleotide sequence in the MPZ gene; and the second nucleic acid comprising the polynucleotide sequence encoding the codon-optimized MPZ DNA, The pharmaceutical combination of any one of claims 58 to 73, which is delivered to the subject at different times.

77. 1. A composition for treating a subject suffering from abnormal expression of a mutant myelin protein zero (MPZ) gene, the composition comprising a nucleic acid comprising a polynucleotide sequence encoding MPZ DNA or codon-optimized MPZ DNA comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 1, 3, and 6; The composition, wherein the treatment comprises delivering an effective amount of the nucleic acid to the subject, thereby expressing a functional MPZ protein in the subject.

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