Polynucleotide therapy for Charcot-Marie-Tooth disease

Abstract

Hereditary peripheral neuropathy, also known as Charcot-Marie-Tooth disease (CMT), is one of the most common genetic disorders of the nervous system, affecting approximately 1 in 2500 people. The present disclosure relates to a therapeutic strategy, including methods and compositions, for treating Charcot-Marie-Tooth disease (CMT) by targeting the pre-mRNA of PMP22 using antisense oligonucleotides (ASOs).

Description

[Technical field]

[0001] Related Applications This international application claims the benefit of U.S. Provisional Application No. 63 / 331,044, filed April 14, 2022, and U.S. Provisional Application No. 63 / 331,045, filed April 14, 2022, both of which are incorporated herein in their entireties.

[0002] Reference to Electronically Submitted Sequence Listing The contents of the sequence listing submitted electronically in ST26 format with this application (file name: UMC_226824.xml; size: 242,797 bytes; and creation date: April 13, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to therapeutic strategies, including methods and compositions, for treating Charcot-Marie-Tooth disease (CMT) by targeting PMP22 mRNA with antisense oligonucleotides (ASOs). [Background technology]

[0004] Hereditary peripheral neuropathy, also known as Charcot-Marie-Tooth disease (CMT), is one of the most common genetic disorders of the nervous system, affecting approximately 1 in 2500 individuals (Krajewski et al., 2000; Braathen, 2012). The most common form of CMT, CMT1A, is a demyelinating neuropathy caused by a gene duplication of the peripheral myelin protein 22 (PMP22) gene (SEQ ID NO:1) (Patel et al., 1992; Timmerman et al., 1992; Valentijn et al., 1992; Matsunami et al., 1992). As its name suggests, PMP22 is a structurally essential component of the myelin sheath that surrounds axons (Lee et al., 2014; Mittendorf et al., 2017; Snipes et al., 1992). Functionally, myelin acts as a biological insulator facilitating the efficient transmission of electrical impulses along axons. In the CNS, myelin is produced by glial cells, whereas in the peripheral nervous system (PNS), Schwann cells (SCs) are responsible for the production of PMP22 and myelin. Excessive PMP22 leads to the development of the autosomal dominant disorder CMT1A. However, maintaining sufficient PMP22 expression is equally important, as its reduction leads to a distinct neuropathic disorder called hereditary neuropathy with predisposition to pressure palsies (HNPP). In summary, PMP22 is an essential component of the myelin sheath, and maintaining the delicate homeostatic balance of this gene is paramount for the development of effective therapeutics.

[0005] CMT is caused by mutations in over 90 different genes, the most common of which is a 1.4 Mb duplication of human chromosome 17, classified as CMT1A. The peripheral myelin protein 22 (PMP22) gene, which encodes the major myelin protein, peripheral myelin protein 22, is present within the 1.4 Mb duplication. PMP22 is an integral myelin membrane protein that alters lipid organization / distribution and is induced in Schwann cells during the initiation of peripheral nerve myelination by Schwann cells.

[0006] Rodent studies have demonstrated that overexpression of PMP22 is sufficient to cause demyelinating neuropathy (Magyar et al., 1996; Sereda et al., 1996; Huxley et al., 1996). Conditional knockout POC studies have demonstrated that reduction of PMP22 overexpression leads to remyelination (Perea et al., 2001). Interestingly, allelic loss of PMP22 by deletion of the same 1.4 Mb region results in a distinct neuropathy known as hereditary neuropathy with pressure palsy (Chance et al., 1993), confirming that gene dosage is a determining factor in such neuropathy. Finally, elevated levels of PMP22 protein have been identified in dermal and sural nerves in CMT1A patients.

[0007] Although several approaches have been proposed to reduce PMP22 expression, no treatment is currently available for patients. For example, in rodent models, PMP22 overexpression can be reduced by high doses of ascorbic acid (Cortese et al., 2020). However, in clinical trials, treatment with ascorbic acid did not reduce PMP22 mRNA levels in skin biopsies from CMT1A patients (Eichinger et al., 2018; Gautier et al., 2021; Kagiava et al., 2018). Progesterone antagonists and GABAB agonists have also been found to reduce PMP22 mRNA expression (Lee et al., 2020; Massade and Charbel, 2020). However, they are less promising due to their various effects on gene expression regulation in Schwann cells and possibly other cell types, which may complicate the long-term treatment of genetic diseases.

[0008] CMT1A is a monogenic disease. The gene for this disease has been identified. Expression of PMP22 can be inhibited by various molecular mechanisms. To date, a panel of 2'-O-2-methoxyethyl phosphorothioate-based (2'MOE) ASOs has been developed and analyzed as a means to block PMP22 expression (Zhao et al., 2018). In this report by Zhao et al., ASOs were identified that reduced PMP22 expression in several important cellular and in vivo contexts, including K-562 cells and the C22 transgenic mouse model. Importantly, the presence of the human PMP22 gene is a prerequisite for each of these experimental contexts. In C22 mice, treatment with ASOs reduced PMP22 expression and significantly improved CMT phenotypes, including neuronal pathology, myelination degree, and CMAP / MNCV.

[0009] Other approaches to modifying PMP22 RNA translation have been proposed, such as forming molecules that interfere with the 3'UTR of the human PMP22 gene, knocking down the entire process (see, for example, World Wide Web at www.jci.org / articles / view / 96499; U.S. Pat. No. 11,136,577). Patent application WO2020 / 132558 A1 describes the use of gapmers to enhance hybridization to a target portion of RNA and induce RNase H cleavage to achieve gene silencing.

[0010] These approaches have in common that they either completely eliminate the expression of the full-length RNA of PMP22 or destroy this RNA at some stage of the genome cycle. Although such approaches can be effective, overdosing patients with too much of the drug can lead to secondary diseases and symptoms, as in the above cases where too much PMP22 has been removed.

[0011] Therefore, there remains a need to develop effective treatments for CMT diseases.

[0012] Summary of the Invention The present disclosure relates to molecules (small molecules, antisense oligonucleotides, antibodies, etc.) that bind to regions of the pre-mRNA of human PMP22 involved in exon splicing. The molecules bind to one (or more) of these critical regions of the pre-mRNA prior to splicing and induce an exon skipping event during translation / transcription, resulting in the production of an mRNA that resembles the full-length version of the native mRNA but lacks one or more exons or portions thereof. The resulting exon-skipped mRNA is stable and measurable in vitro, in vivo, and in situ.

[0013] Provided herein are compositions comprising antisense oligonucleotides (ASOs) that comprise or consist of a complementary region that is complementary to a target region of a PMP22 pre-mRNA or is complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides. In certain embodiments, binding of the complementary region of the ASO to the target region of the PMP22 pre-mRNA in a cell induces exon skipping during RNA transcription. In certain embodiments, induction of exon skipping reduces the production of full-length PMP22 mRNA. In certain embodiments, induction of exon skipping produces exon-skipped PMP22 mRNA.

[0014] Further provided herein is a method for reducing the expression level of full-length PMP22 mRNA in a cell. Such a method comprises administering to a cell an exon skipping-inducing composition comprising the antisense oligonucleotide (ASO) of the present disclosure. In certain embodiments, exon-skipped PMP22 mRNA is produced. In certain embodiments, the amount of PMP22 protein produced in the cell is reduced.

[0015] Further provided herein is a method for producing exon-skipped pre-mRNA of PMP22. Such method comprises administering to a cell an exon skipping-inducing composition comprising any of the antisense oligonucleotides (ASOs) disclosed herein. In certain embodiments, the expression level of full-length mRNA of PMP22 in the cell is reduced. In certain embodiments, the amount of functional PMP22 protein produced in the cell is reduced.

[0016] Further provided herein are methods of treating Charcot-Marie-Tooth disease, comprising administering to a subject in need thereof an exon skipping-inducing composition comprising any of the antisense oligonucleotides (ASOs) disclosed herein.

[0017] Further provided herein are compositions comprising antisense oligonucleotides (ASOs) that comprise or consist of a complementary region that is complementary to a target region of a PMP22 pre-mRNA or is complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides. In certain embodiments, the target region of a PMP22 pre-mRNA comprises an intron / exon junction of one of the coding exons. [Brief description of the drawings]

[0018] [Figure 1] This diagram shows how PMP22 pre-mRNA (top panel) is converted into full-length mRNA (middle panel) containing all amino acids encoded by the exons, and into a functional protein (bottom panel). Introns between each exon are indicated by horizontal double lines, and their base-paired sequences are removed during splicing. [Diagram 2]FIG. 1 shows the PMP22 gene process upon introduction of an antisense oligonucleotide (ASO) that hybridizes to a portion of the pre-mRNA and induces a skipping event during conversion of the pre-mRNA to mRNA. When the exon skipping antisense oligonucleotide is present in a cell during RNA transcription, a skipped exon product is formed, resulting in a non-functional protein. [Diagram 3] (A), (B), and (C) are schematic diagrams showing structural changes of pre-mRNA during splicing. [Figure 4] (A), (B), (C) and (D) are schematic diagrams showing multiple strategies to bind pre-mRNA and induce exon skipping. [Figure 5A] FIG. 1 shows the sequence (all uppercase) around the 5'-region of exon 3 of the PMP22 gene, as well as the corresponding upstream intron, and shows several different representative 25mer-oligonucleotide ASO sequences (under the top sequence) of the present disclosure that are at the 5' end of the exon and can cause exon 3 skipping during pre-mRNA to mRNA conversion, with the final ASO method being available to those skilled in the art in a variety of ways (both solid-phase and liquid-phase, and e.g., 5' to 3' or 3' to 5' orientation), as long as it contains the sequence of the present disclosure (or contains any mismatches presented herein) and hybridizes to the target region or part of the target region. Three representative morpholino antisense oligo sequences, for which data are presented, designed (and synthesized) to bind to intron / exon junctions are depicted. SEQ ID NO:2~34, SHC-006 25-mer (SEQ ID NO:71), SHC-001 24-mer (SEQ ID NO:72), SHC-005 25-mer (SEQ ID NO:73). [Figure 5B]FIG. 1 shows the sequence (all uppercase) around the 3'-region of exon 3 of the PMP22 gene, as well as the corresponding downstream intron, and shows several different representative 25mer-oligonucleotide ASO sequences (below the top sequence) of the present disclosure that are at the 3' end of the exon and can cause exon 3 skipping during pre-mRNA to mRNA conversion, with the final ASO method available to the skilled artisan in a variety of ways (both solid and liquid phase, and e.g., 5' to 3' orientation or 3' to 5' orientation) as long as they contain the sequences of the present disclosure (or contain any mismatches as presented herein) and hybridize to the target region or part of the target region. Three representative morpholino antisense oligo sequences, for which data are presented, designed (and synthesized) to bind to intron / exon junctions are depicted. SEQ ID NO: 35-70, SCH-010 21-mer (SEQ ID NO: 74), SCH-012 20-mer (SEQ ID NO: 75). [Figure 6] FIG. 6 shows the results of PCR amplification and gel electrophoresis analysis of selected exon 3 skipping ASOs from FIG. 5, along with a scrambled injection control (which has no effect on PMP22 pre-mRNA or mRNA). [Figure 7] FIG. 1 shows RT-PCR of C3 mice (liver) treated with SHC-012 (SEQ ID NO:75), scrambled (2x) ASO or water control. In addition to the GAPDH control, full-length human PMP22 mRNA is detectable, demonstrating exon 3 skipping induced by SHC-012 (confirmed by sequence) as shown. [Figure 8]FIG. 1 shows PCR results (and corresponding changes in PMP22 full length mRNA abundance) in multiple tissues following a single subcutaneous injection of SHC-012 (SEQ ID NO:75) (animals were sacrificed 2 days later). Data in this figure represent survival rates, specifically, for each tissue type, the total amount of PMP22 full length mRNA in each tissue type was quantified and compared to SHC-012 animals, with a survival rate of 20% representing 80% of pre-mRNA being inhibited from assembling full length mRNA and becoming exon-skipped mRNA. [Figure 9] FIG. 1 shows the amount of time it takes different treatment groups (applied with SHC-012; SEQ ID NO:75), scrambled animals and wild type mice to walk on the dowel apparatus. For all groups, each group contained 3 animals. Histograms represent the mean and standard deviation, and P values ​​(to determine the confidence level versus the scrambled control) were calculated for each treatment group (p<0.05 for all groups). All data are averaged for each animal at 12 weeks of age. [Figure 10] FIG. 14 shows the amount of time spent on the rotarod apparatus by scrambled, wild-type and treatment groups after the first injection at 5 weeks of age. For all groups, each group contained 3 animals. Histograms represent the mean and standard deviation, and P values ​​(to determine the confidence level versus the scrambled control) were calculated for each treatment group. All data are averaged across animals at 12 weeks of age. [Figure 11A]FIG. 1 shows the sequence (all uppercase) surrounding the 5'-region of exon 4 of the PMP22 gene, as well as the corresponding upstream introns, which are at the 5' end of the exon and may cause skipping of exon 4 during pre-mRNA to mRNA conversion. It depicts three representative morpholino antisense oligonucleotide sequences designed (and synthesized) to bind to intron / exon junctions for which data are presented: SEQ ID NOs: 76-110, SHC-029 21-mer (SEQ ID NO: 146), SHC-028 20-mer (SEQ ID NO: 147), SHC-027 20-mer (SEQ ID NO: 148). [Figure 11B] FIG. 1 shows the sequence (all uppercase) surrounding the 3'-region of exon 4 of the PMP22 gene, as well as the corresponding downstream intron, which is at the 3' end of the exon and may cause skipping of exon 4 upon conversion of pre-mRNA to mRNA, and depicts several different 25mer-oligonucleotide ASO sequences (below the top sequence) of the present disclosure, for which data are presented, designed (and synthesized) to bind to intron / exon junctions: SEQ ID NOs: 111-145, SCH-031 21-mer (SEQ ID NO: 149), SCH-030 20-mer (SEQ ID NO: 150), SCH-030 20-mer (SEQ ID NO: 151). [Figure 12] FIG. 12 shows the results of PCR amplification and gel electrophoresis analysis of exon 4 skipping ASOs selected from FIG. 11. [Figure 13] FIG. 1 shows quantification results from gel results for three of the exon 4 skipping compounds by two different techniques. [Figure 14]FIG. 1 shows three different ASO compounds (each as shown) that were designed and tested to cross-link the 5' and 3' ends of exon 3 (top) and target regions that are non-contiguous and have different effects on pre-mRNA conformation than those previously described: SEQ ID NOs: 152-162 and 233-238, SHC-043 (SEQ ID NO: 156), SHC-044 (SEQ ID NO: 159), SHC-045 (SEQ ID NO: 162), SHC-046 (SEQ ID NO: 235), and SHC-047 (SEQ ID NO: 238). [Figure 15] FIG. 15 shows the results of PCR amplification and subsequent gel electrophoresis analysis for selected exon 3 skipping ASOs from FIG. 14. [Figure 16A] FIG. 1 shows the sequence (all uppercase) surrounding the 5'-region of exon 2 of the PMP22 gene, as well as the corresponding upstream intron, and the 3' end of the exon. SEQ ID NOs: 163-197. [Figure 16B] FIG. 1 shows the sequence (all uppercase) surrounding the 3'-region of exon 2 of the PMP22 gene, as well as the corresponding downstream intron, the 5' end of the exon. SEQ ID NOs: 198-232. [Figure 17] FIG. 1 shows images of the sciatic nerve from WT, untreated and treated animals (top) and the peroneal portion of the nerve (bottom). [Figure 18] FIG. 1 shows a high magnification TEM image of a portion of the peroneal nerve. [Figure 19] (Top) Electrophysiological plots of sciatic stomach slices from sedated mice are shown, (bottom) mean MUNE and CMAP of three measured animals from each group are shown. [Figure 20] Results for treatment groups are shown (also showing groups of scrambled and wild-type animals for comparison at 4 months of age), with each data set in the histogram being the average of dowel walking time over 4 days at the end of each month. [Figure 21]FIG. 1 shows the results of a dowel walking time experiment (3-month-old wild-type animals are also plotted for comparison). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition The entire subject matter and / or language of the appended claims is hereby incorporated by reference in its entirety into this specification, to the extent that explanatory assistance is required.

[0020] Anyone reading this description will understand that the exemplary embodiments described and claimed herein may be suitably practiced even in the absence of any feature, element or step that is specifically disclosed or not disclosed herein.

[0021] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0022] It should be noted that the terms "a" and "an" refer to one or more. For example, "a compound" should be understood to refer to one or more compounds. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0023] Also, "and / or" as used herein should be taken as a specific disclosure of each of the subject components or elements with or without the other. That is, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A (single)," and "B (single)." Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include the following embodiments: A, B, and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (single), B (single), and even C (single).

[0024] Wherever an embodiment is described herein with the term "comprising," it is understood that alternative analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0025] Numerical ranges are inclusive of the stated values ​​of the range. When values ​​are listed, such as 1, 2, 3, or 4, any range between those values, for example 1-3, 1-4, 2-4, etc., is specifically included in the disclosure, even if not expressly stated as "and any range therebetween."

[0026] The headings provided herein are merely for ease of reference and are not intended to limit the various aspects or aspects of the present disclosure, which can be understood by reference to the specification as a whole, i.e., the terms set forth immediately below are more fully defined by reference to the specification as a whole.

[0027] As used herein, the term "identity", e.g., "percent identity", of an amino acid sequence or nucleotide sequence disclosed herein refers to the relationship between two or more nucleotide sequences or two or more amino acid sequences. If a position in a sequence is occupied by the same nucleic acid base or amino acid at the corresponding position in the compared sequence, the sequences are said to be "identical" at that position. The "sequence identity" percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid occurs to obtain the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the comparison range, and multiplied by 100 to obtain the "sequence identity" percentage. The "sequence identity" percentage is determined by comparing two optimally aligned sequences over the comparison range. In order to optimally align sequences for comparison purposes, when the reference sequence remains constant, a portion of the nucleotide or amino acid sequence in the comparison range may contain additions or deletions, called gaps. An optimal alignment is an alignment that, even with gaps, produces the maximum number of "identical" positions between the reference sequence and the compared sequence. The percentage of "sequence identity" between two sequences can be determined, for example, using the "BLAST" program available from the National Center for Biotechnology Information, which is a combination of the BLASTN program (for nucleotide sequence comparison) and the BLASTP program (for amino acid sequence comparison) based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993).

[0028] The term "polypeptide" as used herein is intended to encompass the singular "polypeptide" and the plural "polypeptides" and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. That is, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to refer to a chain of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and modification with non-standard amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specific nucleic acid sequence. It may be generated by any method, such as chemical synthesis.

[0029] As used herein, "protein" may refer to a single polypeptide, i.e., a single amino acid chain as defined above, but it may also refer to two or more polypeptides associated, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions to produce a multimeric protein.

[0030] An "isolated" polypeptide, or a fragment, variant, or derivative thereof, refers to a polypeptide that is not in its natural environment. A particular level of purification is not required. For example, an isolated polypeptide can be removed from its native or natural environment. In the present disclosure, recombinantly produced polypeptides and proteins expressed in a host cell are considered to be isolated, as are recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0031] The term "polynucleotide" includes both the singular and plural nucleic acids and refers to an isolated nucleic acid molecule or construct, such as a pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds, such as amide bonds as found in peptide nucleic acids (PNAs). The term "nucleic acid" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present within a polynucleotide. An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. In the present disclosure, a recombinant polynucleotide encoding a polypeptide subunit contained within a vector, for example, is considered to be isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or polynucleotides that have been purified (partially or substantially) in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of a polynucleotide. Isolated polynucleotides or nucleic acids also include those molecules that have been produced synthetically. The polynucleotide or nucleic acid may also be or comprise regulatory elements such as a promoter, a ribosome binding site, a transcription terminator, and the like.

[0032] As used herein, a "coding region" refers to a portion of a nucleic acid that contains a codon that is translated into an amino acid. A "stop codon" (TAG, TGA, or TAA), although not translated into an amino acid, can be considered to be part of the coding region, but adjacent sequences, such as, for example, promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. Two or more coding regions can be present across a single polynucleotide construct, such as a single vector, or across separate polynucleotide constructs, such as separate (different) vectors. Also, any vector can contain a single coding region or two or more coding regions, for example, a single vector can separately encode a selectable marker gene and a gene of interest. Also, a vector, polynucleotide, or nucleic acid can encode a heterologous coding region, either fused or unfused to a nucleic acid encoding a polypeptide subunit or fusion protein as provided herein. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains.

[0033] As used herein, the term "exon" refers to a portion of a DNA or RNA sequence where an amino acid sequence is synthesized.

[0034] As used herein, the term "intron" refers to a portion of a DNA or RNA sequence where no amino acid sequence is synthesized.

[0035] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide may typically include a promoter and / or other transcription or translation regulatory elements operably associated with one or more coding regions. An operably associated or linked region includes a coding region for a gene product, e.g., a polypeptide, that can be associated with one or more regulatory sequences such that expression of the gene product is influenced or controlled by the regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and its associated promoter) are "operably associated" or "operably linked" if induction of promoter function results in transcription of an mRNA encoding a desired gene product, and if the nature of the linkage of the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or to transcribe the DNA template. In other words, a promoter region and a nucleic acid are operably associated if the promoter is capable of affecting transcription of a nucleic acid encoding a polypeptide. The promoter may also be a cell-specific promoter that directs substantial transcription of the DNA only in a given cell. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, transcription termination signals, and the like, can also be operably associated with the polynucleotide to direct cell-specific transcription.

[0036] A variety of transcriptional regulatory regions are known to those skilled in the art. They include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, including, but are not limited to, promoter and enhancer segments derived from cytomegalovirus (immediate early promoter in conjunction with intron A), Simian Virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcriptional regulatory regions include actin, heat shock proteins, bovine growth hormone, rabbit beta globin, and other sequences that can control gene expression in eukaryotic cells. Suitable transcriptional regulatory regions further include tissue-specific promoters and enhancers.

[0037] Likewise, various translational regulatory elements are known to those skilled in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, elements derived from picornaviruses (in particular, internal ribosome entry sites, also called CITE sequences, or IRES).

[0038] In other embodiments, the polynucleotide can be RNA, for example, in the form of pre-mRNA or messenger RNA (mRNA).

[0039] Polynucleotide and nucleic acid coding regions may be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides disclosed herein. According to the signal hypothesis, proteins secreted from mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the transport of the growing protein chain from the rough endoplasmic reticulum has begun. As will be appreciated by those skilled in the art, polypeptides secreted from vertebrate cells usually have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the complete or "full-length" polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide or a functional derivative thereof that retains the ability of the signal peptide to be operably associated with the polypeptide to direct the secretion of the polypeptide is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0040] A "vector" is a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may contain a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. A vector may also have one or more selectable marker genes and other genetic elements known in the art. Examples of types of vectors include plasmids, phages, viruses, and retroviruses.

[0041] A "transformed" cell, or a "host" cell, is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation includes techniques such as transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration methods, by which a nucleic acid molecule can be introduced into such a cell. A transformed cell, or a host cell, can be a bacterial cell or a eukaryotic cell.

[0042] The term "expression" as used herein refers to the process by which a gene produces a biochemical, e.g., a polypeptide. This process includes any manifestation of the functional presence of a gene in a cell, including, but not limited to, gene knockdown, transient expression, and stable expression. It includes, but is not limited to, transcription of a gene into pre-mRNA and messenger RNA (mRNA), and translation of such mRNA into a polypeptide(s). If the final desired product is a biochemical, then expression includes the production of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Other gene products described herein include nucleic acids that have undergone post-transcriptional modifications, e.g., polyadenylation, or polypeptides that have undergone post-translational modifications, e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, and proteolytic cleavage.

[0043] As used herein, the term "engineered" includes the manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or any combination of these techniques, etc.).

[0044] The term "pharmaceutical composition" refers to a preparation or mixture suitable for administration to a subject, i.e., a preparation or mixture in a form capable of activating the in vivo activity of the active ingredient, and containing no additional ingredients that are unacceptably toxic to the subject to which the composition may be administered. Such compositions may be sterile. For example, a pharmaceutical composition may contain an oligomeric compound and a sterile aqueous solution.

[0045] As used herein, a "pharmaceutical acceptable carrier or diluent" is one suitable for administration. Such carriers include those that allow the pharmaceutical composition to be formulated, for example, as a tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, lozenge, etc., to be orally ingested by a subject. In certain embodiments, the pharmaceutical acceptable carrier or diluent is sterile water, sterile saline, sterile buffer solution, or sterile artificial cerebrospinal fluid.

[0046] As used herein, a "pharmaceutical acceptable salt" refers to a physiologically and pharma- ceutical acceptable salt of a compound that retains the desired in vivo activity of the parent compound and does not impart undesired toxicological effects.

[0047] As used herein, "antisense compound" refers to a compound capable of achieving at least one antisense activity. In certain embodiments, antisense compounds are antisense oligonucleotides (ASOs), and optionally have one or more additional structures, such as conjugate groups or terminal groups. In certain embodiments, antisense compounds are engineered and synthesized to include non-naturally occurring backbone structures (such as sugar and / or phosphate backbone modifications). In certain embodiments, antisense compounds have morpholino backbones.

[0048] As used herein, "antisense activity" refers to any detectable and / or measurable change resulting from hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity refers to a reduction in the amount or expression of a target nucleic acid or protein encoded by such a target nucleic acid, compared to the target nucleic acid level or target protein level in the absence of the antisense compound.

[0049] For purposes of this disclosure, the complete human PMP22 gene was downloaded from the University of California, Santa Cruz, genome.ucsc.edu database. As shown in the figures, sequence regions that are introns or non-coding portions of the genome are written in lower case, following standard nomenclature. Regions of the sequence that code for amino acids are written in upper case. For the databases used and corresponding sequences shown in the figures, the UCSC databases mentioned above were used, with the option to download sequences from Human Assembly Dec.2013 (GRCh38 / hg38) with the protein coding option chosen. Those skilled in the art will understand that other databases are acceptable and available, or, if they become acceptable and available, will include minor sequence variations in this description.

[0050] As used herein, the term "complementary" with respect to oligonucleotides refers to the ability of two single-stranded nucleic acids or portions of a single strand to hybridize into a double-stranded sequence by hydrogen bonding between complementary bases. Complementary bases include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids do not need to be complementary at all positions. Some mismatches are permissible. As used herein, "fully complementary" or "100% complementary" with respect to oligonucleotides means that two oligostrands have complementary bases at all corresponding positions. In certain embodiments, only 70% or more of the bases at all corresponding positions of complementary oligonucleotides are complementary.

[0051] As used herein, "hybridization," hybridization, and the like, refer to the pairing or annealing of complementary oligonucleotides and / or nucleic acids. The most common hybridization mechanism, without being limited to a specific mechanism, involves hydrogen bonding, including Watson-Crick, Hoogsteen, or reversed Hoogsteen bonding, between complementary nucleotides.

[0052] As used herein, "oligomeric compound" refers to an oligonucleotide, which may have one or more additional structures, such as a conjugate group, a terminal group, etc. An oligomeric compound may or may not be paired with a second oligomeric compound that is complementary to the first oligomeric compound. A "single-stranded oligomeric compound" refers to an unpaired oligomeric compound. The term "oligomeric duplex" refers to a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a "double-stranded oligomeric compound."

[0053] As used herein, "exon skipping" can refer to skipping an entire exon or a portion of an exon.

[0054] As used herein, an "exon skipping compound" refers to any compound that binds to a pre-mRNA species and induces the transcription of a stable and measurable, but not full-length, mRNA (i.e., an exon-skipped mRNA).

[0055] Although a gene or locus may be identified by a particular reference sequence, e.g., the human PMP22 gene (SEQ ID NO:1), it should be understood that a gene corresponding to a reference sequence may include various allelic forms or mutations in the sequence, which would still be considered by one of skill in the art to be the same gene.

[0056] The nucleic acid sequences described herein and each corresponding SEQ ID NO are understood to be free of any modifications to a sugar moiety, internucleoside linkage, or nucleobase. That is, any nucleic acid of the disclosure, including any oligonucleotide, can contain, independently of each other, one or more modifications to a sugar moiety, internucleoside linkage, or nucleobase.

[0057] overview Provided herein is a direct therapeutic strategy that targets PMP22 RNA with antisense oligonucleotides (ASOs), including methods using ASOs that bind to and interact with pre-mRNAs in the PMP22 gene cascade, and effectively "splice out" specific portions of the mRNA by binding to the junction between the spacer region of the pre-mRNA and the exon region that is converted into the complete mRNA. In certain embodiments, the ASOs do not prevent the remaining exons of the PMP22 gene from being expressed into amino acids. By removing or partially removing one or more exons from the mRNA, the resulting modified amino acid sequence is shorter or different from the naturally occurring PMP22 amino acid sequence, and the resulting protein has a loss of function and / or accelerated degradation (i.e., reduced / reduced functional PMP22 protein) compared to the naturally occurring protein. In other embodiments, the splicing event causes the resulting alternatively spliced ​​mRNA (exon-skipped mRNA) to be degraded at a faster rate within the cell than the full-length mRNA, thereby reducing protein production.

[0058] In embodiments in which exon-skipped mRNA is produced by the ASO of the present disclosure, it is possible to monitor the ratio of full-length mRNA to exon-skipped mRNA over time, both in the relevant target tissue and in the corresponding blood levels. This allows the molecular activity of the drug to be specifically measured in a subject (e.g., in a patient, such as a laboratory animal and / or a human patient, over time). The amount of exon-skipped mRNA may correlate with various downstream effects of the drug that may result in a favorable outcome for the patient, such as affecting functional protein levels and reducing the phenotypic response of the patient or laboratory animal to the drug. Thus, embodiments of the present disclosure may be applied as diagnostic methods to confirm the activity of the drug in a particular subject, modify the amount of the drug given to the patient to obtain a desired response, and / or modify the frequency of administration of the drug to obtain a desired response.

[0059] In cells, normal full-length mRNA is produced, resulting in a fully functional PMP22 protein (Figure 1). In Figure 1, exons (2-5) within the marked initiation region contain the nucleic acid base pairs that code for the amino acids that make up the functional full-length PMP22 protein. The upper part of Figure 1 shows the pre-mRNA, which contains not only the exon region but also the intron region. The intron is spliced ​​out of the pre-mRNA to produce the mRNA shown below the pre-mRNA. Then, translation occurs, and the protein is produced from exons 2-5 between the initiation and termination regions shown. CMT1A patients have two copies of the PMP22 gene from birth, which leads to overproduction of full-length PMP22 mRNA and full-length PMP22 protein, which is the main cause of the disease.

[0060] Previous approaches have attempted to alter PMP22 protein levels by "knocking down" or "silencing" the mRNA. Others have used drug-like molecules to target the pre-mRNA or the regulatory portion of the mRNA to stop any production. Others have proposed gapmer-like strategies to promote cleavage of the mRNA by RNase H, destroying the mRNA and thus reducing PMP22 production. These approaches have the distinct disadvantage that activity can only be monitored by measuring the total amount of full-length mRNA or protein in comparison to control animals or patients. Because at least a certain amount of PMP22 protein must be produced for normal physical health, excessive removal of PMP22 protein can cause a number of problematic symptoms, including (but not limited to) a distinct neuropathic disorder known as hereditary neuropathy with predisposition to pressure palsies, loss of peripheral nerve function, worsening physical symptoms, and even death. Therefore, removing a large amount of PMP22 protein without removing all of the protein present in the patient's cells is important to achieve a good clinical outcome. This precise goal would likely not be achievable with the same dosing schedule for every patient and every lifestyle, since numerous factors affect the total production and stability over time of PMP22 protein in different patients, including age, weight, lifestyle, natural history, stage of disease progression, etc. Total mRNA reduction approaches make it impossible to monitor molecular activity on a patient-by-patient basis, since there are large natural differences in mRNA and protein levels among patients due to a variety of physical or behavioral factors.

[0061] One embodiment of the present disclosure is shown in Figure 2. This embodiment relates to any compound that can interact with the pre-mRNA shown in Figure 2 to remove one (or more) exons while leaving the remaining exons in place during translation of the pre-mRNA into mRNA, generating a stable and measurable new exon-skipped mRNA that resembles the full-length mRNA but does not encode a complete functional PMP22 protein. A compound that removes exon 3 is shown in Figure 2, but other examples for exon 2 and exon 4 are described below.

[0062] "Exon skipping" events during splicing can result in a fully linked non-functional protein or can result in out-of-frame translation.

[0063] In other words, disclosed herein is a method that can compare the amount of full-length PMP22 mRNA that results in an effective protein with the presence of exon-skipped mRNA that results in a non-functional protein in a given subject, and can utilize the natural and actual changes in background mRNA and protein levels to perform such monitoring. In certain embodiments, this can be performed in animal studies, which not only simplifies these studies but also provides more information within a given study. And, in certain embodiments, this can be performed in human patients undergoing treatment.

[0064] This approach differs from other conventional drug approaches for genetic diseases, which are simply impossible or impractical to monitor due to the lack of internal controls in patient samples. That is, factors such as sample collection, patient differences, and analytical method variability make such testing impossible or impractical. In contrast, the disclosed approach provides two measurable markers in the patient sample, and the ratio of the two markers is a determinant of drug activity. In certain embodiments, the amount of exon-skipped mRNA in an animal or patient sample is used to track therapeutic activity. In another embodiment, the amount of exon-skipped mRNA is compared to the amount of full-length mRNA, and the ratio tracks therapeutic activity.

[0065] In the case of CMT1A, it is desirable to target the production of PMP22 protein. Moreover, it is highly desirable to carry out with modification of the patient's dosing schedule. The dosing conditions may be highly dependent on many patient factors, including weight, age, disease progression, lifestyle, etc. Conventional methods for treating CMT1A disease (and other monogenic and polygenic diseases) cannot accurately track the production of mRNA, i.e., the total (or partial) production of protein from that mRNA.

[0066] In the most classical drug development, the overall strategy is to correlate drug effect with changes in phenotypic response by examining drug effect against delivery concentration in multiple cell and animal studies. The drug is then tested in humans (usually implementing multiple dosing strategies, mainly focusing on safety and toxicity profiling) so that a single recommended dose can be found for the entire patient population of interest. This often results in a drug that, while showing reasonable efficacy in the clinical setting, often shows no measurable phenotypic effect at all in some patient populations. These non-responding patients are often given doses that are simply ineffective (not enough or too much drug) based on semi-relevant previous studies.

[0067] When the ASO of the present disclosure is introduced into a cell (or a subject, such as an animal undergoing in vivo therapy, a human, etc.), a portion of the ASO interacts with the pre-mRNA and causes an exon (or multiple exons) to be skipped during transcription. The mRNA with the "skipped" exon ("exon-skipped mRNA") is still produced, but produces a non-functional protein. One important advantage of such an approach is that the exon-skipped mRNA molecules can be quantified as a measure of drug activity at the molecular level, and therefore are not dependent on certain other (always present) environmental or experimental parameters that may confound the results obtained.

[0068] Provided herein is a new class of therapeutics that induces a molecular response in a subject and allows both unaffected original full length mRNA and exon skipped mRNA to be monitored during the course of treatment and correlated with downstream protein production. In certain embodiments, samples can be taken from the target tissue of interest. In certain embodiments, samples can be taken from another (more accessible) part of the body, such as plasma. Such an approach allows the efficacy of the therapeutic compositions of the present disclosure to be monitored in real patients during animal testing and clinical deployment.

[0069] PCR, for example, is a sensitive and highly specific technique for analyzing RNA abundance both in the target tissue of interest and at the corresponding plasma levels, but monitoring by PCR can be problematic because RNA is less stable and the amount of RNA present in the blood can be significantly lower than the amount and nature of RNA in tissues. Monitoring protein production in target tissues and at the corresponding plasma levels can be even more problematic because protein levels can be even more variable based on environmental factors such as patient activity and differences in native protein stability between target tissues and the rest of the body during biodistribution.

[0070] An important aspect of the present disclosure is the unique approach of targeting the intron-exon junctions of pre-mRNA prior to transcription. Intron-exon junctions are highly susceptible to ASO targeting (compared to non-overlapping regions of introns or exons only) for a number of reasons. First, ASO binding to a target is highly dependent on the availability of steric space in the RNA to accommodate the ASO. Oligonucleotides in cells have complex 3D structures that, due to their unique positioning, are "open" and accessible to the binding sites of the proposed drugs. Second, any ASO binding to a genetic target may "compete" with proteins and enzymes (and other molecules) for the binding site and displace said proteins. Even if the ASO compound binds to its target, it will not function effectively if it is displaced by a functional enzyme (e.g., during transcription, translation, etc.). Surprisingly, it was found that the junction sites are much more accessible in terms of active binding and competitive strength (to maintain binding) than non-junction sites.

[0071] An overview of the targeting approach is shown in Figures 3 and 4. Figure 3A is a schematic diagram of exon 1, an intron region, and exon 2 (referred to as pre-mRNA). During conversion of pre-mRNA to mRNA, prior to the removal of the intron and the joining of consecutive exons, the pre-mRNA undergoes conformation in the presence of snRNPs to generate an intermediate structure shown in Figure 3B, in which the 3' end of exon 1 is close to the 5' end of exon 2. Once this complex conformation is formed, a biomolecule (such as an enzyme) can act on it (in the presence of snRNPs and other cofactors) to remove the intron region of the pre-mRNA and chemically join two adjacent exons together (depicted as exon 1 and exon 2 in Figure 3, but other consecutive exons in a gene may be joined in a similar manner). The present disclosure relates to a technique for binding molecules to selective regions in such genomes to block specific exon-intron-exon junctions and allow other (partial or total) transcription to occur. This region (dotted circle 1z in Figure 3B) is shown, and its conformation is important for the efficient splicing process. This region is important due to a number of factors, including the enzyme that recognizes the conformation, the chemical nature of the conformation, and the proximity of the ends of the two exons to be joined. When splicing occurs, the two ends of exon 1 and exon 2 join together (Figure 3C) to form the mRNA.

[0072] In certain embodiments, antisense oligonucleotides (ASOs) are introduced that specifically bind or hybridize to the region of the intron and / or exon itself targeted for exon skipping. In such embodiments, the introduced ASO contains a sequence complementary to the pre-mRNA in the region near the described proximal site (1z in FIG. 3), thereby preventing exon inclusion and producing a final mRNA product that does not contain the targeted exon, but does contain other exons in the target gene that were not bound by the drug. The presence of the ASO in the nucleus of the cell (where RNA translation occurs) creates an equilibrium with the pre-mRNA, which is directly influenced by the amount of drug present, the amount of pre-mRNA present, the efficiency of binding to the target region, the strength of attachment after binding, etc. In certain embodiments of the present disclosure, the amount of drug introduced is such that not all pre-mRNA present in the cell will result in exon skipping. That is, in any given tissue sample, both full-length and exon-skipped PMP22 mRNA will be produced, and the amount of skipping that occurs will depend on factors such as the chemical composition of the drug and the amount of drug introduced. There are other factors that may affect the rate of exon skipping. In particular, certain embodiments of the present disclosure relate to molecules that do not completely eliminate the production of any full-length mRNA. ASOs complementary to a continuous region of pre-mRNA may target the 3' end of an exon, the 5' end of an exon, or other regions of an intron or exon, provided that they induce a measurable exon skipping event that produces a truncated non-full-length exon-skipped mRNA.

[0073] The length of the ASO shown in FIG. 4B can be various lengths depending on the desired effect. In the present disclosure, ASOs are disclosed that specifically target the pre-mRNA of PMP22, and therefore the minimum length of the ASO base is preferably specific to the mRNA of PMP22 and not to other regions of the human genome. Also, if the ASO is too long, it may be problematic to use, since long strands such as DNA and RNA may fold on themselves and may not have room to bind to the target pre-mRNA. Furthermore, if the ASO is too long, manufacturing concerns must also be considered. Thus, in certain embodiments, the length of the ASO is from about any of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides to about any of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In certain embodiments, the length of the ASO is about 12-30 nucleotides. In certain embodiments, the length of the ASO is about 15-25 nucleotides. In certain embodiments, the length of the ASO is about 18-25 nucleotides.

[0074] The ASO of the present disclosure may not be a perfect match (i.e., 100% complementary) with all target pre-mRNA bases used for hybridization. For example, in certain embodiments, an 18-mer ASO may be designed and synthesized such that one or more bases are not complementary to the target pre-mRNA, but still hybridize / have antisense activity. For example, in certain embodiments, two or more ASO / target region base pairs may be considered non-complementary "mismatches." In certain embodiments, the ASO comprises a complementary region that is complementary to the target region of the pre-mRNA, or is complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides. In certain embodiments, the ASO comprises a complementary region that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% complementary to the target region of the pre-mRNA.

[0075] Another embodiment of the present disclosure is shown in FIG. 4B, where an ASO is used that does not bind to a contiguous portion of the pre-mRNA, but spans two separate sites of the pre-mRNA. For example, the ASO shown in FIG. 4B may contain bases complementary to the 3' end of exon 1 and bases complementary to the 5' end of exon 2. The ASO may bind once the conformation is formed (as shown in FIG. 4B) and interfere with the actual splicing event. The ASO may also bind to affect the overall conformation and its corresponding chemistry. Many other ASO forms are envisioned, provided that they induce exon skipping in one or more exons present. In some embodiments, the ASO overlaps both a portion of an intron and an exon. In other embodiments, the ASO overlaps only the exon portion of the target region. In other embodiments, the ASO overlaps only a portion of the intron near the target region.

[0076] Another embodiment is shown in Figure 4C, where an antibody specific for a given intron / exon junction is added to cause an exon skipping event. Yet another embodiment is shown in Figure 4D, where a small molecule is used to induce an exon skipping event.

[0077] Importantly, the molecules described in this patent are specific to certain parts of the genome and do not randomly bind to intron / exon junction regions, which may cause unwanted side effects and disrupt normal non-targeted biological processes. In certain embodiments, the molecules act to prevent the formation and stabilization of the depicted conformation. In other embodiments, the molecules bind to the region and thereby physically or chemically block cofactors and enzymes from completing the splicing event.

[0078] The methods and compositions disclosed herein may allow ASOs to be thoroughly tested in animals to understand the various effects of drug administration before developing a safe and effective treatment strategy for human patients. Other factors related to drug administration (such as body weight or disease progression) may also be tested in animals, allowing patients to be "stratified" or "classified" during clinical trials and given different doses depending on the specific characteristics of each individual patient. It may also be possible to monitor patients' mRNA levels as a companion diagnostic during treatment, allowing different doses to be administered to different patients based on their molecular response to drug treatment.

[0079] In certain embodiments, the use of morpholino antisense oligonucleotides (or other uncharged backbone chemistry oligonucleotides) enhances the binding strength of the ASO when hybridized to the target region of the pre-mRNA. The uncharged backbone structure does not compete for base pairing through ionic repulsion (as with 2'Me-O ASOs and miRNA drugs), and is therefore less susceptible to displacement by transcriptional proteins (and other cofactors) upon binding of the ASO, leading to a significant enhancement. Morpholino backbones have been well studied in the literature and are used by Sarepta Therapeutics in certain FDA-approved ASO products for other uses (such as eteplirsen).

[0080] Other non-natural amino acid backbones are also contemplated for the synthesis of the ASOs of the present disclosure. For example, 2'Me-O modifications may be made to the sugars and backbones of the ASOs to stabilize the ASOs against enzymatic degradation. Many backbone chemistries, sugar modifications, terminal modifications, etc. may be applied in the methods of the present disclosure to form stable ASOs with antisense activity (see all examples listed in US 2019 / 0062741 Al). In certain embodiments, any ASO structure that hybridizes to a complementary pre-mRNA to enable exon skipping is encompassed.

[0081] Certain embodiments of the present disclosure provide compositions comprising antisense oligonucleotides (ASOs) that comprise or consist of a complementary region that is complementary to the target region of the pre-mRNA of PMP22 or is complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides. In certain embodiments, the complementary region is 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% complementary to the target region of the pre-mRNA. It is understood that the ASO does not need to be complementary to the entire length of the target region, but only to a sufficient portion of the target region to hybridize therewith, i.e., a given target region can be longer than the complementary region of the ASO, or even the entire ASO, i.e., the complementary region of the ASO can be complementary to a subset of the target region sequence. In certain embodiments, the complementary region and the target region are the same length. As described in detail elsewhere herein, binding of the complementary region of the ASO to the target region of the pre-mRNA of PMP22 in a cell induces exon skipping during RNA transcription. Binding of the ASO to the target region may reduce the production of full-length mRNA of PMP22. It may also result in the production of exon-skipped mRNA of PMP22. Binding of the ASO to the target region may result in a reduction in functional PMP22 protein and / or the production of non-functional PMP22 protein. Both the reduction in full-length mRNA and the production of exon-skipped mRNA may be detected and measured. Also, the reduction in functional protein and / or the production of non-functional protein may be detected and measured. Also, correlations and / or ratios of full-length and exon-skipped mRNA to functional and non-functional proteins may be determined or calculated for purposes as described in detail elsewhere herein.

[0082] In certain embodiments, the ASO comprises or consists of a complementary region of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides that are complementary to the target region of the pre-mRNA of PMP22 or are complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides. Those skilled in the art will appreciate that the length of the complementary region of the ASO may vary depending, for example, on the target region sequence of the pre-mRNA of PMP22 and / or the particular application or administration conditions. Illustrative examples of ASOs that comprise a complementary region of "consecutive nucleotides" are disclosed in Example 1 and Example 4 below (e.g., Figures 5A and 5B, Figures 11A and 11B).

[0083] In certain embodiments, the target region of the PMP22 pre-mRNA comprises two separate segments of the PMP22 pre-mRNA, as described in Example 5. In certain embodiments, the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11, or 12 nucleotides that is complementary to a first segment of the contiguous sequence of the target region of the PMP22 pre-mRNA, or is complementary except for 1, 2, or 3 mismatched nucleotides, and the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11, or 12 nucleotides that is complementary to a second segment of the contiguous sequence of the target region of the PMP22 pre-mRNA, or is complementary except for 1, 2, or 3 mismatched nucleotides. That is, the complementary region of the ASO hybridizes to a first segment and a second segment of the PMP22 pre-mRNA and spans a non-complementary / non-hybridizing region of the PMP22 pre-mRNA.

[0084] In certain embodiments of the ASOs of the present disclosure, regardless of whether the target region of the PMP22 pre-mRNA is one contiguous segment or two separate segments, the ASO comprises or consists of a complementary region of about any of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 to about any of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides that are complementary to the target region of the PMP22 pre-mRNA or are complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides. In certain embodiments, the ASO comprises or consists of a complementary region of 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides that are complementary to a target region of the PMP22 pre-mRNA or are complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides.

[0085] In certain embodiments of any ASO of the present disclosure, regardless of whether the target region of the pre-mRNA is one contiguous segment or two separate segments, the ASO has a length of from about any of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, or 75 to about any of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 75, or 100 nucleotides.

[0086] In certain embodiments, the ASO is a modified and / or synthetic oligonucleotide as known in the art and / or defined elsewhere herein. For example, the ASO can be a phosphorodiamidate morpholino oligomer (PMO).

[0087] Although the ASO causes exon skipping of the PMP22 pre-mRNA, in certain embodiments, downstream exons other than those derived from the skipped exon may still be expressed as in the full-length PMP22 pre-mRNA, however, in certain embodiments, exon skipping results in premature termination of protein translation and / or causes the downstream exon to be out of frame.

[0088] It has been found that exon skipping and the production of exon-skipped mRNA of PMP22 has certain advantages over conventional methods that do not disclose the production of exon-skipped mRNA of PMP22. That is, in certain embodiments, the target region of the pre-mRNA of PMP22 spans an intron / exon junction with at least one coding exon (e.g., exon 2, exon 3, exon 4, and exon 5 of PMP22). The targeted intron / exon junction can be at the 3' and / or 5' end of the exon. For example, in certain embodiments, the target region of the pre-mRNA of PMP22 comprises the 3' end of the exon. Also, in certain embodiments, the target region of the pre-mRNA of PMP22 comprises the 5' end of the exon. In certain embodiments, the target region of the pre-mRNA of PMP22 spans an intron / exon junction that comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an intron and a portion of an exon (e.g., Figures 5A and 5B, Figures 11A and 11B). In certain embodiments, the target region of the pre-mRNA of PMP22 spans an intron / exon junction that comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an intron and a portion of an exon. Similarly, in certain embodiments, the target region of the pre-mRNA of PMP22 spans an intron / exon junction that comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an exon and a portion of an intron (e.g., Figures 5A and 5B, Figures 11A and 11B). In certain embodiments, the target region of the pre-mRNA of PMP22 consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an exon and a portion of an intron. In certain embodiments, the exon portion of the intron / exon junction comprises exon 3 of PMP22 (FIGS. 5A and 5B). In certain embodiments, the exon portion of the intron / exon junction comprises exon 4 of PMP22 (FIGS. 11A and 11B).

[0089] In certain embodiments, the target region of the pre-mRNA of PMP22 comprises the 5' end of exon 3. For example, the target region of the pre-mRNA of PMP22 comprises or consists of SEQ ID NO:2, or a portion or subset / fragment thereof.

[0090] In certain embodiments, the target region of the pre-mRNA of PMP22 comprises the 3' end of exon 3. For example, the target region of the pre-mRNA of PMP22 comprises or consists of SEQ ID NO:35, or a portion or subset / fragment thereof.

[0091] In certain embodiments, the target region of the pre-mRNA of PMP22 comprises the 5' end of exon 4. For example, the target region of the pre-mRNA of PMP22 comprises or consists of SEQ ID NO:76, or a portion or subset / fragment thereof.

[0092] In certain embodiments, the target region of the pre-mRNA of PMP22 comprises the 3' end of exon 4. For example, the target region of the pre-mRNA of PMP22 comprises or consists of SEQ ID NO:111, or a portion or subset / fragment thereof.

[0093] In certain embodiments, the target region of the pre-mRNA of PMP22 comprises the 5' end of exon 2. For example, the target region of the pre-mRNA of PMP22 comprises or consists of SEQ ID NO:163, or a portion or subset / fragment thereof.

[0094] In certain embodiments, the target region of the pre-mRNA of PMP22 comprises the 3' end of exon 2. For example, the target region of the pre-mRNA of PMP22 comprises or consists of SEQ ID NO:198, or a portion or subset / fragment thereof.

[0095] In certain embodiments, the ASO comprises or consists of a complementary region that is complementary to SEQ ID NO:2 (exon 3, 5' end), SEQ ID NO:35 (exon 3, 3' end), SEQ ID NO:76 (exon 4, 5' end), SEQ ID NO:111 (exon 4, 3' end), SEQ ID NO:163 (exon 2, 5' end) and / or SEQ ID NO:198 (exon 2, 3' end), or is complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides. In certain embodiments, the ASO comprises or consists of the nucleotide sequence of SEQ ID NO:3-34 (exon 3, 5' end), SEQ ID NO:37-70 (exon 3, 3' end), SEQ ID NO:77-110 (exon 4, 5' end), SEQ ID NO:112-145 (exon 4, 3' end), SEQ ID NO:164-197 (exon 2, 5' end) or SEQ ID NO:199-232 (exon 2, 3' end), or a subset / fragment thereof sufficient to hybridize to the PMP22 pre-mRNA. In certain embodiments, the ASO comprises or consists of a nucleotide sequence of SEQ ID NOs:3-34 (exon 3, 5' end), SEQ ID NOs:37-70 (exon 3, 3' end), SEQ ID NOs:77-110 (exon 4, 5' end), SEQ ID NOs:112-145 (exon 4, 3' end), SEQ ID NOs:164-197 (exon 2, 5' end) or SEQ ID NOs:199-232 (exon 2, 3' end) with one, two or three nucleotide substitutions, or a subset / fragment thereof sufficient to hybridize to the PMP22 pre-mRNA.

[0096] In certain embodiments, the ASO is selected from the group consisting of SEQ ID NO:71 (SHC-006 25-mer), SEQ ID NO:72 (SHC-001 24-mer), SEQ ID NO:73 (SHC-005 25-mer), SEQ ID NO:74 (SHC-010 21-mer), SEQ ID NO:75 (SHC-012 20-mer), SEQ ID NO:146 (SHC-029 21-mer), SEQ ID NO:147 (SHC-028 20-mer), SEQ ID NO:148 (SHC-027 20-mer), SEQ ID NO:149 (SHC-031 21-mer), SEQ ID NO:150 (SHC-030 20-mer), SEQ ID NO:151 (SHC-032 20-mer), SEQ ID NO:152 (SHC-033 20-mer), SEQ ID NO:153 (SHC-034 20-mer), SEQ ID NO:154 (SHC-035 20-mer), SEQ ID NO:155 (SHC-036 20-mer), SEQ ID NO:156 (SHC-037 20-mer), SEQ ID NO:157 (SHC-038 20-mer), SEQ ID NO:158 (SHC-039 20-mer), SEQ ID NO:159 (SHC-040 20-mer), SEQ ID NO:160 (SHC-041 20-mer), SEQ ID NO:161 (SHC-042 20-mer), SEQ ID NO:162 (SHC-043 20-mer), SEQ ID NO:163 (SHC-044 20-mer), SEQ ID NO:164 (SHC-045 20- 20-mer), comprising or consisting of the nucleic acid sequence of SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:235, or SEQ ID NO:238. In certain embodiments, the ASO is selected from the group consisting of SEQ ID NO:71 (SHC-006 25-mer), SEQ ID NO:72 (SHC-001 24-mer), SEQ ID NO:73 (SHC-005 25-mer), SEQ ID NO:74 (SHC-010 21-mer), SEQ ID NO:75 (SHC-012 20-mer), SEQ ID NO:146 (SHC-029 21-mer), SEQ ID NO:147 (SHC-028 20-mer), SEQ ID NO:148 (SHC-027 20-mer), SEQ ID NO:149 (SHC-031 21-mer), SEQ ID NO:150 (SHC-030 20-mer), SEQ ID NO:151 (SHC-032 20-mer), SEQ ID NO:152 (SHC-033 20-mer), SEQ ID NO:153 (SHC-034 20-mer), SEQ ID NO:154 (SHC-035 20-mer), SEQ ID NO:155 (SHC-036 20-mer), SEQ ID NO:156 (SHC-037 20-mer), SEQ ID NO:157 (SHC-038 20-mer), SEQ ID NO:158 (SHC-039 20-mer), SEQ ID NO:159 (SHC-040 20-mer), SEQ ID NO:160 (SHC-041 20-mer), SEQ ID NO:161 (SHC-042 20-mer), SEQ ID NO:162 (SHC-043 20-mer), SEQ ID NO:163 (SHC-044 20-mer), SEQ ID NO:164 (SHC-045 20- 20-mer), SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:235, or SEQ ID NO:238, wherein one, two or three nucleotides are substituted.

[0097] Provided herein is a method for reducing the amount of full-length PMP22 mRNA expression in a cell, comprising administering to the cell a composition comprising an antisense oligonucleotide (ASO) of the present disclosure. In certain embodiments, reducing the amount of full-length PMP22 mRNA comprises targeting an intron-exon junction in the pre-mRNA of PMP22, as described in detail elsewhere herein. As used elsewhere herein, "administering to a cell" is understood to cover any situation in which an ASO contacts a cell such that the cell can take up the ASO and thereby exert its antisense activity. For example, administering to a cell includes exposing a cell to the ASO in an in vitro experiment, e.g., to cells grown in tissue culture. Administering to a cell includes providing the ASO to a subject, e.g., a research animal in an in vivo experiment, such that at least one cell of the subject is contacted with the ASO by local administration, systemic administration, etc. Administering to a cell includes providing the ASO to a patient, e.g., treating a human patient, such that at least one cell of the patient is contacted with the ASO, either locally, systemically, etc. That is, the cells of interest can be present in a tissue, an organ, a body site, a biological fluid, the entire organism, etc.

[0098] In certain embodiments of reducing the amount of full-length PMP22 mRNA, the amount of full-length PMP22 mRNA in a cell is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75% or 95% in response to the ASO. As described in the Examples below, the amount of full-length PMP22 mRNA and / or its reduction in any method of the present disclosure may be compared to the amount of full-length PMP22 mRNA in an untreated cell, subject, patient, etc. to which the ASO composition is not administered. In certain embodiments, since it is not contemplated or desirable to completely remove full-length PMP22 mRNA (or functional PMP22 protein), the amount of full-length PMP22 mRNA in a cell is reduced by no more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in response to the ASO. In certain embodiments, the amount of full length PMP22 mRNA in the cell is reduced by about any of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% to about any of 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% depending on the ASO. As described in detail elsewhere herein, in certain embodiments, exon-skipped mRNA of PMP22 is produced. That is, in certain embodiments, the amount / reduction of full length PMP22 mRNA can be compared to the amount of exon-skipped PMP22 mRNA to determine correlations, calculate ratios, etc. In certain embodiments, the amount of functional PMP22 protein produced in the cell is reduced. Such reduction in functional protein in any of the methods of the present disclosure can be determined relative to untreated control cells, subjects, patients, etc. to which the ASO composition is not administered. In certain embodiments, the amount of functional PMP22 protein in the cell is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75% or 95% in response to the ASO. As noted above, in some embodiments, complete removal of PMP22 protein is not contemplated or desired.Thus, in some embodiments, the amount of functional PMP22 protein in a cell is reduced by no more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in response to the ASO. In certain embodiments, the amount of functional PMP22 protein in a cell is reduced by about any of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or 75% to about any of 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in response to the ASO.

[0099] Provided herein is a method for producing an exon-skipped pre-mRNA of PMP22, comprising administering to a cell a composition comprising an antisense oligonucleotide (ASO) of the present disclosure. In certain embodiments, the method comprises targeting an intron-exon junction in the pre-mRNA of PMP22, as described in detail elsewhere herein. In certain embodiments, the amount of full-length PMP22 mRNA expression in the cell is reduced. In certain embodiments, the amount of full-length PMP22 mRNA in the cell is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75% or 95% depending on the ASO. In certain embodiments, since complete removal of the full length PMP22 mRNA (or functional PMP22 protein) is not contemplated or desired, the amount of full length PMP22 mRNA in the cell is reduced by no more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in response to the ASO. In certain embodiments, the amount of full length PMP22 mRNA in the cell is reduced by no more than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% to about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in response to the ASO. In certain embodiments, the amount / reduction of full length PMP22 mRNA can be compared to the amount of exon-skipped PMP22 mRNA produced, allowing correlations to be made, ratios to be calculated, etc. Also, in certain embodiments, the amount of functional PMP22 protein produced in cells is reduced. In certain embodiments, the amount of functional PMP22 protein in cells is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75% or 95% in response to ASO. As mentioned above, in some embodiments, it is not contemplated or desirable to completely remove PMP22 protein. Thus, in some embodiments, the amount of functional PMP22 protein in cells is reduced by no more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in response to ASO.In certain embodiments, the amount of functional PMP22 protein in the cell is reduced by about any of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or 75% to about any of 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% depending on the ASO.

[0100] Provided herein is a method of treating Charcot-Marie-Tooth disease, e.g., Charcot-Marie-Tooth type 1A, comprising administering to a subject in need thereof a composition comprising an antisense oligonucleotide (ASO) of the present disclosure. In certain embodiments, the subject is a model system, such as a research animal, for Charcot-Marie-Tooth disease. In certain embodiments, the subject is a human patient. In certain embodiments, the composition is administered orally, locally, systemically, e.g., subcutaneously, perineurally, etc. In certain embodiments, the method comprises targeting an intron-exon junction in the pre-mRNA of PMP22, as described in detail elsewhere herein. In certain embodiments, an exon-skipped mRNA of PMP22 is produced. In certain embodiments, the amount of PMP22 full-length mRNA may be compared / reduced, as described in detail elsewhere herein. In certain embodiments, the amount of PMP22 full-length mRNA in the cell is reduced. In certain embodiments, the amount of full length PMP22 mRNA in a cell is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 95% in response to the ASO. In certain embodiments, the amount of full length PMP22 mRNA in a cell is reduced by no more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO, since complete removal of full length PMP22 mRNA (or functional PMP22 protein) is not contemplated or desired. In certain embodiments, the amount of full-length PMP22 mRNA in the cell is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% to about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO. In certain embodiments, the amount of functional PMP22 protein in the cell is reduced. In certain embodiments, the amount of functional PMP22 protein in the cell is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 95% in response to the ASO.As mentioned above, in some embodiments, it is not contemplated or desirable to completely remove the PMP22 protein. Thus, in some embodiments, the amount of functional PMP22 protein in a cell is reduced by about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or less in response to the ASO. In certain embodiments, the amount of functional PMP22 protein in a cell is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or 75% to about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in response to the ASO.

[0101] In certain embodiments, the ASO is administered as a pharma- ceutically acceptable salt, hi certain embodiments, the ASO is administered in a pharma- ceutically acceptable carrier or diluent.

[0102] In certain embodiments, at least one symptom of the disease is alleviated. In certain embodiments, at least one symptom of the disease is slowed down. In certain embodiments, the treatment method does not cause side effects or has fewer or less severe side effects compared to other CMT treatments. In certain embodiments, the correlation or ratio between the amount of exon-skipped mRNA produced of PMP22 and the amount / reduction of full-length mRNA of PMP22 can adjust the dosage of ASO treatment to improve its effect and / or reduce side effects.

[0103] Further provided herein are compositions comprising antisense oligonucleotides (ASOs) that comprise or consist of a complementary region that is complementary to a target region of the pre-mRNA of PMP22 that comprises an intron / exon junction of one of the coding exons (i.e., exon 2, exon 3, exon 4, or exon 5 of PMP22), or is complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides. It is understood that the ASO need not be complementary to the entire length of the target region, but is complementary to a sufficient portion of the target region to hybridize therewith, i.e., a given target region may be longer than the complementary region of the ASO, or even longer than the entire ASO. In certain embodiments, the ASO comprises or consists of a complementary region of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 contiguous nucleotides that are complementary to a target region of the PMP22 pre-mRNA or are complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides. In certain embodiments, the ASO comprises or consists of a complementary region of about any of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 to about any of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 contiguous nucleotides that are complementary to a target region of a PMP22 pre-mRNA or are complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides. In certain embodiments, the ASO comprises or consists of a complementary region of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides that are complementary to a target region of the PMP22 pre-mRNA or are complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides. In certain embodiments, the target region of the PMP22 pre-mRNA comprises the 3' end of an exon. In certain embodiments, the target region of the PMP22 pre-mRNA comprises the 5' end of an exon.In certain embodiments, the target region of the pre-mRNA of PMP22 comprises an intron / exon junction comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an intron and a portion of an exon. In certain embodiments, the target region of the pre-mRNA of PMP22 comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an intron and a portion of an exon. Similarly, in certain embodiments, the target region of the pre-mRNA of PMP22 comprises an intron / exon junction comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an exon and a portion of an intron. In certain embodiments, the target region of the pre-mRNA of PMP22 comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an exon and a portion of an intron. In certain embodiments, the exon portion of the intron / exon junction comprises exon 3 of PMP22. In certain embodiments, the exon portion of the intron / exon junction comprises exon 4 of PMP22. In certain embodiments, the ASO is a modified and / or synthetic oligonucleotide as known to those of skill in the art and / or as disclosed herein. For example, in certain embodiments, the ASO is a phosphorodiamidate morpholino oligomer (PMO). In certain embodiments, the target region of the pre-mRNA of PMP22 comprises or consists of SEQ ID NO:2 (exon 3, 5' end), SEQ ID NO:35 (exon 3, 3' end), SEQ ID NO:76 (exon 4, 5' end), SEQ ID NO:111 (exon 4, 3' end), SEQ ID NO:163 (exon 2, 5' end), and / or SEQ ID NO:198 (exon 2, 3' end).

[0104] In certain embodiments, depending on the age of the patient and the severity of the symptoms, a tailored treatment may be applied (e.g., Example 9). For younger subjects and / or less severe symptoms, a small dose of the composition of the present disclosure may be enough to completely affect the axonal improvement. However, as shown in, for example, Example 9, the 5 mg / kg group for 30 days showed only a small improvement compared to the 50 mg / kg group (groups 2 and 4) tested on day 30. Meanwhile, the dowel walking time of group 5 improved significantly after the second treatment and subsequent test at 60 days. This suggests that older subjects and / or subjects with higher levels of symptoms or longer disease progression times may likely require higher dosages or may require a loading regimen (several injections for the first few months of treatment, then returning to the standard course of treatment) more than those who do not. For other subjects with no disease progression, a loading period may not be necessary. This finding is quite surprising from a biological point of view, since the PMP22 mRNA levels (shown in Table 4) of animals that were normalized over 30 days seem to show similar effects regardless of the dose given. The animals that were tested and sacrificed only one week ago (Group 1) would not have had time to normalize their PMP22 mRNA levels. Also, Group 1 did not show any improvement in performance, suggesting that although the PMP22 levels were affected, the myelin sheath surrounding the axons had not yet begun to improve.

[0105] Thus, in certain embodiments, a dosing regimen is set for each subject based on the progression of symptoms or age. In certain embodiments, the therapeutic amount of the composition of the present disclosure administered to a subject is based on both the progression of the disease and the total body weight. In another embodiment, the progression of the disease is based on the patient's age, the patient's physical ability, or both. In another embodiment, subjects with more severe symptoms and / or older ages are given the treatment described herein at a loading dose for a period of time, and then changed to a lower dose. The loading period may simply involve giving the same amount of drug to the subject more frequently, or may involve administering a higher dose of the composition initially. After a period of time (which may be based on time, or on physical improvement or improvement of some biomarker), the dosage and / or frequency of administration may be reduced. EXAMPLES

[0106] Example 1 - Exon 3 Skipping A number of compounds (FIG. 5) were designed to bind to the PMP22 pre-mRNA and promote exon skipping (FIG. 2). Some of these compounds were designed to interact with the 5' end of the intron / exon region, while others interact with the 3' region. In FIG. 5A and FIG. 5B, the target region in the PMP22 pre-mRNA is illustrated at the top, and the specific 25-mer ASO described herein is illustrated below. The nucleotide bases shown are in lower case (for intron regions) and upper case (for protein-coding exon regions, e.g., g and G are the same structure). These compounds were designed to bind to the pre-mRNA and induce exon skipping. Although the ASOs in FIG. 5 are designed to be 25 base pairs in length, one of skill in the art will recognize that the complementary region of the ASO can be of various lengths, longer or shorter, and still be effective as long as it is capable of specifically hybridizing to the target region of the PMP22 pre-mRNA. For example, some of the ASOs tested are shorter in length. In FIG. 5A, the sequence of PMO SHC-001 (SEQ ID NO:72) is ctaagagagatcGTTACCTAGCAC, which is 22 base pairs in length and is a subset of the 25-mer (SEQ ID NO:15) shown in FIG.

[0107] Many of these compounds were chemically synthesized with a morpholino backbone and tested in both cellular assays and mouse models to confirm their mechanism of action and activity by determining their effectiveness in inhibiting PMP22 mRNA production and producing a measurable exon skipping product. As noted above, these compounds were available in a range of lengths (not all compounds were a full 25 bases in length, some were a subset of the 25 base pair ASO shown).

[0108] The compounds in Figure 5 were screened in a HEK293 cell assay in which the cells express the human PMP22 gene. The cells were grown and the compounds to be tested were added. After a 48 hour incubation period, the transfected cells were harvested and total RNA was subjected to end-point RT-PCR. Figure 6 shows the cell assay results for compounds SHC-006 (SEQ ID NO:71), SHC-001 (SEQ ID NO:72), SHC-005 (SEQ ID NO:73), SHC-010 (SEQ ID NO:74), and SHC-012 (SEQ ID NO:75).

[0109] Each of these compounds effectively reduced the total amount of full-length PMP22 mRNA (i.e., resulting in lower production of PMP22 protein) and produced a stable exon-skipped mRNA product that was confirmed by sequencing to contain the full-length PMP22 mRNA minus exon 3. Similar results were obtained using two additional cellular contexts, K-562 cells (a myeloid leukemia cell line) and CLR-3392 cells, a human Schwann cell line (data not shown). Although these cells are less easily transfected than HEK293 cells, similar levels of exon skipping were observed due to reduced transfection efficiency.

[0110] Schwann cells are the main producer of PMP22 and therefore the primary target for any potential therapy for CMT1A. Schwann cells are heavily myelinated and their biochemistry is highly influenced by surrounding tissues, making cellular testing extremely difficult (although feasible) compared to other target cells. However, we herein disclose an optimal method for doing this and develop a robust assay to induce PMP22 exon skipping in pre-mRNA.

[0111] A relatively simple RT-PCR assay to induce exon skipping of exon 3 from human PMP22 provides a clear molecular readout from both full-length and exon-skipped mRNA DNA that allows for the examination of "target binding" and molecular effects. This is advantageous from a drug development perspective, as the concomitant decrease in full-length PMP22 and increase in exon-skipped PMP22 can be easily monitored in RT-PCR reactions with internal controls using a reliable target.

[0112] Example 2 In vivo activity was tested using C3 mice (3 copies of human PMP22). C3 mice were bred and genotyped according to published procedures from Jackson Labs. At 5 weeks, animals were injected subcutaneously with a single 6.7 mg dose of SHC-012 (SEQ ID NO:75) or scrambled control PMO. Untreated C3 mice were included as controls. 24 hours after injection, animals were sacrificed and tissues (sciatic nerve, kidney, liver, brain, spinal cord, etc.) were harvested and analyzed for reduction / expression of PMP22 mRNA and exon skipping versions of PMP22 mRNA. PMP22 expression was examined by RT-PCR (3% agarose gel). GAPDH was included in the reactions as a control to allow comparison between lanes (Figure 7). Because the liver will naturally receive large amounts of peripherally delivered therapeutics, we examined hepatic PMP22 expression and found a decrease in full-length PMP22 expression, along with an accumulation of exon-skipped PMP22 products (Figure 7). In SHC-012 animals, there was a significant decrease in full-length PMP22 mRNA, along with the presence of measurable amounts of exon 3-skipped PMP22 mRNA (confirmed by sequencing).

[0113] Additional tissues from these animals were analyzed by the same PCR and gel analysis (Figure 8), and we confirmed the overall reduction in these tissues by comparing full-length and exon-skipped amounts. This evidence indicates that a single injection (in the dorsal fold) results in reduction and / or exon skipping of PMP22 in some tissues, such as the peripheral nerve tissue (sciatic nerve) in C3 mice.

[0114] Importantly, a clear reduction in full-length PMP22 expression was observed when peripheral nerve tissue (e.g., a surrogate for Schwann cells in vivo) was examined.The C3 mice express the human genomic PMP22 gene, and key regulatory sequences within and surrounding its various exons can be targeted with ASOs.

[0115] Example 3 - Long-term animal studies C3 mice (3 human PMP22 genes) were chosen as an animal model because they have been shown to exhibit CMT-like behavior, are well studied, and, most importantly for this project, express the human genomic PMP22 gene (Huxley C, Passage E, Manson A, Putzu G, Figarella-Branger D, Pellissier JF, Fontes M. Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNA. Human molecular genetics. 1996;5(5):563-9. Epub 1996 / 05 / 01. doi:10.1093 / hmg / 5.5.563. PubMed PMID:8733121). This is important because ASOs depend on sequence identity within exon and intron regions.

[0116] Two groups of animals were studied: 5-week-old animals (which showed some behavioral changes at an early symptomatic disease stage) and 3-day-old animals (which were treated before measurable CMT behavioral changes). All animals were injected with SCH-012. At week 5, C3 animals (n=3) were injected subcutaneously once a week for 5 weeks with 3.3, 17 or 50 mg / kg SCH-012 or scrambled control PMO. A second group of C3 mice was also tested, with initial injections (SQ in upper back) of 1 mg / kg and 10 mg / kg per animal once a week (once a week for 12 weeks) from 3 days of age (to simulate pre-disease in humans). Untreated WT mice (n=4) were included as controls and were tested in the same way as the other mice. No adverse reactions were observed and all animals remained healthy throughout the study period, gaining weight at similar rates across the dose range and showing no obvious signs of toxicity. As expected, all animals recovered smoothly and rapidly with no complications from each injection and showed no immediate or delayed locomotor, behavioral or neurological deficits due to the injection procedure.

[0117] All animals began the testing procedure at 4 weeks of age (approximately 4 times per week) using a standard rotarod mechanical system and dowel walking 1 week prior to the initial injection to allow for a training and acclimation period. For the dowel walking, a cylindrical 10 mm wooden dowel was suspended and the mouse was asked to walk (unassisted and unguided) from one end of the apparatus to the other. The time taken to walk, the number of slips during the walk, as well as any falls during the walk were recorded. The initial training period was not used in the data analysis. Following injection, animals were continued to be tested daily to monitor disease progression for overall balance, leg power, and tail stability, which have been shown to be measurable phenotypic responses to disease progression in this model (Huxley C, Passage E, Manson A, Putzu G, Figarella-Branger D, Pellissier JF, Fontes M. Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNA. Human molecular genetics. 1996;5(5):563-9. Epub 1996 / 05 / 01. doi:10.1093 / hmg / 5.5.563. PubMed PMID:8733121). As previously reported, untreated animals began to show significant gait impairment at 6-8 weeks of age.

[0118] All data (from both test groups) were analyzed and directly compared for all animals at 12 weeks of age, including the time required to walk the dowel, the number of slips, falls, hesitations, etc., and as the experiment progressed, untreated animals walked more slowly over time, slipping less, but clearly "grabbing" the dowel and walking more cautiously. Video evidence very clearly demonstrated significant behavioral differences between treated and untreated animals. Figure 9 shows a comparison of performance (phenotypic) data between the test animals and controls and wild types by comparing the amount of time it took to walk the dowel unassisted. The number of slips (and falls) on the dowel showed a similar trend. Five consecutive days of testing were analyzed at approximately 12 weeks of age, and the mean and standard deviation of the time to walk the dowel and the number of slips were calculated for all animals within the treatment group. Rotarod testing was also performed, but with much higher test-to-test variability. With the understanding that complete elimination of PMP22 would be detrimental (based on cellular data and previous knowledge of the biodistribution of similar molecules), the concentrations were tentatively selected to be low (3 mg / kg), medium (17 mg / kg), and high (50 mg / kg). At 17 mg / kg (5 weeks of age), all animals performed significantly better than the control group (highly evident from the video data), and at P3, animals in both groups performed similarly to wild-type animals.

[0119] In this study, animals were also tested on a commercially available rotarod system and the performance data are presented in Figure 10.

[0120] For both experiments, p-values ​​(relative to scrambled control animals) were calculated and are shown in FIGS.

[0121] From the data in Examples 2 and 3, it is clear that modification of PMP22 mRNA with SCH-012 as described in this disclosure exhibits the desired phenotypic effects in the animals used, i.e., improving locomotion and alleviating certain symptoms due to overproduction of PMP22 protein.

[0122] Surprisingly, this approach is much more effective than traditional knockdown approaches in reducing symptoms in CMT1A mouse models. For example, Figure 1D in Zhao et al.'s paper (The Journal of Clinical Investigation, Volume 128, No 1., January 2018, pp 359-368) shows that for the C22 humanized CMT1A mouse model (containing 7 copies of the human PMP22 gene), weekly injections of 100 mg / kg of the most effective ASO targeting the 3'UTR of the human PMP22 gene were required to obtain statistically significant results in the rotarod test. No statistical difference was obtained at 50 mg / kg weekly. For animals of the same starting age, our analysis showed significant improvement even in animals injected with a low dose of 3.3 mg / kg weekly. For animals starting treatment at a younger age, only 1 mg / kg was required (see Figures 9 and 10). Although the C3 animal model used in the present invention has only three copies of the PMP22 gene, whereas the C22 animals have seven copies (2.3 times more target RNA), 50 mg / kg is 15 times higher than the 3.3 mg / kg animals and 50 times higher than the 1 mg / kg animals. Clearly, the exon skipping approach described here offers a significant advantage over the 3'UTR strategy in terms of efficacy per dose.

[0123] Amelioration of significant disease-related pathology in axons is an important surrogate marker of efficacy. To determine whether the observed molecular changes in PMP22 translated into amelioration of axonal pathology, various axons from wild-type, treated and scramble-treated C3 mice were examined. The 17 mg / kg treatment group animals, wild-type animals and scramble control C3 animals were sacrificed at 20 weeks of age (6 weeks after cessation of treatment in the 17 mg / kg treatment group). Nerves (sciatic, peroneal, ulnar and tibial nerves) were removed from each animal and nerve sections were prepared for transmission electron microscopy (TEM) analysis.

[0124] Figure 17 (top) shows images of the sciatic nerve of WT, untreated and treated animals, and Figure 17 (bottom) shows images of the peroneal portion of the nerve.

[0125] FIG. 18 shows high magnification TEM images of a portion of the peroneal nerve from each animal.

[0126] Analysis of sections showed that SHC-012 treatment significantly improved the pathology of several nerves (shown here as the sciatic nerve) compared to scrambled-treated axons based on demyelination, thickness, structural integrity, and frequency of axonal necrosis. Although wild-type tissue still performed better than axons treated with SHC-012, it is clear that the ASO resulted in improvement of these key structural features of the disease. Similar results were observed when analyzing sections of the ulnar and tibial nerves, suggesting that the subcutaneous injection used (scruff of the neck on the back of the animal) was sufficient to effectively distribute the molecule throughout the animal's body and have a positive effect.

[0127] Next, the functional impact of such improvements was examined based on the improvement of myelin sheath. Electrophysiological tests were performed to measure the functional recovery of treated C3 mice. Improvements in CMAP and MUNE were detected in treated C3 mice (and control untreated C3 mice) of the same treatment group as above (Figure 19).

[0128] Figure 19 (top) shows electrophysiological plots of sciatic stomach slices from sedated mice. The table at the bottom of Figure 19 shows the average MUNE and CMAP for the three animals measured in each group. "Dowel walking time" (described below), a measure of general health, balance and mobility, is also included, showing that improvement in dowel walking ability tracks with improvement in electrophysiological measurements.

[0129] A notable specification of the envisioned CMT1A treatment is that each patient would only need to be treated 2-4 times per year. This is possible because PMOASO is nuclease resistant and extremely stable after uptake into cells. To examine the duration and persistence of SHC-012 activity, animals in the 1 mg / kg and 10 mg / kg treatment groups described above were monitored for 5 months after the last treatment (Figure 20). The half-life of the PMO molecule (once it reaches cells such as Schwann cells) is approximately 3-4 months in most tissues. Thus, it is hypothesized that the therapeutic effect will be long-lasting. Of note, C3 animals continued to perform well in the dock walking test 5 months after treatment, with little or no decline in activity 4 months (the study is ongoing) after the last treatment.

[0130] Results for both treatment groups are shown in Figure 20 (groups of 4-month-old scrambled and wild-type animals are shown for comparison). Each data set in the histogram is the average of the dowel walking time over 4 days at the end of each month; the last data set plotted is for 7-month-old animals.

[0131] Example 4 A number of ASOs were designed that skip exon 4 of the PMP22 RNA (FIGS. 11A and 11B). ASOs of 25 base pairs in length are shown, although shorter subsets are contemplated and disclosed. ASOs were designed for both the 5' and 3' regions. With reference to FIG. 3, such ASOs were designed for the 3' and 5' ends of exon 4. Selected ASOs were tested in a cellular assay (SHC-027 (SEQ ID NO:148), SHC-028 (SEQ ID NO:147), SHC-029 (SEQ ID NO:146), SHC-030 (SEQ ID NO:150), SHC-031 (SEQ ID NO:149) and SHC-032 (SEQ ID NO:151)) are shown in FIG. A large amount of exon 4 skipped product (and a reduction in full-length PMP22 mRNA) was observed and confirmed by sequencing.

[0132] It is important to be able to quantify the presence of both the exon-skipped product and full-length PMP22 (Figure 12). The bands in the gel were analyzed and the "activity" of the compounds was calculated by two methods (Figure 13). In the first method, the amount of full-length PMP22 mRNA in a standard sample (not shown in the figure) was compared to the resulting amount of full-length PMP22 mRNA. This is the only method possible with traditional knockout and RNA silencing methods (since only one measurable mRNA is present in the sample). The data from this method, due to experimental variability, resulted in the results shown in the first row at the bottom of Figure 13, i.e., SHC-027 had a negative activity (%) and SHC-032 had very low activity (<10%). Both compounds could be erroneously identified as ineffective or only slightly effective. However, by comparing the amount of full-length mRNA with the amount of exon-skipped mRNA (the lower band in the gel as confirmed by sequencing), we were able to determine the relative activity of the compounds very accurately (adjusting for the difference in sequence length between these two types of mRNA). Note that even though each lane on the gel is a completely separate run of cell assays (these are injections from different assays, not multiple injections from the same well), the standard deviation of this method is much smaller than the traditional PMP22 production reduction method. For the quantification method (full-length comparison) in the top row, the values ​​are simply the measured peak divided by the full-length standard peak. For the ratio method, the exon-skipped peak was divided by the total mRNA peak adjusted for size.

[0133] Example 5 A number of phosphorodiamidate morpholino oligomer PMOs (using morpholino backbones) were designed and synthesized that contain bridging moieties as described above, rather than a continuous sequence for the PMP22 pre-mRNA. The top of Figure 14 shows the 5' and 3' sequences of exon 3, flanked by an intro. There is a gap at the top (a space with the word " / " below it), indicating that the portion of exon 3 joining position 20 (G) and position 86 (C) has been omitted from the illustration. As shown, PMOs SHC-043 (SEQ ID NO:156), SHC-044 (SEQ ID NO:159), and SHC-045 (SEQ ID NO:162) were designed for both the 3' and 5' portions. These PMOs are continuous molecules (as can be seen in Figure 14) but hybridize to non-contiguous portions of the PMP22 pre-mRNA. Other bridge-type ASOs are also envisioned, provided they can hybridize to PMP22 pre-mRNA and induce both the production of exon-skipped mRNA products and the reduction of full-length mRNA.

[0134] These compounds were applied to cellular assays (as described above) to confirm their performance, and the results are shown in Figure 15, again showing both full-length and exon-skipped products that can be individually quantified to determine activity.

[0135] Example 6 A number of ASOs were designed that induce exon skipping of exon 2 of the PMP22 pre-mRNA (Figure 16). As disclosed elsewhere herein, shorter ASOs and mismatch ASOs are also envisioned. These ASOs were designed against the 3' and 5' ends of exon 2.

[0136] Example 7 In CMT1A, it is desirable to be able to monitor the activity of a given drug at the molecular level over time without having to sacrifice animals (during research trials) or perform extensive biopsies on human patients to access the tissue of interest and determine whether the pre-mRNA, mRNA, or protein of PMP22 is being affected. CMT1A is a disease in which the tissues in which the levels of PMP22 should be affected are peripheral nerve tissues and the myelin sheath sheets surrounding the tissues, and PMP22 protein is produced in large quantities primarily in Schwann cells in this region. Thus, to effectively measure the effect of a drug on Schwann cells, it is necessary to perform extensive tissue biopsies and / or sacrifice animals to evaluate such tissues. Such measurements are not practical or possible in clinical trials of drugs or in human patients undergoing treatment. Thus, measuring activity over time is not possible in humans and is difficult in animals. Laboratory experiments often involve large groups of animals being tested and their tissues examined with periodic sacrifice.

[0137] Although the majority of PMP22 pre-mRNA and mRNA are present in cells and tissues of interest, both substances are present in very small but detectable amounts in the bloodstream or other biological fluids that can be accessed without extensive biopsy or dissection. The substance in blood (although not synthesized there) is present in dead cells within the body, in RNA that leaks nonspecifically into the bloodstream. Other substances may be present in damaged cells or nonspecific cell waste. With traditional drug approaches in which total mRNA is reduced according to the drug's mechanism of action, it is unrealistic to measure even small decreases in total mRNA in the bloodstream due to the drug's effect, given the large background variability due to natural fluctuations (due to exercise, diet, metabolism, disease, injury, etc.). Such fluctuations in natural total mRNA can be so large even over a 24-hour period that it is unrealistic to quantify small decreases due to the drug. In certain embodiments, in the absence of an exon-skipping drug, the amount of exon-skipped PMP22 in the bloodstream is close to zero. In other words, if the amount of exon-skipped mRNA in the bloodstream is quantified, it will be directly proportional to the activity of the drug. For further quantification, the ratio of the amount of full-length PMP22 mRNA to the amount of exon-skipped mRNA in the bloodstream is compared at any time point to confirm and quantify the drug activity. For example, after one day of giving the drug to the patient, the drug has enough time to effectively distribute throughout the body and enter the desired tissues and cells. At that point, de novo production of PMP22 protein at the cellular level should be near maximum for a given drug composition and dose. The concentration of exon-skipped mRNA in the blood can be monitored periodically from the blood over a long period of time and correlated with the drug activity. More preferably, both full-length and exon-skipped mRNA are quantified and the ratio is correlated with the drug activity. This method provides an internal control for the measurements, simplifies the assay measurements, and improves accuracy.This value will decrease over time (indicating the formation of new drug-free cells or a decline in drug activity), at which point exon skipping can be re-equilibrated to the desired value by administering an additional dose of the selected drug.

[0138] The disclosed approach of measuring exon-skipped mRNA in blood also allows for adjustment of initial doses for different patients, and for initial dose escalation in clinical trials. In CMT1A (and other diseases), the variability of actual measurements (relative to net molecular biological activity) can be significant from patient to patient. Inaccurate results can lead to underdosing patients (i.e., falling outside the drug's active range) or to significantly overdosing patients (potentially causing dangerous side effects). Due to a number of factors, two different patients may require different amounts of drug to achieve the desired level of reduction in PMP22. For example, if it is desired to block 50% of PMP22 protein production, a smaller patient may require less drug than a heavier patient. Patients with different metabolisms and lifestyles may require different amounts of drug for each treatment to achieve optimal molecular changes. These measurements are made possible by generating stable, non-naturally occurring mRNA products in animals and patients (as described in this disclosure).

[0139] Example 8 An important consideration in the treatment of CMT is how to make the treatment effective in older patients who may have had more axonal damage than others due to the disease's long-term progression. C3 animals were grown to 3 months of age and monitored for their ambulatory ability to walk the hanging dowel as previously described. Prior to treatment, the amount of time it took to walk the dowel was monitored. The animals were then treated weekly with 17 mg / kg SCH-012 for 6 weeks, and then grown for an additional 6 weeks (without treatment) and their ambulatory ability was monitored again.

[0140] The results of this experiment are shown in Figure 21 (3 month old wild type animals are also plotted for comparison). As can be seen, this group of animals performed extremely poorly at 3 months of age (mean dowel walking time = 18.1 seconds, standard deviation = 7.1 seconds). After treatment and a recovery period, the animals improved significantly to a mean walking time = 7.1 seconds, standard deviation 2.4 seconds. Each bar on the graph shown represents the average value for these time points, with the animals measured over 4 days (every 2 days).

[0141] Example 9 Another group of C3 animals was allowed to grow untreated until 12 months of age. Wild-type animals were included in the study because at 12 months of age, wild-type animals also begin to show some decline in walking and balance abilities. At 12 months of age, C3 animals were treated (Table 1). Treatment groups 1, 2, 3, 5, 6, and 7 included 5 animals per group, and groups 4 and 5 included 4 animals. [Table 1]

[0142] Table 2 shows raw test performance results. For dowel walking time, the same apparatus was used as before, and the time (in seconds) for each animal was recorded on two consecutive days, with both days used to calculate group averages. For grip strength, each data set is the forepaw grip strength (25 Newton tension setting in grams) of the entire group tested on one day. [Table 2]

[0143] Table 3 shows the average percent improvement (compared to day 1 for each group individually) for the data in Table 2. For example, for the dowel walking time in group 2 (since a reduction in time is an improvement in walking), the animals in this group showed an average improvement in walking ability of 26% from pre-treatment to post-treatment. The percent improvement at each time point for animals in groups 4 and 5, who had two performance testing days (days 30 and 60), is compared to the percent improvement of the initial pre-test data. For example, the grip strength of animals in group 4 improved by 90% after the first treatment (from 1st to 2nd) and by 256% at day 60 compared to pre-treatment values. A positive increase in grip strength represents an improvement. [Table 3]

[0144] In this example, after sacrifice time points shown in Table 1, nerves were removed and subjected to quantitative PCR analysis to determine the total amount of human PMP22 mRNA present in the nerve. If SHC-012 is effective, there should be a decrease in the amount of human PMP22 mRNA compared to the scrambled treated animals. Table 4 shows the data for all animals. For percent reduction, the amount of PMP22 mRNA in the scrambled group was determined and the percent reduction for each treatment group (relative to the scrambled control) was calculated. [Table 4]

[0145] The performance data above were also monitored in wild-type animals to see if values ​​were affected by different treatment days. The values ​​in Table 3 show a small effect, but this is likely simply due to test variability (walking time improved 10%, grip strength deteriorated 7%). *****

[0146] Particular embodiments of the present disclosure may be defined in any of the following numbered paragraphs:

[0147] [Paragraph 1] A composition comprising an antisense oligonucleotide (ASO), wherein the ASO comprises or consists of a complementary region that is complementary to a target region of a pre-mRNA of PMP22 or is complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides; A composition in which the complementary region of the ASO binds to a target region of PMP22 pre-mRNA in a cell, inducing exon skipping during RNA transcription, thereby suppressing the production of full-length PMP22 mRNA and producing exon-skipped PMP22 mRNA.

[0148] [Paragraph 2] the ASO comprises or consists of a complementary region of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides that is complementary to a target region of the PMP22 pre-mRNA or is complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides; or The composition of paragraph 1, wherein the target region of PMP22 pre-mRNA comprises two separate segments of the PMP22 pre-mRNA, and optionally the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11 or 12 nucleotides that is complementary to a first segment of the contiguous sequence of the target region of PMP22 pre-mRNA or is complementary except for 1, 2 or 3 mismatched nucleotides, and the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11 or 12 nucleotides that is complementary to a second segment of the contiguous sequence of the target region of PMP22 pre-mRNA or is complementary except for 1, 2 or 3 mismatched nucleotides.

[0149] [Paragraph 3] The ASO comprises or consists of a complementary region of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 to about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides that are complementary to a target region of the pre-mRNA of PMP22 or are complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides; Optionally, the composition of paragraph 1 or 2, wherein the ASO comprises or consists of a complementary region of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides that are complementary to a target region of the PMP22 pre-mRNA or are complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides.

[0150] [paragraph 4] the ASO is a modified and / or synthetic oligonucleotide; Optionally, the composition of any one of paragraphs 1 to 3, wherein the ASO is a phosphorodiamidate morpholino oligomer (PMO).

[0151] [Paragraph 5] A composition described in any one of paragraphs 1 to 4, wherein the downstream exon is still expressed.

[0152] [Paragraph 6] A composition described in any one of paragraphs 1 to 4, wherein exon skipping renders the downstream exon out of frame.

[0153] [paragraph 7] The target region of the PMP22 pre-mRNA spans an intron / exon junction of one of the coding exons, Optionally, the exonic portion of the intron / exon junction includes exon 3 of PMP22, and / or Optionally, the composition of any one of paragraphs 1 to 6, wherein the exon portion of the intron / exon junction comprises exon 4 of PMP22.

[0154] [Paragraph 8] A composition described in any one of paragraphs 1 to 7, wherein the target region of the PMP22 pre-mRNA includes the 5' end of the exon.

[0155] [Paragraph 9] A composition described in any one of paragraphs 1 to 7, wherein the target region of the PMP22 pre-mRNA includes the 3' end of the exon.

[0156] [Paragraph 10] The target region of the pre-mRNA of PMP22 spans an intron / exon junction that includes or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an intron and a portion of an exon; Optionally, the composition of any one of paragraphs 7 to 9, wherein the target region of the pre-mRNA of PMP22 consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides of an intron and a portion of an exon.

[0157] [Paragraph 11] The target region of the pre-mRNA of PMP22 spans an intron / exon junction that includes or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of an exon and a portion of an intron, Optionally, the composition of any one of paragraphs 7 to 10, wherein the target region of the pre-mRNA of PMP22 consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides of an exon and a portion of an intron.

[0158] [Paragraph 12] A composition described in any one of paragraphs 1 to 11, wherein the target region of the pre-mRNA of PMP22 comprises or consists of SEQ ID NO:2 (exon 3, 5' end), SEQ ID NO:35 (exon 3, 3' end), SEQ ID NO:76 (exon 4, 5' end), SEQ ID NO:111 (exon 4, 3' end), SEQ ID NO:163 (exon 2, 5' end) and / or SEQ ID NO:198 (exon 2, 3' end), or a part or subset / fragment thereof.

[0159] [paragraph 13] the ASO comprises or consists of a complementary region that is complementary to, or is complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides of, SEQ ID NO:2 (exon 3, 5' end), SEQ ID NO:35 (exon 3, 3' end), SEQ ID NO:76 (exon 4, 5' end), SEQ ID NO:111 (exon 4, 3' end), SEQ ID NO:163 (exon 2, 5' end) and / or SEQ ID NO:198 (exon 2, 3' end); Optionally, the ASO comprises or consists of the nucleotide sequence of SEQ ID NOs:3-34 (exon 3, 5' end), SEQ ID NOs:37-70 (exon 3, 3' end), SEQ ID NOs:77-110 (exon 4, 5' end), SEQ ID NOs:112-145 (exon 4, 3' end), SEQ ID NOs:164-197 (exon 2, 5' end) or SEQ ID NOs:199-232 (exon 2, 3' end), or a fragment thereof sufficient to hybridize to a PMP22 pre-mRNA; or 13. The composition of paragraph 12, wherein the ASO comprises or consists of the nucleotide sequence of SEQ ID NO:3-34 (exon 3, 5' end), SEQ ID NO:37-70 (exon 3, 3' end), SEQ ID NO:77-110 (exon 4, 5' end), SEQ ID NO:112-145 (exon 4, 3' end), SEQ ID NO:164-197 (exon 2, 5' end) or SEQ ID NO:199-232 (exon 2, 3' end), except for having one, two or three nucleotide substitutions, or a fragment thereof sufficient to hybridize to the pre-mRNA of PMP22.

[0160] [Paragraph 14] ASO, SEQ ID NO:71(SHC-006 25-mer), SEQ ID NO:72(SHC-001 24-mer), SEQ ID NO:73(SHC-005 25-mer), SEQ ID NO:74(SHC-010 21-mer), SEQ ID NO:75(SHC-012 20-mer), SEQ ID NO:146(SHC-029 21-mer), SEQ ID NO:147(SHC-028 20-mer), SEQ ID NO:148(SHC-027 20-mer), SEQ ID NO:149(SHC-031 21-mer), SEQ ID NO:150(SHC-030 20-mer), SEQ ID NO:151(SHC-032 20-mer), SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO: 235, or comprising or consisting of the nucleic acid sequence of SEQ ID NO:238, Alternatively, the ASO has one, two or three nucleotide substitutions, SEQ ID NO:71(SHC-006 25-mer), SEQ ID NO:72(SHC-001 24-mer), SEQ ID NO:73(SHC-005 25-mer), SEQ ID NO:74(SHC-010 21-mer), SEQ ID NO:75(SHC-012 20-mer), SEQ ID NO:146(SHC-029 21-mer), SEQ ID NO:147(SHC-028 20-mer), SEQ ID NO:148(SHC-027 20-mer), SEQ ID NO:149(SHC-031 21-mer), SEQ ID NO:150(SHC-030 20-mer), SEQ ID NO:151(SHC-032 20-mer), SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO: 235, or The composition described in paragraph 1, comprising or consisting of the nucleic acid sequence of SEQ ID NO:238.

[0161] [Paragraph 15] A method for reducing the expression level of full-length mRNA of PMP22 in a cell, comprising administering to the cell a composition comprising the antisense oligonucleotide (ASO) described in any one of paragraphs 1 to 14, Optionally, an exon-skipped mRNA of PMP22 is produced, and / or Optionally, the method reduces the amount of functional PMP22 protein produced in the cell.

[0162] [paragraph 16] In response to the ASO, the amount of full-length PMP22 mRNA in the cell is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75% or 95%; and / or The method of paragraph 15, wherein the amount of PMP22 mRNA in the cell is reduced by about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or less in response to the ASO.

[0163] [Paragraph 17] The method of paragraphs 15 or 16, wherein the amount of full-length PMP22 mRNA in the cell is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or 75% to about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% depending on the ASO.

[0164] [Paragraph 18] in response to the ASO, the amount of functional PMP22 protein in the cell is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75% or 95%; and / or The method of any one of paragraphs 15 to 17, wherein the amount of functional PMP22 protein in the cell is reduced by about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or less in response to the ASO.

[0165] [Paragraph 19] The method of paragraphs 17 or 18, wherein the amount of functional PMP22 protein in the cell is reduced by about any of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or 75% to about any of 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% depending on the ASO.

[0166] [Paragraph 20] A method according to any one of paragraphs 15 to 19, comprising targeting a junction between an intron and an exon in the pre-mRNA of PMP22.

[0167] [Paragraph 21] A method for producing exon-skipped pre-mRNA of PMP22, the method comprising administering to a cell a composition comprising an antisense oligonucleotide (ASO) described in any one of paragraphs 1 to 14.

[0168] [Paragraph 22] The expression level of full-length PMP22 mRNA in cells is reduced, Optionally, the method of paragraph 21, wherein the amount of functional PMP22 protein produced in the cell is reduced.

[0169] [Paragraph 23] The method of paragraph 21 or 22, wherein the method comprises targeting a junction between an intron and an exon in the pre-mRNA of PMP22.

[0170] [Paragraph 24] A method for treating Charcot-Marie-Tooth disease, comprising administering to a subject in need of said treatment a composition comprising an antisense oligonucleotide (ASO) described in any one of paragraphs 1 to 14.

[0171] [Paragraph 25] The method of Paragraph 24, wherein the ASO is administered as a pharma- ceutically acceptable salt.

[0172] [Paragraph 26] The method of paragraphs 24 or 25, wherein the ASO is administered in a pharma- ceutically acceptable carrier or diluent.

[0173] [Paragraph 27] A method according to any one of paragraphs 24 to 26, in which at least one symptom of the disease is alleviated.

[0174] [Paragraph 28] A method according to any one of Paragraphs 24 to 26, in which the rate of progression of at least one symptom of the disease is reduced.

[0175] [Paragraph 29] The method of any one of Paragraphs 24 to 28, wherein the dosing regimen for administering the composition is based on the age of the subject.

[0176] [Paragraph 30] The method of any one of Paragraphs 24 to 29, wherein the dosage regimen for administering the composition is based on the progression of the subject's symptoms.

[0177] [Paragraph 31] The method of any one of Paragraphs 24 to 30, wherein the dosing regimen for administering the composition is based on the total body weight of the subject.

[0178] [Paragraph 32] The method of any one of Paragraphs 24 to 31, wherein the dosing regimen for administering the composition is based on the physical capacity of the subject.

[0179] [Paragraph 33] A method according to any one of Paragraphs 24 to 32, wherein the subject is given a higher dose or loading dose of the composition for a period of time based on the severity of the symptoms and / or advanced age, which dose is later changed to a lower dose.

[0180] [paragraph 34] A composition comprising an antisense oligonucleotide (ASO), the ASO comprising or consisting of a complementary region that is complementary to a target region of a pre-mRNA of PMP22, or that is complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides; A composition, wherein the target region of the pre-mRNA of PMP22 comprises an intron / exon junction of one of the coding exons.

[0181] [Paragraph 35] the ASO comprises or consists of a complementary region of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides that is complementary to a target region of the PMP22 pre-mRNA or is complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides; or The composition of paragraph 34, wherein the target region of PMP22 pre-mRNA comprises two separate segments of the PMP22 pre-mRNA, and optionally the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11 or 12 nucleotides that is complementary to a first segment of the contiguous sequence of the target region of PMP22 pre-mRNA or is complementary except for 1, 2 or 3 mismatched nucleotides, and the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11 or 12 nucleotides that is complementary to a second segment of the contiguous sequence of the target region of PMP22 pre-mRNA or is complementary except for 1, 2 or 3 mismatched nucleotides.

[0182] [paragraph 36] the ASO comprises or consists of a complementary region of about any of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 to about any of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 contiguous nucleotides that is complementary to a target region of the PMP22 pre-mRNA or is complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides; Optionally, the composition described in paragraph 34 or 35, wherein the ASO comprises or consists of a complementary region of 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides that are complementary to a target region of a PMP22 pre-mRNA or are complementary except for 1, 2, 3, 4 or 5 mismatched nucleotides.

[0183] [Paragraph 37] the exonic portion of the intron / exon junction includes exon 3 of PMP22; and / or 37. The composition of any one of paragraphs 34 to 36, wherein the exon portion of the intron / exon junction comprises exon 4 of PMP22.

[0184] [Paragraph 38] A composition described in any one of Paragraphs 34 to 37, wherein the target region of the PMP22 pre-mRNA comprises the 3' end of the exon.

[0185] [Paragraph 39] A composition described in any one of Paragraphs 34 to 37, wherein the target region of the PMP22 pre-mRNA comprises the 5' end of the exon.

[0186] [paragraph 40] the target region of the pre-mRNA of PMP22 comprises an intron / exon junction comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides of an intron and a portion of an exon; Optionally, the composition of any one of paragraphs 34 to 39, wherein the target region of the pre-mRNA of PMP22 comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides of an intron and a portion of an exon.

[0187] [paragraph 41] the target region of the pre-mRNA of PMP22 contains an intron / exon junction that includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides of an exon and a portion of an intron, Optionally, the composition of any one of paragraphs 34 to 40, wherein the target region of the PMP22 pre-mRNA may comprise or consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides of an exon and a portion of an intron.

[0188] [paragraph 42] the ASO is a modified and / or synthetic oligonucleotide; Optionally, the composition of any one of paragraphs 34 to 41, wherein the ASO is a phosphorodiamidate morpholino oligomer (PMO).

[0189] [Paragraph 43] A composition described in any one of Paragraphs 34 to 42, wherein the target region of the pre-mRNA of PMP22 comprises or consists of SEQ ID NO:2 (exon 3, 5' end), SEQ ID NO:35 (exon 3, 3' end), SEQ ID NO:76 (exon 4, 5' end), SEQ ID NO:111 (exon 4, 3' end), SEQ ID NO:163 (exon 2, 5' end) and / or SEQ ID NO:198 (exon 2, 3' end).

[0190] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0191] References Krajewski KM,Lewis RA,Fuerst DR,Turansky C,Hinderer SR,Garbern J,Kamholz J,Shy ME.Neurological dysfunction and axonal degeneration in Charcot-Marie-Tooth disease type 1A.Brain :a journal of neurology.2000;123( Pt 7):1516-27.Epub 2000 / 06 / 27.doi:10.1093 / brain / 123.7.1516.PubMed PMID:10869062. Braathen GJ.Genetic epidemiology of Charcot-Marie-Tooth disease.Acta Neurol Scand Suppl.2012(193):iv-22.Epub 2012 / 11 / 01.doi:10.1111 / ane.12013.PubMed PMID:23106488. Patel PI,Roa BB,Welcher AA,Schoener-Scott R,Trask BJ,Pentao L,Snipes GJ,Garcia CA,Francke U,Shooter EM,Lupski JR,Suter U.The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A.Nature genetics.1992;1(3):159-65.Epub 1992 / 06 / 01.doi:10.1038 / ng0692-159.PubMed PMID:1303228. Timmerman V,Nelis E,Van Hul W,Nieuwenhuijsen BW,Chen KL,Wang S,Ben Othman K,Cullen B,Leach RJ,Hanemann CO,et al.The peripheral myelin protein gene PMP-22 is contained within the Charcot-Marie-Tooth disease type 1A duplication.Nature genetics.1992;1(3):171-5.Epub 1992 / 06 / 01.doi:10.1038 / ng0692-171.PubMed PMID:1303230. Valentijn LJ,Bolhuis PA,Zorn I,Hoogendijk JE,van den Bosch N,Hensels GW,Stanton VP,Jr.,Housman DE,Fischbeck KH,Ross DA,et al.The peripheral myelin gene PMP-22 / GAS-3 is duplicated in Charcot-Marie-Tooth disease type 1A.Nature genetics.1992;1(3):166-70.Epub 1992 / 06 / 11.doi:10.1038 / ng0692-166.PubMed PMID:1303229. Matsunami N,Smith B,Ballard L,Lensch MW,Robertson M,Albertsen H,Hanemann CO,Muller HW,Bird TD,White R,et al.Peripheral myelin protein-22 gene maps in the duplication in chromosome 17p11.2 associated with Charcot-Marie-Tooth 1A.Nature genetics.1992;1(3):176-9.Epub 1992 / 06 / 01.doi:10.1038 / ng0692-176.PubMed PMID:1303231. Lee S,Amici S,Tavori H,Zeng WM,Freeland S,Fazio S,Notterpek L.PMP22 is critical for actin-mediated cellular functions and for establishing lipid rafts.The Journal of neuroscience :the official journal of the Society for Neuroscience.2014;34(48):16140-52.Epub 2014 / 11 / 28.doi:10.1523 / JNEUROSCI.1908-14.2014.PubMed PMID:25429154;PMCID:PMC4244477. Mittendorf KF,Marinko JT,Hampton CM,Ke Z,Hadziselimovic A,Schlebach JP,Law CL,Li J,Wright ER,Sanders CR,Ohi MD.Peripheral myelin protein 22 alters membrane architecture.Sci Adv.2017;3(7):e1700220.Epub 2017 / 07 / 12.doi:10.1126 / sciadv.1700220.PubMed PMID:28695207;PMCID:PMC5498104. Snipes GJ,Suter U,Welcher AA,Shooter EM.Characterization of a novel peripheral nervous system myelin protein(PMP-22 / SR13).The Journal of cell biology.1992;117(1):225-38.Epub 1992 / 04 / 01.doi:10.1083 / jcb.117.1.225.PubMed PMID:1556154;PMCID:PMC2289391. Magyar JP,Martini R,Ruelicke T,Aguzzi A,Adlkofer K,Dembic Z,Zielasek J,Toyka KV,Suter U.Impaired differentiation of Schwann cells in transgenic mice with increased PMP22 gene dosage.The Journal of neuroscience :the official journal of the Society for Neuroscience.1996;16(17):5351-60.Epub 1996 / 09 / 01.PubMed PMID:8757248;PMCID:PMC6578876. Sereda M,Griffiths I,Puhlhofer A,Stewart H,Rossner MJ,Zimmerman F,Magyar JP,Schneider A,Hund E,Meinck HM,Suter U,Nave KA.A transgenic rat model of Charcot-Marie-Tooth disease.Neuron.1996;16(5):1049-60.Epub 1996 / 05 / 01.doi:10.1016 / s0896-6273(00)80128-2.PubMed PMID:8630243. Huxley C,Passage E,Manson A,Putzu G,Figarella-Branger D,Pellissier JF,Fontes M.Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNA.Human molecular genetics.1996;5(5):563-9.Epub 1996 / 05 / 01.doi:10.1093 / hmg / 5.5.563.PubMed PMID:8733121. Perea J,Robertson A,Tolmachova T,Muddle J,King RH,Ponsford S,Thomas PK,Huxley C.Induced myelination and demyelination in a conditional mouse model of Charcot-Marie-Tooth disease type 1A.Human molecular genetics.2001;10(10):1007-18.Epub 2001 / 05 / 02.doi:10.1093 / hmg / 10.10.1007.PubMed PMID:11331611. Chance PF,Alderson MK,Leppig KA,Lensch MW,Matsunami N,Smith B,Swanson PD,Odelberg SJ,Disteche CM,Bird TD.DNA deletion associated with hereditary neuropathy with liability to pressure palsies.Cell.1993;72(1):143-51.Epub 1993 / 01 / 15.doi:10.1016 / 0092-8674(93)90058-x.PubMed PMID:8422677. Zhao HT,Damle S,Ikeda-Lee K,Kuntz S,Li J,Mohan A,Kim A,Hung G,Scheideler MA,Scherer SS,Svaren J,Swayze EE,Kordasiewicz HB.PMP22 antisense oligonucleotides reverse Charcot-Marie-Tooth disease type 1A features in rodent models.The Journal of clinical investigation.2018;128(1):359-68.Epub 2017 / 12 / 05.doi:10.1172 / JCI96499.PubMed PMID:29202483;PMCID:PMC5749515. Boutary,Suzan et al.「Squalenoyl siRNA PMP22 nanoparticles are effective in treating mouse models of Charcot-Marie-Tooth disease type 1 A.」 Communications biology vol.4,1 317.9 Mar.2021,doi:10.1038 / s42003-021-01839-2. Cornett,Kayla M D et al.「Natural history of Charcot-Marie-Tooth disease during childhood.」Annals of neurology vol.82,3(2017):353-359.doi:10.1002 / ana.25009 18.Cortese,Andrea et al.「Targeted next-generation sequencing panels in the diagnosis of Charcot-Marie-Tooth disease.」Neurology vol.94,1(2020):e51-e61.doi:10.1212 / WNL.0000000000008672. Eichinger,Katy et al.「The Charcot-Marie-Tooth Functional Outcome Measure(CMT FOM).」Neurology vol.91,15(2018):e1381-e1384.doi:10.1212 / WNL.0000000000006323. Gautier, Benoit et al. "AAV2 / 9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1A." Nature communications vol. 12, 1 2356. 21 Apr. 2021, doi:10.1038 / s41467-021-22593-3 21. Kagiava, A et al. "Intrathecal gene therapy in mouse models expressing CMT1X mutations." Human molecular genetics vol. 27, 8 (2018): 1460-1473. doi:10.1093 / hmg / ddy056. Lee, Ji-Su et al. "Targeted PMP22 TATA-box editing by CRISPR / Cas9 reduces demyelinating neuropathy of Charcot-Marie-Tooth disease type 1A in mice." Nucleic acids research vol. 48, 1 (2020): 130-140. doi:10.1093 / nar / gkz1070. Massade, Liliane, and Charbel Massaad. "Antisense RNA Targeting PMP22 for the Treatment of Charcot Marie-Tooth 1A Disease." EPO Patent 18306241. 3. 1 Apr. 2020. Morelli,Kathryn H et al.「Allele-specific RNA interference prevents neuropathy in Charcot-Marie-Tooth disease type 2D mouse models.」The Journal of clinical investigation vol.129,12(2019):5568-5583.doi:10.1172 / JCI130600. Reynolds,Angela et al.「Rational siRNA design for RNA interference.」Nature biotechnology vol.22,3(2004):326-30.doi:10.1038 / nbt936 26.Sandelius,Asa et al.「Plasma neurofilament light chain concentration in the inherited￥peripheral neuropathies.」Neurology vol.90,6(2018):e518-e524.doi:10.1212 / WNL.0000000000004932. Schorling,Elisabeth et al.「Cost of illness in Charcot-Marie-Tooth neuropathy:Results from Germany.」Neurology vol.92,17(2019):e2027-e2037.doi:10.1212 / WNL.0000000000007376 Stavrou,Marina et al.「Emerging Therapies for Charcot-Marie-Tooth Inherited Neuropathies.」International journal of molecular sciences vol.22,11 6048.3 Jun.2021,doi:10.3390 / ijms22116048. Stavrou,Marina et al.「Genetic mechanisms of peripheral nerve disease.」Neuroscience letters vol.742(2021):135357.doi:10.1016 / j.neulet.2020.135357. Tozza,S et al.「Motor performance deterioration accelerates after 50 years of age in Charcot-Marie-Tooth type 1A patients.」European journal of neurology vol.25,2(2018):301-306.doi:10.1111 / ene.13494. Wang,Hongge et al.「MicroRNAs as Biomarkers of Charcot-Marie-Tooth Disease Type 1A.」Neurology vol.97,5(2021):e489-e500.doi:10.1212 / WNL.0000000000012266. Wang,Hongge et al.「Transmembrane protease serine 5:a novel Schwann cell plasma marker for CMT1A.」Annals of clinical and translational neurology vol.7,1(2020):69-82.doi:10.1002 / acn3.50965. Passage E, Norreel JC, Noack-Fraissignes P, Sanguedolce V, Pizant J, Thirion X, Robaglia-Schlupp A, Pellissier JF, Fontes M. Ascorbic acid treatment corrects the phenotype of a mouse model of Charcot-Marie-Tooth disease. Nat Med. 2004;10(4):396-401. doi:10.1038 / nm1023. PubMed PMID: 15034573. Gess B, Baets J, De Jonghe P, Reilly MM, Pareyson D, Young P. Ascorbic acid for the treatment of Charcot-Marie-Tooth disease. Cochrane Database Syst Rev. 2015(12):CD011952. Epub 2015 / 12 / 15. doi:10.1002 / 14651858.CD011952. PubMed PMID: 26662471; PMCID: PMC6823270. Pareyson D, Reilly MM, Schenone A, Fabrizi GM, Cavallaro T, Santoro L, Vita G, Quattrone A, Padua L, Gemignani F, Visioli F, Laura M, Radice D, Calabrese D, Hughes RA, Solari A, Cmt T, groups C-T. Ascorbic acid in Charcot-Marie-Tooth disease type 1A(CMT-TRIAAL and CMT-TRAUK): a double-blind randomised trial. Lancet Neurol. 2011;10(4):320-8. doi:10.1016 / S1474-4422(11)70025-4. PubMed PMID: 21393063; PMCID: PMC3154498. Pareyson D,Schenone A,Fabrizi GM,Santoro L,Padua L,Quattrone A,Vita G,Gemignani F,Visioli F,Solari A,Group C-T.A multicenter,randomized,double-blind,placebo-controlled trial of long-term ascorbic acid treatment in Charcot-Marie-Tooth disease type 1A(CMT-TRIAAL):the study protocol [EudraCT no.:2006-000032-27].Pharmacological research :the official journal of the Italian Pharmacological Society.2006;54(6):436-41.Epub 2006 / 10 / 13.doi:10.1016 / j.phrs.2006.09.001.PubMed PMID:17029975. Meyer zu Horste G,Prukop T,Liebetanz D,Mobius W,Nave KA,Sereda MW.Antiprogesterone therapy uncouples axonal loss from demyelination in a transgenic rat model of CMT1A neuropathy.Ann Neurol.2007;61(1):61-72.doi:10.1002 / ana.21026.PubMed PMID:17262851. Sereda MW,Meyer zu Horste G,Suter U,Uzma N,Nave KA.Therapeutic administration of progesterone antagonist in a model of Charcot-Marie-Tooth disease(CMT-1A).Nat Med.2003;9(12):1533-7.doi:10.1038 / nm957.PubMed PMID:14608378. Prukop T, Stenzel J, Wernick S, Kungl T, Mroczek M, Adam J, Ewers D, Nabirotchkin S, Nave KA, Hajj R, Cohen D, Sereda MW. Early short-term PXT3003 combinational therapy delays disease onset in a transgenic rat model of Charcot-Marie-Tooth disease 1A (CMT1A). PLoS One. 2019;14(1):e0209752. doi:10.1371 / journal.pone.0209752. PubMed PMID: 30650121; PMCID: PMC6334894. [Pharnext, the employer of Serguei Nabirotchkin, Rodolphe Hajj and Daniel Cohen. DC, RH, MWS, TP and KAN submitted a patent based on this work: Full patent name: Early treatment of CMT disease Number: US2018 / 0000813. MWS, TP and KAN act as consultants to Pharnext. PXT3003 achieved a Phase 3 trial in CMT1A adult patients. There are no further patents related to this study, or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.] Sociali G,Visigalli D,Prukop T,Cervellini I,Mannino E,Venturi C,Bruzzone S,Sereda MW,Schenone A.Tolerability and efficacy study of P2X7 inhibition in experimental Charcot-Marie-Tooth type 1A(CMT1A) neuropathy.Neurobiol Dis.2016;95:145-57.doi:10.1016 / j.nbd.2016.07.017.PubMed PMID:27431093. Verhamme C,King RH,ten Asbroek AL,Muddle JR,Nourallah M,Wolterman R,Baas F,van Schaik IN.Myelin and axon pathology in a long-term study of PMP22-overexpressing mice.Journal of neuropathology and experimental neurology.2011;70(5):386-98.Epub 2011 / 04 / 14.doi:10.1097 / NEN.0b013e318217eba0.PubMed PMID:21487305. Landis SC,Amara SG,Asadullah K,Austin CP,Blumenstein R,Bradley EW,Crystal RG,Darnell RB,Ferrante RJ,Fillit H,Finkelstein R,Fisher M,Gendelman HE,Golub RM,Goudreau JL,Gross RA,Gubitz AK,Hesterlee SE,Howells DW,Huguenard J,Kelner K,Koroshetz W,Krainc D,Lazic SE,Levine MS,Macleod MR,McCall JM,Moxley RT,3rd,Narasimhan K,Noble LJ,Perrin S,Porter JD,Steward O,Unger E,Utz U,Silberberg SD.A call for transparent reporting to optimize the predictive value of preclinical research.Nature.2012;490(7419):187-91.Epub 2012 / 10 / 13.doi:10.1038 / nature11556.PubMed PMID:23060188;PMCID:3511845. FDA.Current Good Manufacturing Practice for Phase 1 Investigational Drugs.2008. FDA.CMC Regulatory Considerations for Oligonucleotide Drug Products:FDA Perspective.2019. FDA.IND Submissions for Individualized Antisense Oligonucleotide Drug Products:Administrative and Procedural Recommendations.2020. Fledrich R,Schlotter-Weigel B,Schnizer TJ,Wichert SP,Stassart RM,Meyer zu Horste G,Klink A,Weiss BG,Haag U,Walter MC,Rautenstrauss B,PAULus W,Rossner MJ,Sereda MW.A rat model of Charcot-Marie-Tooth disease 1A recapitulates disease variability and supplies biomarkers of axonal loss in patients.Brain :a journal of neurology.2012;135(Pt 1):72-87.Epub 2011 / 12 / 23.doi:10.1093 / brain / awr322.PubMed PMID:22189569. Gautier B,Hajjar H,Soares S,Berthelot J,Deck M,Abbou S,Campbell G,Ceprian M,Gonzalez S,Fovet CM,Schutza V,Jouvenel A,Rivat C,Zerah M,Francois V,Le Guiner C,Aubourg P,Fledrich R,Tricaud N.AAV2 / 9-mediated silencing of PMP22 prevents the development of pathological features In a rat model of Charcot-Marie-Tooth disease 1 A.Nature communications.2021;12(1):2356.Epub 2021 / 04 / 23.doi:10.1038 / s41467-021-22593-3.PubMed PMID:33883545;PMCID:PMC8060274. Duchesne M,Danigo A,RICHARD L,Vallat JM,Attarian S,Gonnaud PM,Lacour A,Pereon Y,Stojkovic T,Nave KA,Bertrand V,Nabirotchkin S,Cohen D,Demiot C,Magy L.Skin Biopsy Findings in Patients With CMT1A:Baseline Data From the CLN-PXT3003-01 Study Provide New Insights Into the Pathophysiology of the Disorder.Journal of neuropathology and experimental neurology.2018;77(4):274-81.doi:10.1093 / jnen / nly001.PubMed PMID:29408953. Svaren J,Moran JJ,Wu X,Zuccarino R,Bacon C,Bai Y,Ramesh R,Gutmann L,Anderson DM,Pavelec D,Shy ME.Schwann cell transcript biomarkers for hereditary neuropathy skin biopsies.Annals of neurology.2019;85(6):887-98.Epub 20190422.doi:10.1002 / ana.25480.PubMed PMID:30945774;PMCID:PMC7050273. Wang H,Davison M,Wang K,Xia TH,Call KM,Luo J,Wu X,Zuccarino R,Bacha A,Bai Y,Gutmann L,Feely SME,Grider T,Rossor AM,Reilly MM,Shy ME,Svaren J.MicroRNAs as Biomarkers of Charcot-Marie-Tooth Disease Type 1A.Neurology.2021;97(5):e489-e500.Epub 20210524.doi:10.1212 / WNL.0000000000012266.PubMed PMID:34031204;PMCID:PMC8356381. Park NY,Kwak G,Doo HM,Kim HJ,Jang SY,Lee YI,Choi BO,Hong YB.Farnesol Ameliorates Demyelinating Phenotype in a Cellular and Animal Model of Charcot-Marie-Tooth Disease Type 1A.Curr Issues Mol Biol.2021;43(3):2011-21.Epub 20211113.doi:10.3390 / cimb43030138.PubMed PMID:34889893. Morrow JM,Evans MRB,Grider T,Sinclair CDJ,Thedens D,Shah S,Yousry TA,Hanna MG,Nopoulos P,Thornton JS,Shy ME,Reilly MM.Validation of MRC Centre MRI calf muscle fat fraction protocol as an outcome measure in CMT1A.Neurology.2018;91(12):e1125-e9.Epub 20180817.doi:10.1212 / WNL.0000000000006214.PubMed PMID:30120135;PMCID:PMC6161551. Scharner J,Qi S,Rigo F,Bennett CF,Krainer AR.Delivery of GalNAc-Conjugated Splice-Switching ASOs to Non-hepatic Cells through Ectopic Expression of Asialoglycoprotein Receptor.Mol Ther Nucleic Acids.2019;16:313-25.doi:10.1016 / j.omtn.2019.02.024.PubMed PMID:30965276;PMCID:PMC6453860. Brennan KM,Bai Y,Shy ME.Demyelinating CMT――what‘s known,what’s new and what‘s in store?Neuroscience letters.2015;596:14-26.Epub 20150124.doi:10.1016 / j.neulet.2015.01.059.PubMed PMID:25625223. Morrow JM,Sinclair CD,Fischmann A,Machado PM,Reilly MM,Yousry TA,Thornton JS,Hanna MG.MRI biomarker assessment of neuromuscular disease progression:a prospective observational cohort study.Lancet neurology.2016;15(1):65-77.Epub 2015 / 11 / 10.doi:10.1016 / S1474-4422(15)00242-2.PubMed PMID:26549782;PMCID:PMC4672173. Fridman V,Sillau S,Acsadi G,Bacon C,Dooley K,Burns J,Day J,Feely S,Finkel RS,Grider T,Gutmann L,Herrmann DN,Kirk CA,Knause SA,Laura M,Lewis RA,Li J,Lloyd TE,Moroni I,Muntoni F,Pagliano E,Pisciotta C,Piscosquito G,Ramchandren S,Saporta M,Sadjadi R,Shy RR,Siskind CE,Sumner CJ,Walk D,Wilcox J,Yum SW,Zuchner S,Scherer SS,Pareyson D,Reilly MM,Shy ME,Inherited Neuropathies Consortium-Rare Diseases Clinical Research N.A longitudinal study of CMT1A using Rasch analysis based CMT neuropathy and examination scores.Neurology.2020;94(9):e884-e96.Epub 2020 / 02 / 13.doi:10.1212 / WNL.0000000000009035.PubMed PMID:32047073;PMCID:PMC7238948.

Claims

1. A composition comprising an antisense oligonucleotide (ASO), wherein the ASO includes or comprises a complementary region that is complementary to the target region of PMP22 premRNA, or complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides. A composition in which, within a cell, the complementary region of the ASO binds to the target region of the pre-mRNA of the PMP22, thereby inducing exon skipping during RNA transcription, which suppresses the production of full-length mRNA of the PMP22 and produces exon-skipped mRNA of the PMP22.

2. The ASO contains or consists of a complementary region of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides that are complementary to the target region of the premRNA of PMP22, or complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides, or The composition according to claim 1, wherein the target region of the premRNA of PMP22 comprises two separate segments of the premRNA of PMP22, and optionally, the ASO comprises or consists of complementary regions of at least about 6, 8, 9, 10, 11, or 12 nucleotides that are complementary to a first segment of the contiguous sequence of the target region of the premRNA of PMP22, or complementary to it with the exception of 1, 2, or 3 mismatched nucleotides, and the ASO comprises or consists of complementary regions of at least about 6, 8, 9, 10, 11, or 12 nucleotides that are complementary to a second segment of the contiguous sequence of the target region of the premRNA of PMP22, or complementary to it with the exception of 1, 2, or 3 mismatched nucleotides.

3. The ASO contains or consists of a complementary region of nucleotides from approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 nucleotides to approximately 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides, which are complementary to the target region of the premRNA of PMP22, or complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides. The composition according to claim 1, wherein the ASO optionally includes or comprises a complementary region of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides that are complementary to the target region of the premRNA of PMP22, or complementary to it with the exception of 1, 2, 3, 4, or 5 mismatched nucleotides.

4. The ASO is a modified and / or synthetic oligonucleotide, The composition according to claim 1, wherein the ASO is optionally a phosphorodiamidate morpholino oligomer (PMO).

5. The composition according to claim 1, wherein the downstream exons are still expressed.

6. The composition according to claim 1, wherein the downstream exons are moved out of the frame by the exon skipping.

7. The target region of the PMP22 premRNA spans one intron / exon junction among the coding exons, Optionally, the exon portion of the intron / exon junction includes exon 3 of PMP22, and / or The composition according to claim 1, wherein the exon portion of the intron / exon junction optionally includes exon 4 of PMP22.

8. The composition according to claim 1, wherein the target region of the PMP22 premRNA includes the 5' end of an exon.

9. The composition according to claim 1, wherein the target region of the PMP22 premRNA includes the 3' end of an exon.

10. The target region of the PMP22 premRNA spans an intron / exon junction containing or consisting of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the intron and a portion of the exon. The composition according to claim 7, wherein the target region of the PMP22 premRNA optionally consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the intron and a portion of the exon.

11. The target region of the PMP22 premRNA spans an intron / exon junction comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the exon and a portion of the intron, The composition according to claim 7, wherein the target region of the PMP22 premRNA optionally consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the exon and a portion of the intron.

12. The composition according to claim 1, wherein the target region of the premRNA of PMP22 includes or comprises SEQ ID NO: 2 (exon 3, 5' end), SEQ ID NO: 35 (exon 3, 3' end), SEQ ID NO: 76 (exon 4, 5' end), SEQ ID NO: 111 (exon 4, 3' end), SEQ ID NO: 163 (exon 2, 5' end), and / or SEQ ID NO: 198 (exon 2, 3' end), or a part or subset / fragment thereof.

13. The ASO contains or consists of complementary regions that are complementary to SEQ ID NO: 2 (exon 3, 5' end), SEQ ID NO: 35 (exon 3, 3' end), SEQ ID NO: 76 (exon 4, 5' end), SEQ ID NO: 111 (exon 4, 3' end) and / or SEQ ID NO: 163 (exon 2, 5' end) and / or SEQ ID NO: 198 (exon 2, 3' end), or complementary except for 1, 2, 3, 4, or 5 mismatched nucleotides. Optionally, the ASO may contain or consist of nucleotide sequences of SEQ ID NO: 3-34 (exon 3, 5' end), SEQ ID NO: 37-70 (exon 3, 3' end), SEQ ID NO: 77-110 (exon 4, 5' end), SEQ ID NO: 112-145 (exon 4, 3' end), SEQ ID NO: 164-197 (exon 2, 5' end), or SEQ ID NO: 199-232 (exon 2, 3' end), or fragments thereof sufficient to hybridize to the premRNA of PMP22, or The composition according to claim 12, wherein the ASO comprises or consists of nucleotide sequences of SEQ ID NO: 3-34 (exon 3, 5' end), SEQ ID NO: 37-70 (exon 3, 3' end), SEQ ID NO: 77-110 (exon 4, 5' end), SEQ ID NO: 112-145 (exon 4, 3' end), SEQ ID NO: 164-197 (exon 2, 5' end), or SEQ ID NO: 199-232 (exon 2, 3' end), or fragments thereof sufficient to hybridize to premRNA of PMP22, except that the ASO has one, two, or three nucleotide substitutions.

14. The aforementioned ASO SEQ ID NO: 71 (SHC-006 25-mer), SEQ ID NO: 72 (SHC-001 24-mer), SEQ ID NO: 73 (SHC-005 25-mer), SEQ ID NO: 74 (SHC-010 21-mer), SEQ ID NO: 75 (SHC-012 20-mer), SEQ ID NO: 146 (SHC-029 21-mer), SEQ ID NO: 147 (SHC-028 20-mer), SEQ ID NO: 148 (SHC-027 20-mer), SEQ ID NO: 149 (SHC-031 21-mer), SEQ ID NO: 150 (SHC-030 20-mer), SEQ ID NO: 151 (SHC-032 20-mer), SEQ ID NO: 156, SEQ ID NO: 159, SEQ ID NO: 162, SEQ ID NO: 235, or It contains or consists of the nucleic acid sequence of SEQ ID NO:

238. Alternatively, the ASO may have one, two, or three nucleotide substitutions, SEQ ID NO: 71 (SHC-006 25-mer), SEQ ID NO: 72 (SHC-001 24-mer), SEQ ID NO: 73 (SHC-005 25-mer), SEQ ID NO: 74 (SHC-010 21-mer), SEQ ID NO: 75 (SHC-012 20-mer), SEQ ID NO: 146 (SHC-029 21-mer), SEQ ID NO: 147 (SHC-028 20-mer), SEQ ID NO: 148 (SHC-027 20-mer), SEQ ID NO: 149 (SHC-031 21-mer), SEQ ID NO: 150 (SHC-030 20-mer), SEQ ID NO: 151 (SHC-032 20-mer), SEQ ID NO: 156, SEQ ID NO: 159, SEQ ID NO: 162, SEQ ID NO: 235, or The composition according to claim 1, comprising or consisting of the nucleic acid sequence of SEQ ID NO:

238.

15. A composition comprising an antisense oligonucleotide (ASO) according to any one of claims 1 to 14, used in the treatment of Charcot-Marie-Tooth disease, Selectively, PMP22 exon-skipped mRNA is produced, and / or A composition that selectively reduces the amount of functional PMP22 protein produced within the cell.

Images