Antisense RNA targeting PMP22 for treatment of charcot-marie-tooth 1a disease

Abstract

To provide a solution for the treatment of CMT-1A disease.SOLUTION: The present invention relates to antisense RNAs targeting PMP22 and capable of inhibiting the expression of PMP22 in the cells by 40% to 60%, and a pharmaceutical composition comprising the antisense RNAs. The antisense RNAs are preferably siRNA and are preferably provided in the form of nanoparticles. The present invention also relates to the use of these antisense RNAs targeting PMP22 for the treatment of Charcot-Marie-Tooth 1A disease.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention relates to the treatment of Charcot-Marie-Tooth 1A (CMT-1A) disease.

Background Art

[0002] Background CMT-1A neuropathy is due to alterations in peripheral nerve myelin, resulting in demyelination and causing a severe and disabling disease. The myelin sheath that surrounds the peripheral nerve axons is formed by Schwann cells. They are essential for the rapid and accurate transmission of electrical impulses by saltatory conduction. The dominant subtype of Charcot-Marie-Tooth disease is CMT-1A, accounting for more than 50% of all patients. It is associated with a duplication of the middle part of chromosome 17p11.2, resulting in overexpression of the gene encoding the 22 kDa peripheral myelin protein (PMP22), which is the cause of the disease characterized by the loss of muscle and sensory functions.

[0003] Several attempts have been made to cure CMT-1A disease or at least improve its disabling symptoms.

[0004] One promising molecule was ascorbic acid. In an animal model of CMT-1A overexpressing PMP22, ascorbic acid treatment resulted in a substantial improvement in the disease phenotype and a decrease in the expression of PMP22. Unfortunately, clinical trials testing the effect of ascorbic acid supplementation in CMT-1A patients had no beneficial effect when compared to placebo. Therefore, there is no clinical evidence to date to support ascorbic acid treatment in adults with CMT-1A.

[0005] Anti-progesterone therapy was found to significantly increase muscle strength and prevent axonal loss in PMP22 transgenic rats, although it did not affect myelin sheath thickness. Unfortunately, currently available progesterone antagonists are too toxic to be safely administered to patients.

[0006] Neurotrophin-3 (NT3), a neurotrophic factor known to promote axonal growth, was tested in two animal models and in a pilot study involving eight CMT-1A patients and had desirable results.

[0007] However, there is currently no effective drug for CMT-1A, and supportive treatment is limited to physical therapy, orthotics, surgical treatment of skeletal and soft tissue abnormalities, and symptomatic drug treatment.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0010] Therefore, there is a need to provide a solution for the treatment of CMT-1A disease.

Means for Solving the Problem

[0011] Description of the Present Invention The inventors have found that antisense RNAs targeting PMP22, particularly those using RNA interference (RNAi), are very efficient for the treatment of Charcot-Marie-Tooth 1A. The inventors have actually shown that by administering small interfering RNAs (siRNAs) targeting PMP22 via the intravenous route, in a mouse model of Charcot-Marie-Tooth 1A disease in which PMP22 is overexpressed 1.5-fold in these mice, it is possible to recover the same locomotor movement and strength as wild-type mice (see Examples).

[0012] Importantly, the inventors selected antisense RNAs, particularly siRNAs, that are capable of inhibiting the expression of PMP22 only partially, preferably by 40% to 60%, to counteract the 50% overexpression of PMP22 resulting from the 1.5 Mb duplication on chromosome 17p11.2. The PMP22 protein must actually remain in the cells in an amount equivalent to that found in healthy individuals.

[0013] Furthermore, to demonstrate that siRNA PMP22 tightly regulates PMP22 expression, the inventors showed that administration of siRNA targeting the PMP22 gene to wild-type mice decreased the PMP22 protein level and, thus, induced neuropathy in these mice (data not shown).

[0014] The antisense RNAs according to the present invention, particularly siRNA PMP22, advantageously do not interfere with the expression of the P0 protein, which remains unchanged. The P0 protein is actually involved with PMP22 in the myelination process, and deregulation of the gene encoding the P0 protein is the cause of another type of CMT, such as CMT1B.

[0015] Importantly, the antisense RNA according to the present invention, particularly siRNA PMP22, does not affect the cell viability.

[0016] In order to protect and safely deliver antisense RNAs targeting PMP22, particularly siRNAs targeting PMP22, they can be vectorized and provided, for example, in the form of nanoparticles. For example, the antisense RNA can be conjugated to squalene, thereby forming nanoparticles containing the antisense RNA. These nanoparticles (also referred to as siRNA PMP22-SQ NPs) remain active after bioconjugation with squalene thanks to the modification only in the passenger sense strand. Advantageously, these nanoparticles have a size of approximately 180 nm and a low polydispersity index of 0.14 and are stable for 30 days, thereby enabling intravenous injection.

[0017] Most drugs assayed for the treatment of CMT-1A focus on adenylyl cyclase activity that indirectly affects PMP22 expression, whereas the antisense RNAs according to the present invention, particularly siRNAs, target the PMP22 gene itself and enable improvement of various aspects of the disease, such as motor activity, strength, and axonal regeneration.

[0018] Importantly, the therapeutic effect of the antisense RNAs, particularly siRNAs, provided by the present invention has been demonstrated in an animal model similar to human CMT-1A (particularly in mice having only one or two extra copies of the PMP22 gene) and under conditions replaceable in humans, using particularly intravenous administration of a low dose of antisense RNA (e.g., 2.5 mg / kg). Administering a high dose of siRNA can actually induce off-target effects. Moreover, the antisense RNAs according to the present invention provide a long-term therapeutic effect.

[0019] Therefore, a first object of the present invention is an antisense RNA targeting the mRNA encoding the PMP22 protein.

[0020] The antisense RNA preferably reduces the amount of PMP22 protein in cells by 40% to 60%, more preferably by 40% to 55%.

[0021] The antisense RNA can be selected from the group consisting of siRNA, shRNA, miRNA, dsRNA, and RNA species that can be cleaved in vivo to form siRNA.

[0022] The antisense RNA can be complementary to a part of (i) the sequence of SEQ ID NO: 9, (ii) SEQ ID NO: 11, or (iii) a naturally occurring variant of the sequence of SEQ ID NO: 9 or 11.

[0023] For example, the antisense RNA is (i) - nucleotides 989 to 1007 of the sequence of SEQ ID NO: 11, - nucleotides 970 to 988 of the sequence of SEQ ID NO: 9, - nucleotides 1721 to 1739 of the sequence of SEQ ID NO: 11, - nucleotides 1726 to 1744 of the sequence of SEQ ID NO: 9, - nucleotides 431 to 449 of the sequence of SEQ ID NO: 11, - nucleotides 429 to 447 of the sequence of SEQ ID NO: 9, - nucleotides 1805 to 1823 of the sequence of SEQ ID NO: 11, - nucleotides 1809 to 1827 of the sequence of SEQ ID NO: 9, - nucleotides 921 to 939 of the sequence of SEQ ID NO: 11, or - nucleotides 903 to 921 of the sequence of SEQ ID NO: 9 consisting of, contained in, or overlapping with a part thereof, or (ii) a part homologous to a part of (i) present in a naturally occurring variant and can be a nucleic acid complementary thereto.

[0024] The antisense RNA may comprise at least 10 consecutive nucleotides of a sequence selected from the group consisting of the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0025] The antisense RNA can be an siRNA that includes one or two single-stranded overhangs, such as two single-stranded 3' overhangs.

[0026] The antisense RNA can be an siRNA that comprises (i) a sequence selected from the group consisting of the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and (ii) optionally, one or two single-stranded overhangs, particularly one or two single-stranded 3' overhangs.

[0027] Another object of the present invention is a nanoparticle comprising the antisense RNA as defined above, wherein the antisense RNA is preferably bound to squalene or a derivative thereof. The nanoparticle contains, for example, siRNA, and the sense strand of the siRNA is preferably bound to squalene or a derivative thereof.

[0028] Another object of the present invention is a pharmaceutical composition comprising or consisting of the antisense RNA targeting the mRNA encoding the PMP22 protein as defined above and a pharmaceutically acceptable carrier.

[0029] The pharmaceutically acceptable carrier can be conjugated with the antisense RNA, for example, with the sense strand of the antisense RNA. Such a pharmaceutically acceptable carrier can be a precursor of cholesterol, such as squalene, PEG, phospholipid, or a lipophilic moiety.

[0030] Yet another object of the present invention is the antisense RNA targeting the mRNA encoding the PMP22 protein as defined above for use in the treatment of Charcot-Marie-Tooth 1A (CMT-1A). Thus, the antisense RNA enables the restoration of normal levels of PMP22 expression in patients suffering from Charcot-Marie-Tooth disease type 1A.

[0031] Antisense RNA can be provided in the form of nanoparticles containing the antisense RNA, such as the nanoparticles defined above.

[0032] Antisense RNA can be provided in the form of the pharmaceutical composition defined above.

[0033] Antisense RNA can be administered intravenously, intraperitoneally, subcutaneously, or intranervously, for example, to the sciatic nerve.

[0034] Antisense RNA can be used in combination with at least one other drug useful in the treatment of Charcot-Marie-Tooth 1A.

[0035] PMP22 protein The PMP22 protein, also known as peripheral myelin protein 22, is a transmembrane glycoprotein.

[0036] The human PMP22 protein is encoded by the PMP22 gene.

[0037] The PMP22 protein is mainly expressed in Schwann cells.

[0038] The PMP22 protein is preferably of mammalian origin.

[0039] The term "mammal" includes humans and non-human mammals.

[0040] The expression "non-human mammal" includes, for example, rats, mice, pigs, cats, dogs, rabbits, or primates.

[0041] Charcot-Marie-Tooth 1A (CMT1A) disease is caused by a 1.5 Mb duplication on chromosome 17p11.2 containing the PMP22 coding gene, resulting in the presence of three copies of PMP22 in all individuals with CMT1A.

[0042] The PMP22 protein involved in CMT1A disease is a functional PMP22 protein.

[0043] As used herein, "functional PMP22 protein" means a protein encoded by a gene that can increase PMP22 expression (when duplication occurs at the DNA level) at both the mRNA level (tested by real-time polymerase chain reaction after RNA extraction followed by reverse transcription (RT-qPCR)) and the protein level (tested by Western blot and immunohistochemistry).

[0044] One of ordinary skill in the art can readily determine whether a given PMP22 protein is functional by well-known methods. For example, a proband with a family history combined with a very slow nerve conduction velocity, or DNA diagnosis, RT-qPCR, Western blot, and / or electrophysiological tests can be used.

[0045] The reference sequence for human PMP22 is, for example, the sequence of SEQ ID NO: 10.

[0046] The PMP22 protein can comprise or consist of a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, such as at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 10.

[0047] The reference sequence for the mRNA encoding human PMP22 is the sequence of SEQ ID NO: 9 in which the nucleotide "T" is replaced by "U".

[0048] The mRNA encoding the human PMP22 protein comprises or consists of a sequence that is at least 80% identical to the sequence of SEQ ID NO: 9 in which the nucleotide "T" is replaced by "U", preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, for example at least 96%, at least 97%, at least 98%, or at least 99% identical.

[0049] The reference sequence for the mRNA encoding mouse PMP22 is the sequence of SEQ ID NO: 11 in which the nucleotide "T" is replaced by "U".

[0050] The mRNA encoding the mouse PMP22 protein comprises or consists of a sequence that is at least 80% identical to the sequence of SEQ ID NO: 11 in which the nucleotide "T" is replaced by "U", preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, for example at least 96%, at least 97%, at least 98%, or at least 99% identical.

[0051] As defined herein, an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" to a reference sequence may contain mutations such as deletions, insertions, and / or substitutions when compared to the reference sequence.

[0052] In the case of substitutions, the substitutions preferably correspond to conservative substitutions shown in Table 1 below. In a preferred embodiment, a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence differs from the reference sequence only by conservative substitutions.

[0053]

Table 1

[0054] An amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may correspond to a naturally occurring variant of the reference sequence.

[0055] An amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may correspond to a homologous sequence derived from a mammalian species other than the species from which the reference sequence is derived.

[0056] For example, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may differ from the reference sequence only by conservative substitutions, and / or may correspond to a naturally occurring variant of the reference sequence, and / or may correspond to a homologous sequence derived from a mammalian species other than the species from which the reference sequence is derived.

[0057] By "a sequence that is at least x% identical to a reference sequence", it is intended that the sequence is identical to the reference sequence or differs from the reference sequence by at most 100 - x amino acid changes per 100 amino acids of the reference sequence.

[0058] The determination of alignment and percentage identity can be performed manually or automatically using, for example, the needle program based on the Needleman and Wunsch algorithm described in Needleman and Wunsch (1970) J. Mol Biol. 48:443 - 453, with, for example, the following parameters for polypeptide sequence comparison: comparison matrix: BLOSUM62, gap open penalty: 10 and gap extension penalty: 0.5, end gap penalty: false, end gap open penalty = 10, end gap extension penalty = 0.5; and the following parameters for polynucleotide sequence comparison: comparison matrix: DNAFULL; gap open penalty = 10, gap extension penalty = 0.5, end gap penalty: false, end gap open penalty = 10, end gap extension penalty = 0.5.

[0059] A reference sequence and a nucleic sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" may include mutations such as deletions, insertions, and / or substitutions when compared to the reference sequence.

[0060] In the case of nucleotide substitution, the substitution may correspond to a silent substitution as shown in Table 1 above, for example, or a substitution that leads to a conservative substitution in the translated amino acid sequence when compared to the reference sequence.

[0061] A nucleic sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may correspond to a naturally occurring variant of the reference sequence and / or a homologous sequence derived from a mammalian species other than the reference sequence.

[0062] A nucleic sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may differ from the reference sequence only by substitutions that lead to silent substitutions and / or conservative amino acid substitutions, and / or may correspond to a naturally occurring variant of the reference sequence, and / or may correspond to a homologous sequence derived from a mammalian species other than the reference sequence.

[0063] Antisense RNA targeting mRNA encoding PMP22 protein The present invention relates in particular to antisense RNA targeting mRNA encoding PMP22 protein.

[0064] The PMP22 protein and the mRNA encoding PMP22 are as defined above in particular.

[0065] Antisense RNA targeting mRNA encoding PMP22 protein is hereinafter referred to as "antisense RNA".

[0066] Antisense RNA advantageously reduces the amount of PMP22 protein in cells, particularly in Schwann cells.

[0067] The antisense RNA preferably reduces the amount of PMP22 protein in cells, preferably in Schwann cells, by 40% to 60%, preferably 40% to 55%, more preferably 40% to 50%, such as 40% to 45% or 45% to 50%, and / or reduces the amount of PMP22 mRNA in cells, preferably in Schwann cells, by 40% to 60%, preferably 40% to 55%, preferably 40% to 50%, such as 40% to 45% or 45% to 50%.

[0068] The amount of PMP22 protein expressed in cells can be determined by any method well known to those skilled in the art, such as Western blot, Elisa (enzyme-linked immunosorbent assay) or immunohistochemistry.

[0069] Determining whether the antisense RNA can reduce the amount of PMP22 in cells and, optionally, quantifying the percentage of reduction can be carried out, for example, by assaying the amount of PMP22 protein in cells in the presence and absence of the antisense RNA to be tested (see, for example, the Examples section).

[0070] The amount of mRNA encoding PMP22 present in cells can be determined by any method well known to those skilled in the art, such as RT-PCR.

[0071] Determining whether the antisense RNA can reduce the amount of mRNA encoding PMP22 in cells and, optionally, quantifying the percentage of reduction can be carried out, for example, by assaying the amount of mRNA encoding PMP22 in cells by, for example, RT-PCR in the presence and absence of the antisense RNA to be tested (see, for example, the Examples section).

[0072] Therefore, the antisense RNA is a nucleic acid targeting the mRNA encoding PMP22.

[0073] As used herein, the expression "nucleic acid targeting a given mRNA" means a nucleic acid that can specifically bind to the mRNA. Thus, a nucleic acid targeting a given mRNA comprises or consists of a sequence that is completely complementary to a portion of the sequence of the mRNA. Complementarity enables the specific binding of the nucleic acid targeting the mRNA to the mRNA under intracellular conditions.

[0074] As used herein, the expression "sequence completely complementary to a second sequence" means the complementary counterpart in the reverse orientation of the second sequence.

[0075] Antisense RNAs targeting the mRNA encoding PMP22 can be designed from the sequence of the mRNA, for example, using bioinformatics tools. For example, the sequence of SEQ ID NO: 9 or SEQ ID NO: 11 can be used as a target for designing antisense RNAs. Since the sequences of SEQ ID NO: 9 and SEQ ID NO: 11 are cDNA sequences, the nucleotide "T" has to be replaced by the nucleotide "U" to obtain the mRNA sequence.

[0076] The antisense RNAs defined above can be single-stranded or double-stranded RNAs (ribonucleic acids), such as siRNAs.

[0077] The antisense RNA is preferably completely complementary to a portion of the sequence of the mRNA encoding PMP22 in the subject to be treated.

[0078] The antisense RNAs defined above can contain at least one non-standard nucleotide, such as a nucleotide or deoxyribonucleotide that does not occur naturally.

[0079] The antisense RNAs defined above can contain or consist of an RNA portion and at least one additional portion, such as a deoxyribonucleotide portion.

[0080] The antisense RNA defined above can have a length of 12 to 50 nucleotides, 12 to 35 nucleotides, 12 to 30, 12 to 25, 12 to 22, 15 to 35, 15 to 30, 15 to 25, 15 to 22, or 18 to 22 nucleotides, for example 19, 20, or 21 nucleotides.

[0081] The antisense RNA defined above can include or consist of, for example, 12 to 50 consecutive nucleotides, 12 to 35, 12 to 30, 12 to 25, 12 to 22, 15 to 35, 15 to 30, 15 to 25, 15 to 22, or 18 to 22 nucleotides, for example 19, 20, or 21 nucleotides.

[0082] The antisense RNA defined above can include or consist of 12 to 50 consecutive nucleotides, for example 12 to 35, 12 to 30, 12 to 25, 12 to 22, 15 to 35, 15 to 30, 15 to 25, 15 to 22, or 18 to 22 nucleotides, for example 19, 20, or 21 consecutive nucleotides of a sequence complementary to the mRNA encoding the PMP22 protein.

[0083] The antisense RNA is preferably an RNA interference agent.

[0084] The antisense RNA defined above, particularly the RNA interference agent, can be selected from the group consisting of siRNA, shRNA, miRNA, dsRNA, and RNA species that can be cleaved in vivo to form siRNA.

[0085] "Short interfering RNA" or "siRNA" includes a double-stranded RNA portion and optionally one or two single-stranded overhangs.

[0086] The single-stranded overhang can be a 3' overhang or a 5' overhang.

[0087] The double-stranded RNA portion includes an antisense strand and a sense strand complementary to the mRNA encoding the PMP22 protein.

[0088] The siRNA defined above preferably contains one or two 3' overhangs.

[0089] The siRNA defined above preferably contains or consists of 19, 20, or 21 base pairs.

[0090] The siRNA defined above preferably contains or consists of 19, 20, or 21 base pairs and two 3' overhangs.

[0091] The 3' and / or 5' overhangs can consist of at least one, preferably at least two deoxyribonucleotides T (referred to as "dT").

[0092] For example, the 3' and / or 5' overhangs can consist of two deoxyribonucleotides T.

[0093] "Short hairpin RNA (shRNA)" is a single-stranded RNA having a stem-loop (hairpin) structure. Expression of shRNA in cells can be obtained using a vector.

[0094] "MicroRNA" or "miRNA" is a short non-coding RNA approximately 22 nucleotides in length. -miRNA is a post-transcriptional regulator of target genes and is generally expressed in a very tissue-specific or developmental stage-specific manner. Based on the characteristics of existing miRNA genes, it is possible to design and express artificial miRNAs.

[0095] "dsRNA" is an RNA having the same double-strand as DNA but having uracil instead of thymine. It forms the genetic material of many viruses and is a major component of the interferon system. It induces the immune system against viral infections.

[0096] The antisense RNA defined above may target a part of the sequence of SEQ ID NO: 9, the sequence of SEQ ID NO: 11, or a natural variant thereof, and the nucleotide "T" is replaced by the nucleotide "U" in these sequences.

[0097] In other words, the antisense RNA defined above may be complementary to a part of (i) the sequence of SEQ ID NO: 9, (ii) SEQ ID NO: 11, or (iii) a natural variant of the sequence of SEQ ID NO: 9 or 11.

[0098] An antisense RNA complementary to a part of a DNA sequence means herein that it is complementary to the corresponding RNA sequence in which the nucleotide "T" in the DNA sequence is replaced by the nucleotide "U".

[0099] The antisense RNA defined above may target a part of a sequence that is at least 80% identical to the sequence of SEQ ID NO: 9, preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, for example at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 9. For determining sequence identity, the nucleotide "T" is considered identical to the nucleotide "U".

[0100] In other words, the antisense RNA defined above may target a part of a sequence that is at least 80% identical to the sequence of SEQ ID NO: 11, preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, for example at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 11.

[0101] The antisense RNA defined above is (i) - nucleotides 989 to 1007 of the sequence of SEQ ID NO: 11, - nucleotides 970 to 988 of the sequence of SEQ ID NO: 9, - nucleotides 1721 to 1739 of the sequence of SEQ ID NO: 11, - nucleotides 1726 to 1744 of the sequence of SEQ ID NO: 9, - nucleotides 431 to 449 of the sequence of SEQ ID NO: 11, - nucleotides 429 to 447 of the sequence of SEQ ID NO: 9, - nucleotides 1805 to 1823 of the sequence of SEQ ID NO: 11, - nucleotides 1809 to 1827 of the sequence of SEQ ID NO: 9, - nucleotides 921 to 939 of the sequence of SEQ ID NO: 11, or - nucleotides 903 to 921 of the sequence of SEQ ID NO: 9 consisting of, contained within, or overlapping with a part thereof, or (ii) a part homologous to a part of (i) present in a naturally occurring variant may be complementary to.

[0102] The antisense RNA defined above may comprise at least 10 consecutive nucleotides, preferably at least 12 nucleotides, more preferably at least 15 nucleotides, such as 16, 17, 18, or 19 nucleotides, of a sequence selected from the group consisting of the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0103] The antisense RNA defined above may be an siRNA comprising (i) at least 10 consecutive nucleotides, preferably at least 12 nucleotides, more preferably at least 15 nucleotides, such as 16, 17, 18, or 19 nucleotides, of a sequence selected from the group consisting of the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and (ii) optionally, one or two single-stranded overhangs, such as 3' or 5' overhangs consisting of, for example, two dTs.

[0104] The antisense RNA defined above, preferably siRNA, may comprise or consist of (i) a sequence selected from the group consisting of the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and (ii) optionally, one or two single-stranded overhangs, such as one or two overhangs consisting of 2 dT, at the 3' and / or 5' ends.

[0105] The antisense RNA defined above is preferably siRNA comprising or consisting of (i) a sequence selected from the group consisting of the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and (ii) two 3' overhangs, each of which preferably consists of 2 dT.

[0106] The antisense RNA defined above may contain modified nucleotides, such as chemically modified nucleotides, which increase its stability and / or therapeutic efficiency in vivo.

[0107] For example, the antisense RNA defined above may contain phosphorothioate derivatives, 2'-O-(2-methoxyethyl) oligoribonucleotides, and / or lipid-modified oligonucleotides. When the antisense RNA contains a double-stranded RNA portion, the sense strand (also called the passenger strand) may contain the same modifications as the antisense strand, so that the modifications will not affect the inhibitory effect of the antisense strand. Alternatively, when the antisense RNA contains a double-stranded RNA portion, only the sense strand may contain modifications, such as dibenzocyclooctyne (DBCO) reactive groups at the 5' end of the sense strand, via a spacer such as N-(hexamethylenyl)-6-oxohexane amide spacer (C6).

[0108] The antisense RNA defined above, particularly shRNA, can be cloned into a vector and then sent into cells.

[0109] The antisense RNA defined above can be provided in a pharmaceutical composition.

[0110] Nanoparticles containing at least one antisense RNA The antisense RNA defined above can be provided in the form of nanoparticles containing the antisense RNA.

[0111] The antisense RNA can be carried by the nanoparticles.

[0112] By using the nanoparticles, it is possible to increase the half-life of the antisense RNA, particularly in the case of siRNA.

[0113] Furthermore, the nanoparticles can enable specific delivery to target cells, for example, by essentially targeting the target cells or by binding to a ligand specific to the target cells.

[0114] The target cells are, for example, Schwann cells.

[0115] For example, natural triterpenes such as squalene have the ability to self-associate like nanoparticles in water when linked to siRNA.

[0116] Vectorization using squalenic acid or its derivatives is disclosed, for example, in document WO2006 / 090029.

[0117] In one advantageous embodiment, the sense strand of the siRNA is covalently bound to azidosqualene or its derivative, which further anneals to the antisense strand of the siRNA, thereby forming nanoparticles, particularly after nano-precipitation.

[0118] The sense strand of the antisense RNA defined above, particularly the siRNA defined above, can be conjugated to squalene, for example, by using copper-free click chemistry as previously described (Massaad-Massade et al., Bioconjugate Chem., 2018, 29 (6), pp. 1961-1972, DOI: 10.1021 / acs.bioconjchem.8b00205).

[0119] The nanoparticles containing the siRNA defined above - a step of adding a dibenzocyclooctyne residue to the 5'-end of the sense strand of the siRNA to obtain a modified sense strand, - a step of conjugating squalene to the modified sense strand, particularly via bioconjugation of the azide functional group of squalene to the dibenzocyclooctyne residue, - a step of adding the antisense strand of the siRNA to anneal both strands of the siRNA, - a step of adding acetone and water to precipitate the nanoparticles, optionally, preferably under stirring, particularly by slowly adding one phase (aqueous or organic) to the other, and - a step of evaporating the acetone, optionally, for example, using a nitrogen flow, to obtain an aqueous suspension of pure siRNA-SQ nanoparticles can be obtained by a method comprising.

[0120] The nanoparticles can be purified, for example, by HPLC.

[0121] The nanoparticles preferably have a size lower than 300 nm, more preferably lower than 250 nm, and / or a size larger than 50 nm, more preferably larger than 100 nm.

[0122] For example, the nanoparticles have a size included in 100-200 nm, more preferably 170 nm-190 nm, for example, 180 nm.

[0123] The nanoparticles preferably have a low polydispersity index, particularly a polydispersity lower than 0.3 nm, more preferably lower than 0.2 nm, for example a polydispersity index of 0.14.

[0124] The nanoparticles are preferably stable for at least 20 days, preferably at least 25 days, more preferably at least 30 days.

[0125] The nanoparticles are preferably suitable for intravenous injection.

[0126] The nanoparticles may in particular contain or consist of siRNA comprising (i) a sequence selected from the group consisting of the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and (ii) two 3' overhangs preferably consisting of two dTs.

[0127] Nanoparticles containing antisense RNA can be provided in a pharmaceutical composition.

[0128] Pharmaceutical composition The present invention also relates to a pharmaceutical composition comprising or consisting of an antisense RNA targeting the mRNA encoding the PMP22 protein as defined above, in particular an siRNA targeting the mRNA encoding the PMP22 protein as defined above, and a pharmaceutically acceptable carrier.

[0129] Non-limiting examples of pharmaceutically acceptable carriers that can be conjugated with nucleic acids such as antisense RNA, for example with the sense strand of siRNA, include squalene, PEG, phospholipids, lipophilic moieties, P-glycoprotein inhibitors, etc., precursors of cholesterol.

[0130] Thus, the antisense RNA can be provided in the form of exosomes containing or conjugated with nucleic acids, liposomes containing or conjugated with nucleic acids, and / or nanoparticles containing or conjugated with nucleic acids, for example the nanoparticles as defined above.

[0131] The expression "pharmaceutically acceptable" is intended to encompass any carrier that does not interfere with the biological activity of the active ingredient and is preferably not harmful to the host to which it is administered.

[0132] Pharmaceutically acceptable carriers can be prepared by any method known to those skilled in the art.

[0133] The pharmaceutical composition is preferably a sterile solution or suspension.

[0134] The pharmaceutical composition is preferably suitable for injectable administration.

[0135] The pharmaceutical composition may contain, in the case of siRNA, a nucleic acid encoding an antisense RNA, or, particularly when the antisense RNA is part of an shRNA, a vector containing said nucleic acid.

[0136] The pharmaceutical composition may further contain at least one pharmaceutically acceptable excipient.

[0137] Suitable pharmaceutically acceptable excipients are well known to those skilled in the art. Pharmaceutically acceptable excipients can be routinely selected according to the mode of administration, the solubility and stability of the RNA antisense. For example, a pharmaceutical composition for intravenous administration may contain a sterile aqueous solution, buffers, diluents, and / or other suitable additives.

[0138] The pharmaceutical composition as defined above may contain an amount of RNA antisense suitable for administering from 0.1 mg of RNA antisense / kg of subject to 20 mg of RNA antisense / kg of subject, preferably from 0.2 mg / kg to 15 mg / kg, more preferably from 0.5 mg / kg to 10 mg / kg.

[0139] The pharmaceutical composition as defined above may contain, for example, from 5 mg to 2 g of RNA antisense / kg of subject, preferably from 15 mg to 1 g, more preferably from 30 mg to 500 mg.

[0140] When the antisense RNA binds to squalene or a derivative thereof, thereby forming nanoparticles, the pharmaceutical composition as defined above may contain an amount of nanoparticles suitable for administering from 0.1 mg of nanoparticles / kg of subject to 20 mg of nanoparticles / kg of subject, preferably from 0.2 mg / kg to 15 mg / kg, more preferably from 0.5 to 10 mg / kg.

[0141] When the antisense RNA binds to squalene or a derivative thereof, thereby forming nanoparticles, the pharmaceutical composition as defined above may contain from 5 mg to 2 g of nanoparticles, preferably from 15 mg to 1 g, more preferably from 30 mg to 500 mg.

[0142] In one embodiment, the pharmaceutical composition is presented in unit dosage forms that facilitate accurate dosing. The term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and other non-human mammalian subjects, each unit containing a predetermined quantity of the active substance calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions.

[0143] The present invention also relates to a kit comprising the pharmaceutical and instructions for use regarding the mode of administration as defined above. These instructions for use may indicate, for example, the medical indication, route of administration, dosage, and / or the group of patients to be treated.

[0144] Subject The subject can be a human or a non-human mammal.

[0145] Non-human mammals are, for example, mice, rats, cats, dogs, rabbits, or primates.

[0146] The subject is preferably a human, also referred to as an individual or a patient.

[0147] The subject can be of any age, such as infants, children, youths, adults, the elderly, and of any gender.

[0148] Treatment of Charcot-Marie-Tooth 1A disease As used herein, "treatment of Charcot-Marie-Tooth 1A disease" means at least partially arresting the progression of the disease or reversing the disease.

[0149] Desirable effects of treatment include, for example, - preventing or reducing muscle weakness and / or atrophy in the lower limbs, hand weakness, and / or numbness, thereby normalizing walking and / or preventing or reducing foot drop, - arresting, delaying, or curing muscle weakness and / or atrophy in the lower limbs, hand weakness, and / or numbness, thereby normalizing walking and / or reducing foot drop, and / or - normalizing nerve conduction velocity are included.

[0150] Antisense RNA targeting PMP22 for use in the treatment of Charcot-Marie-Tooth 1A disease The present invention also relates to an antisense RNA targeting the mRNA encoding the PMP22 protein for use in the treatment of Charcot-Marie-Tooth 1A (CMT-1A).

[0151] The present invention also relates to a method for treating Charcot-Marie-Tooth 1A disease, comprising administering to a subject in need thereof an antisense RNA targeting the mRNA encoding the PMP22 protein.

[0152] The antisense RNA targeting the mRNA encoding the PMP22 protein is as defined above, particularly as defined in the section of the same name.

[0153] The antisense RNA may be provided in the form of a pharmaceutical composition.

[0154] The pharmaceutical composition is as defined above, particularly as defined in the section of the same name.

[0155] The treatment of Charcot-Marie-Tooth 1A disease is as particularly defined above.

[0156] The step of administering an antisense RNA, particularly siRNA, targeting the mRNA encoding PMP22 can be carried out using various techniques well-known in the art, including naked administration and / or administration with a pharmaceutically acceptable carrier such as nanoparticles.

[0157] The nanoparticles containing the antisense RNA according to the present invention are as particularly defined above.

[0158] The antisense RNA can be formulated to target Schwann cells.

[0159] The antisense RNA is preferably complementary to the mRNA encoding PMP22 present in a subject suffering from or prone to CMT-1A.

[0160] The antisense RNA targeting the mRNA encoding PMP22 can be administered, for example, intravenously or intraperitoneally or subcutaneously or intraneurally, preferably to the sciatic nerve.

[0161] The antisense RNA targeting the mRNA encoding PMP22 is preferably administered in an "effective amount", i.e., an amount sufficient to treat Charcot-Marie-Tooth 1A disease. It will be understood that this amount will vary depending on both the effectiveness of the antisense RNA and the nature of any carrier used. The determination of the appropriate amount for any given composition is within the scope of skill in the art through a standard series of tests designed to assess appropriate treatment levels.

[0162] The antisense RNA targeting the mRNA encoding PMP22 can be administered at a dose of 0.1 to 20 mg / kg of the subject, preferably 0.2 to 15 mg / kg of the subject, more preferably 0.5 to 10 mg / kg of the subject.

[0163] When an antisense RNA targeting the mRNA encoding PMP22 is bound to squalene or a derivative thereof, 0.1 to 20 mg of nanoparticles / kg subject, preferably 0.2 to 15 mg of nanoparticles / kg subject, more preferably 0.5 to 10 mg of nanoparticles / kg subject can be administered.

[0164] The antisense RNA targeting the mRNA encoding PMP22 can be administered, for example, for at least 3 weeks and / or up to 3 months, for example, once or twice a week, as a single injection or multiple injections.

[0165] The antisense RNA can be used in combination with at least another drug useful in the treatment of Charcot-Marie-Tooth 1A, such as ascorbic acid, neurotrophin 3, and / or curcumin.

[0166] The present invention will be further illustrated in light of the following examples and figures.

[0167] Brief Description of Sequences SEQ ID NO: 1 is the sense strand sequence of an siRNA containing the antisense strand of SEQ ID NO: 12.

[0168] SEQ ID NO: 2 is the sense strand sequence of an siRNA containing the antisense strand of SEQ ID NO: 13.

[0169] SEQ ID NO: 3 is the sense strand sequence of an siRNA containing the antisense strand of SEQ ID NO: 14.

[0170] SEQ ID NO: 4 is the sense strand sequence of an siRNA containing the antisense strand of SEQ ID NO: 15.

[0171] SEQ ID NO: 5 is the sense strand sequence of an siRNA containing the antisense strand of SEQ ID NO: 16.

[0172] SEQ ID NO: 6 is the sense strand sequence of an siRNA containing the antisense strand of SEQ ID NO: 17.

[0173] SEQ ID NO: 7 is the sense strand sequence of siRNA containing the antisense strand of SEQ ID NO: 18.

[0174] SEQ ID NO: 8 is the sense strand sequence of siRNA containing the antisense strand of SEQ ID NO: 19.

[0175] SEQ ID NO: 9 is the cDNA sequence encoding the human PMP22 protein of reference sequence NM_000304.3, which was available on July 30, 2018.

[0176] SEQ ID NO: 10 is the amino acid sequence of the human PMP22 protein encoded by the sequence of SEQ ID NO: 9.

[0177] SEQ ID NO: 11 is a cDNA sequence whose sense strand is the cDNA encoding the mouse PMP22 protein of reference NM_008885.3 in the NCBI database, which was available on July 30, 2018.

[0178] SEQ ID NO: 12 is an RNA sequence that is completely complementary to nucleotides 474 - 492 of the sequence of SEQ ID NO: 11 and partially complementary to nucleotides 472 - 489 of the sequence of SEQ ID NO: 9 (with one mismatch regarding nucleotide 472).

[0179] SEQ ID NO: 13 is an RNA sequence that is completely complementary to nucleotides 923 - 941 of the sequence of SEQ ID NO: 11 and to nucleotides 905 - 923 of the sequence of SEQ ID NO: 9.

[0180] SEQ ID NO: 14 is an RNA sequence that is completely complementary to nucleotides 1562 - 1580 of the sequence of SEQ ID NO: 11 and partially complementary to nucleotides 1565 - 1583 of the sequence of SEQ ID NO: 9 (with five mismatches).

[0181] Array number 15 is an RNA sequence that is completely complementary to nucleotides 989 - 1007 of the sequence of array number 11 and partially complementary (with two mismatches) to nucleotides 970 - 988 of the sequence of array number 9.

[0182] Array number 16 is an RNA sequence that is completely complementary to nucleotides 1721 - 1739 of the sequence of array number 11 and completely complementary to nucleotides 1726 - 1744 of the sequence of array number 9.

[0183] Array number 17 is an RNA sequence that is completely complementary to nucleotides 431 - 449 of the sequence of array number 11 and completely complementary to nucleotides 429 - 447 of the sequence of array number 9.

[0184] Array number 18 is an RNA sequence that is completely complementary to nucleotides 1805 - 1823 of the sequence of array number 11 and completely complementary to nucleotides 1809 - 1827 of the sequence of array number 9.

[0185] Array number 19 is an RNA sequence that is completely complementary to nucleotides 921 - 939 of the sequence of array number 11 and completely complementary to nucleotides 903 - 921 of the sequence of array number 9.

Brief Description of the Drawings

[0186]

Figure 1

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Figure 9

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Figure 11

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Figure 14

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Figure 16

Figure 17

Figure 18

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Figure 22

Mode for Carrying Out the Invention

Examples

[0187] Examples Materials and Methods siRNA and Chemical Modifications The designed sequences of sense and antisense siRNA strands were purchased from Eurogentec, France. They were synthesized, then characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). Single-stranded RNAs were synthesized as 19-mers with two 3'-overhanging 2'-deoxynucleotide residues that provide stabilization against nucleases. To maintain functionality, a dibenzocyclooctyne (DBCO) reactive group was introduced at the 5'-end of the sense strand of each siRNA sequence via an N-(hexamethylenyl)-6-oxohexanamide spacer (C6). To generate siRNAs from the RNA single strands, equimolar amounts of both the sense and antisense strands were annealed in annealing buffer [30 mM HEPES-KOH (pH 7.4), 2 mM magnesium acetate, 100 mM potassium acetate] at 95°C for 3 minutes and then incubated at room temperature for 45 minutes before storage at -20°C.

[0188] Screening of siRNAs against PMP22 To restore basal levels of PMP22 gene expression, eight siRNAs against PMP22 (see Tables 2 and 3) and a siRNA control (siRNA CTRL) scramble sequence were designed by using three different methods (Tafer software, Thermofisher software, and the Reynolds method). The siRNAs were transfected into a Schwann cell model (MSC 80) that endogenously expresses PMP22 by using Lipofectamine iMAX™. The ability of the siRNAs to reduce PMP22 expression was tested at 24, 48, and 72 hours. The expression of both the PMP22 and P0 genes was assayed by real-time PCR. Then, the best siRNA sequence that can inhibit ~50% of PMP22 expression with a long-lasting effect without affecting P0 levels (P0 deregulation has been described to be involved in CMT-1 disease) was tested at various concentrations of 25 nM, 50 nM, and 100 nM to check its ability to reduce PMP22 and cell viability (MTT assay). Scrambled siRNA was used as a control.

[0189]

Table 2

[0190]

Table 3

[0191] Conjugation of siRNAs to squalene and polyisoprenyl chains: Squalene was conjugated to siRNA by the Huisgen cycloaddition of dibenzocyclooctyne (copper-free click chemistry) as previously described (Massaad-Massade et al., Bioconjugate Chem., 2018, 29 (6), 1961-1972, DOI: 10.1021 / acs.bioconjchem.8b00205). Briefly, the sense strand of the oligonucleotide was modified at the 5'-end with a dibenzocyclooctyne residue, a commercially available modification for siRNA. Squalene carried an azide functional group. The conjugate was purified by HPLC and characterized by MALDI-TOF mass spectrometry.

[0192] Annealing of both strands of siRNA PMP22 and siRNA CT was performed after bioconjugation of the sense strand to SQ and then precipitated in acetone / water. While stirring, one phase (aqueous or organic) was slowly added to the other. Acetone was completely evaporated using a nitrogen stream to obtain an aqueous suspension of pure siRNA-SQ nanoconjugates at a concentration of 10 μM. Control siRNA-SQ was prepared using the same protocol. The size of the nanoconjugates was determined by dynamic light scattering and their zeta potential by their electrophoretic mobility. The morphology of these squalene-based nanoconjugates was observed using cryo-transmission electron microscopy. Nanoconjugate stability and drug release were tested in phosphate buffer (PBS) and cell culture medium.

[0193] A detailed protocol for obtaining the siRNA PMP22-SQ conjugate is described below.

[0194] (i) Bioconjugation of siRNA 1 nmol of the 5'-end modified sense strand of siRNA PMP22, DBCO-C6 (1 mg / mL in DNAse / RNAse-free water), and 50 nmol of SQ-N3 (1 mg / mL in DMSO) were mixed in a glass vial containing DMSO (286 μL) and acetone (65 μL). The solution was then incubated at room temperature for 12 h with stirring to obtain the siRNA PMP22-SQ bioconjugate. The next day, the excess acetone was removed under a nitrogen flow for 30 min, followed by a lyophilization step.

[0195] Purification of the bioconjugate from the excess unconjugated SQ was carried out by RP-HPLC on a polymer column described below. The identity of the siRNA PMP22-SQ bioconjugate was confirmed by MALDI-TOF mass spectrometry. The purified product was lyophilized and then solubilized in RNAse-free water at a desired molar concentration of 10 μM. The same protocol was carried out to obtain the siRNA Ct-SQ bioconjugate.

[0196] (ii) Purification of the siRNA PMP22-SQ Bioconjugate by HPLC HPLC purification was carried out using a Thermo Scientific high-performance liquid chromatography system (Ultimate 3000) equipped with a photodiode array detector with a wavelength range of 190 - 800 nm, a pump, and a manual injector. The stationary phase consisted of a non-porous alkylated polystyrene divinylbenzene column (Hamilton PRP-3 10 μm, 4.6 × 250 mm, PEEK, Ref: 79574) protected by a pre-column (Hamilton). Thermo Fisher Chromeleon software was used for data collection with a flow rate of 1.2 mL / min and an injection volume of 100 μL. A gradient of mobile phases A and B was applied. Mobile phase A was composed of 5% acetonitrile, 90% water, and 0.2 M TEAA (5%) at pH 7.0, while mobile phase B consisted of 5% TEAA, 5% water, and 95% acetonitrile. The gradient applied for purification was as follows: 0 - 8 min, a linear gradient of 0% - 24% of phase B; 8 - 16 min, a linear gradient of 24% - 90% of phase B; 16 - 18 min, a linear gradient of 90% - 100% of phase B; 18 - 30 min, 100% of phase B; 30 - 32 min, a linear gradient from 100% of phase B to 100% of phase A; and 32 - 42 min, re-equilibration with 100% of phase A. The siRNA-SQ bioconjugate was purified by manual peak collection. Fractions corresponding to a fraction volume of 2.4 mL were collected for 2 min and then lyophilized. All lyophilized siRNA fractions were reconstituted in DEPC-treated water.

[0197] (iii) MALDI-TOF mass spectrometry The MALDI-TOF / TOF UltrafleXtreme mass spectrometer (Bruker Daltonics, Bremen) was used for all experiments. Mass spectra were acquired in linear positive ion mode. The laser intensity was set just above the ion generation threshold to obtain peaks with the highest possible signal-to-noise (S / N) ratio without significant peak broadening. All data were processed using the FlexAnalysis software package (Bruker Daltonics).

[0198] (iv) Annealing of siRNA-SQ bioconjugate to the antisense siRNA strand Following the manufacturer's protocol and in the same manner as previously mentioned for the generation of siRNA from a single RNA strand, annealing of both strands of siRNA PMP22 and siRNA Ct was performed after bioconjugation of the sense strand to SQ. Specifically, equimolar amounts of both siPMP22C6-SQ bioconjugate and antisense siPMP22 were mixed using annealing buffer [30 mM HEPES-KOH (pH 7.4), 2 mM Mg acetate, 100 mM K acetate], incubated at 95 °C for 3 minutes, and then incubated at room temperature for 45 minutes before storage at -80 °C or directly before precipitation. The same protocol was carried out to obtain the siRNA Ct-SQ bioconjugate.

[0199] (v) Preparation and characterization of siRNA PMP22-SQ nanoparticles siRNA PMP22-SQ and siRNA Ct-SQ nanoparticles (NPs) were prepared by nanoprecipitation in acetone:water (1:2). Under stirring, one phase was slowly added to the other, i.e., 10 nmol of siRNA-SQ was dissolved in 1 ml of DEPC-treated water and the droplets were carefully added on top of 500 μl of acetone under stirring. The two solutions were then kept under stirring for 5 minutes, after which the acetone was completely evaporated using a nitrogen stream to obtain an aqueous suspension of pure siRNA-SQ nanocomplexes at a concentration of 10 μM.

[0200] The hydrodynamic diameter (nm) was measured by a Malven Zeta Sizer NANO for dynamic light scattering (DLS). Samples were analyzed at a concentration of 10 μM in H2O. Three measurements of 5 minutes each were performed for each sample, and the mean diameter ± S.D. of three independent samples was calculated.

[0201] Cryogenic transmission electron microscopy (cryo-TEM) was performed using a JEOL 2100 electron microscope on an electron microscopy platform (IBPS / lnstitut de Biologie Paris-Seine, Universite P. et M. Curie, Paris, FRANCE). A 4 μL droplet of siRNA PMP22-SQ NPs (concentration of 2.2 mg / mL) was deposited onto a carbon-coated copper grid. Excess liquid was removed with blotting filter paper, and the samples were vitrified rapidly by placing them into ethane liquid using a guillotine-like framework. The samples were then transferred to a cryo-sample container. Observation was carried out at an acceleration voltage of 200 kV under a low electron dose. Analysis was performed using Image J software.

[0202] Effect of siRNA PMP22-SQ nanoparticles in a conditional CMT-1A transgenic mouse model This investigation was first carried out in vivo in a mouse model of CMT-1A established by Perea et al. (Hum Mol Genet 10, pp. 1007 - 1018, 2001). In this model, overexpression of PMP22 occurs specifically in Schwann cells of the peripheral nerves, causing demyelination that is the cause of CMT-1A disease. Transgenic mice were purchased from TAAM CNRS after reviviscence of oocyte in two genetic backgrounds, B6 and CBA. At the age of 3 months, molecular and behavioral investigations were performed on JP18 B6 and CBA to examine their gene expression, motor function, and sensory function of PMP22, respectively. This mouse model contains one extra copy of the PMP22 gene. All data were compared between CMT-1A mice and WT mice in the B6 or CBA background. For the JP18 mouse model, mouse pmp22 cDNA was introduced under the control of the PhCMV * -1 promoter, and thus the mice overexpressed pmp22 throughout their lives.

[0203] This investigation was also carried out in a double transgenic mouse model (JP18 / JY13). This model was created by crossing JP18 and JY13 mice and contains two extra copies of the PMP22 gene. In the absence of tetracycline, pmp22 overexpression occurs throughout the mouse lifespan. At 10 days of age, mice were systematically genotyped as previously described by Robertson et al. by using specific primers for PMP22 and the tTA sequence.

[0204] Effect of siRNA administration against PMP22 on the progression of CMT-1A pathology JP18B6 mice were divided into three groups of five each, in addition to a wild-type group as a comparison. One group was given a vehicle of 5% dextrose solution, the second was treated with siRNACTRL-SQ NP, and the third was treated with siRNAPMP22-SQ NP. All treatments were administered by sub-ocular IV injection at a cumulative dose of 2.5 mg / Kg at intervals of 0.5 mg / kg per injection, twice a week (a total of 5 treatments). At the end of the treatment, the mice were sacrificed and the sciatic nerve was harvested for further investigation. All animal experiments were approved by the Institutional Animal Care and Use Committee (CEEA) and the Research Council in a facility registered with the French Ministry of Higher Education and Research (Ministere de I'Enseignement Superieur et de la Recherche; MESR, APAFIS#I 0131-2016112916404689 vl 6) and were conducted in accordance with French laws and regulations under the conditions established by the European Community (Directive 2010 / 63 / UE). Verification was carried out in accordance with ethical standards and the Helsinki Declaration. Every effort was made to minimize animal suffering: treatment administration was carried out under isoflurane anesthesia and the animals were sacrificed by cervical dislocation. All animals were housed in a sterile laminar flow cage system. Food, water, and bedding were sterilized before being placed in the cages. Food and water were provided ad libitum.

[0205] The progression or regression of the disease state was monitored by the kinematic beam walking test and the grip strength test. These tests essentially examine the ability of the animals to remain upright and walk on an elevated and relatively narrow beam, as well as their muscle strength.

[0206] In another similar experiment testing siPMP22-SQ NPs against the more affected CMT1A mouse model, a double transgenic model (JP18 / JY13) with two extra copies of the PMP22 gene was used. At 12 weeks of age, JP18 / JY13 mice were divided into three groups similar to the groups of J18 mice. The same protocol of treatment was carried out, using six mice per group in addition to the wild-type B6 group. Behavioral tests were performed as described for JP18 B6 mice, and then the sciatic nerve was harvested.

[0207] To investigate the long-term effects of siPMP22-SQ NPs, 12-week-old JP18 / JY13 B6 mice were used. The mice were further divided into three groups of six each, in addition to a wild-type B6 group as a control: JP18 / JY13 vehicle, JP18 / JY13 siRNA Ct-SQ NP, and JP18 / JY13 siPMP22-SQ NP. Two cycles of treatment were administered. The first cycle of treatment was a cumulative dose of 2.5 mg / kg of siPMP22-SQ NP and siRNA Ct-SQ NP at an interval of 0.5 mg / Kg per injection, twice a week. Then, the treatment was stopped for 3 weeks to check the recurrence period, and at the end of the first treatment cycle, three mice per group were sacrificed and the sciatic nerves were harvested for further analysis. At the fourth week, a new cycle of treatment was initiated for another cumulative dose of 2.5 mg / kg of siPMP22-SQ NP and siRNA Ct-SQ NP at an interval of 0.5 mg / Kg per injection, twice a week. Behavioral tests were performed before treatment, at 1.5 mg / kg during the first treatment cycle, at 2.5 mg / kg during the first treatment cycle, 2 weeks after stopping the first cycle of treatment, 3 weeks after stopping the first cycle of treatment, at 1.5 mg / kg during the second treatment cycle, and at 2.5 mg / kg during the second treatment cycle. The sciatic nerves were excised for further analysis at the end of the second cycle.

[0208] Behavioral test Beam walking test: Mice were placed on a platform with a rod 3 cm in diameter, 70 cm in length, and approximately 30 cm above the plane. A safety platform for accommodating the animal was set at one end of the rod. First, the mice were acclimated and then trained to cross the beam, after which the time taken to cross, the speed, the number of stops, and the number of errors / slips of the left or right hindaw were recorded for analysis. The animals were recorded for three trials per session before starting the treatment and at the end of the experiment. The behavioral task repeated three times per animal was recorded using a high-resolution digital camera.

[0209] Locomotion: The locomotor apparatus was used to test the motor coordination during walking. Mice crossed a flat 75×5×20 cm ladder with bars (7 mm in diameter) set 2 cm apart. Infrared photocell sensors positioned above and below the bars monitored foot errors. The locomotor apparatus was connected to software that automatically recorded the time taken by the mice to cross the path and the foot errors. Time and errors were assayed in three trials with a 15-minute rest between trials. The test was performed at the end of the treatment. Statistical analysis of the obtained data was performed by calculating the mean of three trials per day over three days for each group. Data were presented as mean ± SD.

[0210] Grip strength test: The neuromuscular strength was assayed by using a grip strength test. This test was performed using a computerized grip meter. The apparatus consisted of a T-shaped metal bar and a rectangular metal bar connected to a force transducer. To measure the force in the forepaws of the mice, each mouse was gently supported by the base of the tail and the animal was made to grasp the T-shaped metal bar with its forepaws. As soon as the mouse grasped the transducer metal bar with its forepaws, the mouse was gently pulled backward by the tail until the grip was released. This procedure was repeated three times and the highest force was automatically recorded by the instrument in grams (g). To measure the force regarding both limbs, each mouse was made to grasp the rectangular metal with its fore and hind limbs. Then, it was gently pulled backward by its tail perpendicular to the axis of the apparatus until the mouse released the grip. The highest force was automatically recorded by the instrument.

[0211] Hot plate test: The sensitivity to heat was assayed using a hind paw withdrawal test. The platform on which the animals were placed was set at 52 °C and the time until they sensed the heat and withdrew their feet was recorded for analysis.

[0212] Electrophysiological investigation The tests were performed using a standard EMG device (Natus / EMG) according to the guidelines of the American Association Of Neuromuscular And Electrodiagnostic Medicine. Anesthesia was induced by inhalation of isoflurane, and the mice were placed in an induction chamber containing 1.5 - 2% isoflurane in pure oxygen. During the entire procedure, anesthesia was maintained at the same level by a face mask. The mice were placed on their ventral sides on a heating pad to maintain their body temperature at 34 - 36 °C. To record compound muscle action potentials, a stimulating needle electrode was inserted at the level of the sciatic notch, an anode electrode was inserted at the upper base of the tail, while a receptor needle or recording needle was inserted into the medial part of the gastrocnemius muscle. A maximum supramaximal rectangular wave pulse of 8 mA was delivered through the stimulating needle and recorded as magnitude through the muscle. For measurement of the sensory nerve conduction velocity, multiple stimulations of the tail nerve were delivered through a stimulating needle placed at two-thirds of the tail length with a distance of 2 - 2.5 cm from the receptor needle. A ground electrode was inserted midway between the stimulating and receptor electrodes. The sensory nerve conduction velocity was calculated from the latency of the stimulation and the distance between the stimulating and receptor electrodes.

[0213] Biological tests The sciatic nerve tissue structure of CMT-1A animals (treated or untreated with siRNA) was examined by electron microscopy, in which the number of myelinated or unmyelinated axons and the thickness of the myelin sheath were assessed by determining the g ratio. Then, the number of mitochondria per axon was calculated to assess axonal involvement. RT-qPCR and Western blot experiments were performed to evaluate the effect of siRNA on PMP22 expression in the sciatic nerve of CMT-1A mice. PMP22 expression was normalized against the control level (WT animals or CMT-1A mice treated with siRNA PMP22). The neuromuscular junction (NMJ) of CMT-1A mice treated or untreated with siRNA PMP22 was analyzed by immunohistochemistry. Muscle weakness in CMT-1A is due to axonal degeneration, leading to denervation and atrophy of the muscle.

[0214] In CMT-1A, the disappearance of neuromuscular junctions is observed. It is important to investigate the NMJ to determine the effectiveness of siRNA therapy. Using confocal and electron microscopy, a comprehensive investigation of the structure of the NJM (nerve innervation, denervation, nerve reinnervation, etc.) is carried out.

[0215] Pharmacokinetics and biodistribution investigation Radio-labeled free or SQ-conjugated siRNA PMP22 (siRNA PMP22-SQ 32 P NP) is intravenously injected into transgenic mice with CMT-1A. Organs and blood are collected at various time points, and the concentrations of free vs. siRNA PMP22-SQ NP are determined by counting radioactivity with a g-counter. Also, it is demonstrated by "Radio-HPLC" analysis that siRNA PMP22 is not degraded once it accumulates in the desired tissue. Pharmacokinetic parameters are calculated (e.g., plasma half-life and clearance). Notably, a proportion exceeding 50% of squalene is transported by LDL and HDL lipoproteins, and thus, the possible interaction between siRNA PMP22-SQ NP and blood components (i.e., LDL, HDL, VLDL, blood cells, etc.) is evaluated as previously described by Sawle et al. (Journal of lipid research 43, pp. 335 - 343, 2002).

[0216] Statistical analysis All data were presented as mean ± standard deviation (SD). Multiple treatments were compared using non-parametric Kruskal-Wallis analysis followed by Dunn's test, or Anova followed by Bonferroni's test, using GraphPad Prism. p < 0.05 was considered the statistically significant level.

[0217] Results Effect of siRNA PMP22 on PMP22 mRNA expression in MSC-80 cells First, the effects of siRNA PMP22(1 - 8) on mRNA PMP22 expression were investigated and compared with its effects on the scrambled sequence, which is the siRNA control for untreated cells. By using the Kruskal - Wallis followed by the Dunn's test, it was found that all siRNAs except siPMP6 significantly down - regulated PMP22 expression at 24 hours and 48 hours. 72 hours after transfection, siPMP4, 5, 7, and 8 were able to reduce PMP22 gene expression by 50% when compared to the control (see Figure 1). In addition, 72 hours after transfection, it was found that siPMP7 was able to not only reduce the gene expression of PMP22 but also its protein expression (see Figure 2).

[0218] Effects of various concentrations of siPMP22 on mRNA PMP22 and P0 and on cell viability Subsequently, the effects of various concentrations (25 nM, 50 nM, and 100 nM) of siPMP7 on the gene expression of PMP22 and P0 by transfection of MSC80 cells at 72 hours and 96 hours were investigated to determine the optimal concentration that can normalize PMP22 expression without affecting the expression of P0, a protein involved in myelin compaction and associated with PMP22 in cell viability. The results of the present inventors indicate that the optimal concentration is 50 nM. siPMP7 (50 nM) reduced the gene expression of PMP22 by 50% each between 72 and 96 hours, while it did not affect the P0 gene expression (see Figure 3). Also, the cell viability test by the MTT assay showed that siPMP7 50 nM had no significant effect on MSC80 viability (see Figure 4). The siRNA control (siCt) did not modify the expression of the two genes investigated and cell viability.

[0219] Subsequently, siPMP7, named siPMP22, was the best candidate to continue the investigation because it had the ability to reduce PMP22 levels by 50%, it did not affect P0 levels, and it had no effect on cell viability in vitro.

[0220] The sequence of siRNA PMP22 is Sense strand: 5'-AUACCAACUGUGUGGACUA-3' (SEQ ID NO: 7) Antisense strand: 5'-UAGUCCACACAGUUGGUAU-3' (SEQ ID NO: 18) as follows.

[0221] Binding of siRNA PMP22 and siRNA CTRL to squalene (SQ) and characterization of siRNA PMP22-SQ nanoparticles Bioconjugated siRNA PMP22-SQ and siRNA CTRL were obtained by Cu-free click chemistry with a yield of more than 90% thanks to the optimized conditions of the reaction detailed in Figure 12. For siRNA PMP22-SQ, the recovered by-products identified by HPLC and analyzed by MALDI-TOF MS indicated that the bioconjugate had a predicted molecular weight of 7628 (data not shown). The resulting bioconjugate was annealed with the antisense strand and nanoprecipitated in RNase-free water. The solution produced the Tyndall effect, suggesting the formation of nanoparticles. Dynamic light scattering analysis (DLS) and cryoTEM images showed the formation of stable nanoparticles of approximately 180 nm with a good polydispersity index (0.14 - 0.2) over 30 days, reflecting a homogeneous solution suitable for IV injection (see Figure 4). For siRNA CTRL, the molecular weight identified by MALDI-TOF MS after bioconjugation and product recovery was 7621 daltons. DLS measurements showed that the size of siRNA CTRL-SQ NPs was stable over a one-month period (size: 255 ± 2 on day 0 and 238 ± 4 on day 30), and the polydispersity index (0.15 ± 0.02 and 0.1 ± 0.01 on day 0 and 30, respectively).

[0222]

Table 4

[0223] Next, these nanoparticle siRNA PMP22-SQ and siRNA CTRL were tested in vitro over time (48 hours and 72 hours) for their efficiency and effect on cell viability. Previously, the inventors have shown that squalene-based siRNA nanoparticles cannot enter cells spontaneously without any cationic compound. Therefore, SQ-based siRNA PMP22 nanoparticles as well as unvectorized siRNA PMP22 were transfected into MSC-80 cells by using Lipofectamine iMAX (registered trademark). After 48 and 72 hours, vectorized siRNA PMP22 was able to inhibit mRNA PMP22 to the same extent as free siRNA PMP22 (see Figure 5) without affecting cell viability (see Figure 6). Furthermore, nanoparticulated or unvectorized siRNA controls did not affect PMP22 mRNA levels or cell viability when cells were transfected (see Figures 5 and 6).

[0224] In summary, this part of the investigation showed that i) the squalenoylation of siRNA PMP22 produced a yield of approximately 100% after bioconjugation to SQ and approximately 85% after HPLC purification; ii) the resulting NPs were reproducible and stable over one month; and iii) the NPs were efficient in vitro: when transfected with Lipofectamine, they inhibited PMP22 to the same extent as naked siRNA PMP22 and had no effect on cell viability .

[0225] In vivo experiments: PMP22 gene expression and behavioral tests in B6 and CBA wild-type and transgenic PMP22 mice First, the expression of PMP22 in the sciatic nerve was examined by comparing transgenic PMP22 mice in the B6 and CBA backgrounds with their corresponding wild-type mice. For both PMP22 mice in the B6 and CBA backgrounds, the inventors found an increase in PMP22 mRNA expression compared to the wild-type. P0 expression was found at the same level in both strands (see FIGS. 7A and 7B, and FIGS. 8A and 8B).

[0226] Fine motor coordination and balance were assayed by beam walking and rotarod assays. The goal of these tests is for the mouse to maintain an upright position and walk across an elevated narrow beam to a safe platform. Interestingly, the beam walking and rotarod assays showed an increase in time and number of errors for both PMP22 mice in the B6 and CBA backgrounds compared to the wild-type, suggesting the onset of CMT-1A disease. Indeed, as seen in FIGS. 7C, 7D, and 8C, a significant decrease in speed on the beam was observed in CMT-1A mice. In fact, CMT-1A mice walked the distance twice as slowly as control mice, and the number of foot slips (errors) of CMT-1A animals increased. The foot withdrawal test, which detects noxious stimuli such as pain caused by stimulation of nociceptors (sensory neurons), was found to be the same for transgenic and wild-type mice, suggesting that the sensory neurons of these transgenic mice were not affected. Therefore, the useful model in the inventors' investigation represents the CMT-1A pathology characterized by i) an increase in PMP22 expression of less than two-fold due to a 1.5 Mb duplication on chromosome 17p11.2, ii) a decrease in motor neuron activity, and iii) no effect on sensory neurons. Peripheral nerve injury tends to induce defects, which can cause rodents to slip to one side.

[0227] JP18 Effect of PMP22-SQ nanoparticles on the behavior of B6 and CBA backgrounds Interestingly, the beam walking test conducted at JP18 in the B6 background showed that the time spent by mice treated with siRNA PMP22-SQ nanoparticles to cross the path was comparable to that of untreated wild-type mice (see Figure 8C; see Figure 8D). Mice treated with vehicle (5% dextrose) or siRNA control-SQ nanoparticles showed no difference in time. Preliminary results in CBA mice showed the same results for the group treated with siRNA PMP22-SQ nanoparticles (see Figure 10).

[0228] In summary, these results indicate that siRNA PMP22-SQ nanoparticles can restore the motor activity of CMT-1A transgenic mice.

[0229] siRNA PMP22-SQ nanoparticles restore motor activity in single and double transgenic mice The walking motor activity of mice was tested by two complementary tests: beam walking and rotarod test before and after treatment. Grip strength was also investigated under the same conditions. The inventors found that JP18 (single transgenic) and JP18 / JY13 (double transgenic) mice treated with siRNA PMP22-SQ nanoparticles had a walking motor ability similar to that of wild-type mice and significantly better walking motor ability than untreated mice (5% dextrose) and mice treated with Ct-SQ nanoparticles (Figures 13 and 14).

[0230] Electrophysiological results siPMP22-SQ NP restores compound muscle action potential (CMAP) (see Figures 15A and 15C) and sensory nerve velocity (see Figures 15B and 15D) 3 weeks after treatment in both single and double transgenic mice.

[0231] Myelination and axonal regeneration The siPMP22-SQ NPs did not modify the g ratio in either single or double transgenic mice (see Figures 16 and 17).

[0232] TEM micrographs of ultrathin sections of the sciatic nerve showed myelin regulation in single and double transgenic mice treated with siPMP22-SQ NPs (data not shown). The nanoparticles were localized in the cytoplasm of Schwann cells, and it was also shown that they could enter the nerve through Ranvier nodes (data not shown).

[0233] Next, the expression of proteins involved in myelination and axonal regeneration was analyzed in a single transgenic mouse model. The levels of the transcription factor SOX10, the myelination factor KROX20, and the axonal regeneration NF marker were reduced in the JP18 5% dextrose and siRNA Ct groups; notably, siPMP22-SQ NP treatment significantly increased their levels compared to the JP18 5% dextrose group (see Figures 18A, 18B, and 19). Similar results were obtained in the double transgenic mouse model: siPMP22-SQ NP treatment increased the myelination transcription factors (SOX10 and KROX20) and enhanced axonal regeneration compared to the JP18 / JY13 5% dextrose group (see Figure 20).

[0234] Long-term effects of siRNA PMP22-SQ NPs on the behavior of double transgenic mice Next, the long-lasting effects of siPMP22-SQ NPs on beam walking, rotarod, and grip strength tests were analyzed. Double transgenic JP18 / JY13 B6 mice received two cycles of treatment at 2.5 mg / Kg per cycle. The treatment was stopped for 21 days between the two cycles to investigate recurrence. The mice were followed up once a week to analyze the recovery and recurrence periods.

[0235] The locomotor activity of the JP18 / JY13 mice recovered from the 1.5 mg / kg dosage (11 days after the first injection), and this effect continued for 15 days after the last treatment (approximately 30 days after the first injection). However, there was a recurrence 37 days after the first injection. From the same dosage in the second cycle of treatment (1.5 mg / kg, 11 days after the first injection of the second cycle), the mice walked like wild-type mice. At the end of the second cycle of treatment, remission was complete (see Figure 21). The same result profile was observed for grip strength (see Figure 22).

[0236] Conclusion CMT-1A disease is the most common type of Charcot-Marie-Tooth (CMT) pathology, accounting for 40 - 50% of CMT cases. This innovative approach aims to develop a new therapy for CMT-1A disease by targeting the overexpression of peripheral myelin protein 22 (PMP22), particularly by siRNA.

[0237] We successfully developed an siRNA against PMP22 that can counteract the 50% overexpression of PMP22 caused by a 1.5 Mb duplication on chromosome 17p11.2, thus restoring normal levels of PMP22 expression. Importantly, the knockdown of PMP22 did not affect MPZ (P0), which is involved with PMP22 in the myelination process (abnormal regulation of MPZ can lead to another type of CMT, CMT1B). Furthermore, the siRNA mentioned did not affect cell viability.

[0238] Next, to protect and safely deliver siRNA targeting PMP22, the "click chemistry squalenoylation method" was developed. The bioconjugation of siRNA PMP22 to squalene was near complete, giving a 95% reaction yield. siRNA PMP22-SQ NPs remained active after bioconjugation to squalene, thanks to the modification only on the passenger sense strand. After nanoprecipitation, the resulting nanoparticles had a size of approximately 180 nm and a low polydispersity index of 0.14 and were stable for 30 days, indicating that these nanoparticles could be intravenously injected.

[0239] Interestingly, in a transgenic mouse model of CMT-1A with 1.5-fold overexpression of PMP22, these siRNA PMP22-SQ nanoparticles injected via the intravenous route were able to restore the motor activity of CMT-1A mice, which was demonstrated to be identical to that of wild-type mice. This demonstration was performed in two genetic backgrounds, B6 and CBA mice, giving similar results. Similar results were obtained using double transgenic JP18 / JY13 B6 mice.

[0240] In CMT-1A, the major form of CMT pathology, there is no equivalent study available in Europe or other regions of the world that describes the restoration of motor activity in mice by knocking down gene expression by 50% with siRNA, thanks to the regulation in fine-tuning PMP22 expression.

Claims

1. An antisense RNA targeting mRNA encoding PMP22 protein for use in the treatment of Charcot-Marie-Tooth 1A (CMT-1A).

2. The antisense RNA according to claim 1 for use, wherein the antisense RNA reduces the amount of PMP22 protein in cells by 40% to 60%.

3. The antisense RNA according to claim 1 or 2 for use, wherein the antisense RNA is selected from the group consisting of siRNA, shRNA, miRNA, dsRNA, and RNA species that can be cleaved in vivo to form siRNA.

4. The antisense RNA according to any one of claims 1 to 3 for use, wherein the antisense RNA is complementary to a part of (i) the sequence of SEQ ID NO: 9, (ii) the sequence of SEQ ID NO: 11, or (iii) a naturally occurring variant of the sequence of SEQ ID NO: 9 or 11.

5. The antisense RNA is (i) - nucleotides 989 to 1007 of the sequence of SEQ ID NO: 11, - nucleotides 970 to 988 of the sequence of SEQ ID NO: 9, - nucleotides 1721 to 1739 of the sequence of SEQ ID NO: 11, - nucleotides 1726 to 1744 of the sequence of SEQ ID NO: 9, - nucleotides 431 to 449 of the sequence of SEQ ID NO: 11, - nucleotides 429 to 447 of the sequence of SEQ ID NO: 9, - nucleotides 1805 to 1823 of the sequence of SEQ ID NO: 11, - nucleotides 1809 to 1827 of the sequence of SEQ ID NO: 9, - nucleotides 921 to 939 of the sequence of SEQ ID NO: 11, or - nucleotides 903 to 921 of the sequence of SEQ ID NO: 9 comprising, contained therein, or overlapping with a part thereof, or (ii) a part homologous to a part of (i) present in a naturally occurring variant The antisense RNA according to claim 4 for use, which is complementary thereto.

6. The antisense RNA according to any one of claims 1 to 5 for use, wherein the antisense RNA comprises at least 10 consecutive nucleotides of a sequence selected from the group consisting of the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO:

19.

7. The antisense RNA according to any one of claims 1 to 6 for use, wherein the antisense RNA is an siRNA containing one or two single-stranded overhangs.

8. The antisense RNA is an siRNA comprising or consisting of (i) a sequence selected from the group consisting of the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and (ii) optionally, one or two single-stranded overhangs, for use according to any one of claims 1 to 7.

9. The antisense RNA for use according to any one of claims 1 to 8, wherein the antisense RNA is provided in the form of a nanoparticle comprising the antisense RNA.

10. The antisense RNA for use according to any one of claims 1 to 9, wherein the antisense RNA is administered intravenously, intraperitoneally, subcutaneously, or intraneurally, preferably to the sciatic nerve.

11. The antisense RNA for use according to any one of claims 1 to 10, wherein the antisense RNA is used in combination with at least one other drug useful in the treatment of Charcot-Marie-Tooth 1A.

12. An antisense RNA targeting mRNA encoding PMP22 protein, wherein the antisense RNA is (i) - nucleotides 970 to 988 of the sequence of SEQ ID NO: 9, - nucleotides 431 to 449 of the sequence of SEQ ID NO: 11, - nucleotides 429 to 447 of the sequence of SEQ ID NO: 9 comprising, contained within, or overlapping with a portion thereof, (ii) - nucleotides 1721 to 1739 of the sequence of SEQ ID NO: 11, - nucleotides 1726 to 1744 of the sequence of SEQ ID NO: 9, - nucleotides 1805 to 1823 of the sequence of SEQ ID NO: 11, - nucleotides 1809 to 1827 of the sequence of SEQ ID NO: 9 comprising or overlapping with a portion thereof, (iii) a nucleic acid complementary to a portion homologous to (i) or (ii) present in a naturally occurring variant is an antisense RNA.

13. The antisense RNA targeting PMP22 according to claim 12, wherein the antisense RNA is selected from the group consisting of siRNA, shRNA, miRNA, dsRNA, and RNA species that can be cleaved in vivo to form siRNA.

14. The antisense RNA targeting PMP22 according to claim 12 or 13, wherein the antisense RNA is an siRNA comprising one or two single-stranded overhangs.

15. The antisense RNA according to any one of claims 12 to 14, which is an siRNA comprising or consisting of (i) a sequence selected from the group consisting of the sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and (ii) optionally, one or two single-stranded overhangs, and targeting PMP22.

16. A nanoparticle comprising the antisense RNA according to any one of claims 12 to 15.

17. The nanoparticle according to claim 16, wherein the antisense RNA is bound to squalene or a derivative thereof.