Antisense RNA targeting PMP22 for the treatment of Charcot-Marie-Tooth 1A disease

Abstract

The present invention relates to antisense RNAs that target PMP22 and can inhibit PMP22 expression in cells by 40% to 60%, and pharmaceutical compositions containing the same. The antisense RNA is preferably siRNA, and is preferably provided in the form of nanoparticles. The present invention also relates to the use of these antisense RNAs that target PMP22 for the treatment of Charcot-Marie-Tooth 1A disease.

Description

[Technical Field]

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

[0002] CMT-1A neuropathy is due to alterations in peripheral nerve myelin, leading to demyelination and severe, disabling disease. The myelin sheaths surrounding peripheral nerve axons are formed by Schwann cells, which are essential for the rapid and accurate transmission of electrical impulses by saltatory conduction. The predominant subtype of Charcot-Marie-Tooth disease is CMT-1A, accounting for more than 50% of all patients. It is associated with an interstitial chromosome duplication of 17p11.2, resulting in overexpression of the gene encoding the 22-kDa peripheral myelin protein (PMP22), which causes disease characterized by loss of muscle and sensory function.

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

[0004] One promising molecule was ascorbic acid. In an animal model of CMT-1A that overexpresses PMP22, ascorbic acid treatment resulted in substantial improvement of the disease phenotype and reduced PMP22 expression. Unfortunately, clinical trials testing the effects of ascorbic acid supplementation in CMT-1A patients have not shown any beneficial effects compared with placebo. Thus, there is currently no clinical evidence supporting ascorbic acid treatment in adults with CMT-1A.

[0005] Antiprogesterone therapy was found to significantly increase muscle strength and prevent axonal loss in PMP22 transgenic rats, although myelin thickness was unaffected. Unfortunately, currently available progesterone antagonists are too toxic to safely administer to patients.

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

[0007] However, there are currently no effective drugs 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] WO2006 / 090029 [Non-patent literature]

[0009] [Non-Patent Document 1] Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453 [Non-patent document 2] Massaad-Massade et al., Bioconjugate Chem., 2018, 29 (6), pp. 1961-1972, DOI:10.1021 / acs.bioconjchem.8b00205 [Non-patent document 3] Reynolds and Tafer, Nature biotech., 2008, (26) 5, pp. 578-83 [Non-patent document 4] Perea et al. (Hum Mol Genet 10, 1007-1018, 2001) [Non-patent document 5] Sawle et al. (Journal of lipid research 43, pp. 335-343, 2002) Summary of the Invention [Problem 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 Invention The present inventors have found that antisense RNA targeting PMP22, particularly using RNA interference (RNAi), is highly effective in treating Charcot-Marie-Tooth 1A. Indeed, the present inventors have shown that intravenous administration of small interfering RNA (siRNA) targeting PMP22 allows recovery of locomotion and strength identical to that of wild-type mice in a mouse model of Charcot-Marie-Tooth 1A disease in which PMP22 is overexpressed 1.5-fold in these mice (see Examples).

[0012] Importantly, we selected antisense RNA, particularly siRNA, capable of only partially inhibiting PMP22 expression, preferably by 40% to 60%, to counter the 50% overexpression of PMP22 resulting from the 1.5 Mb duplication on chromosome 17p11.2. PMP22 protein should indeed remain in cells in amounts comparable to those found in healthy individuals.

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

[0014] The antisense RNA according to the invention, in particular the siRNA PMP22, advantageously does not interfere with the expression of the P0 protein, which remains unchanged. The P0 protein is in fact involved together with PMP22 in the myelination process, and deregulation of the gene encoding the P0 protein is responsible for other types of CMT, such as CMT1B.

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

[0016] In order to protect and safely deliver antisense RNA targeting PMP22, especially siRNA targeting PMP22, they can be vectorized and provided in the form of nanoparticles, for example.For example, antisense RNA can be conjugated with squalene, thereby forming nanoparticles containing antisense RNA.These nanoparticles (also referred to as siRNA PMP22-SQ NP) remain active after bioconjugation with squalene due to the modification of only 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 allowing them to be injected intravenously.

[0017] Whereas most drugs assessed for the treatment of CMT-1A focus on adenylyl cyclase activity, which indirectly affects PMP22 expression, the antisense RNA, particularly siRNA, according to the present invention targets the PMP22 gene itself, making it possible to improve various aspects of the disease, such as motor activity, strength, and axonal regeneration.

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

[0019] Therefore, a first object of the present invention is an antisense RNA that targets 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 may 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 may be complementary to a portion 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 SEQ ID NO:11.

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

[0024] The antisense RNA may comprise at least 10 consecutive nucleotides of a sequence selected from the group consisting of the sequences 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 containing one or two single-stranded overhangs, for example two single-stranded 3' overhangs.

[0026] The antisense RNA may be an siRNA comprising or consisting of (i) a sequence selected from the group consisting 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, in particular one or two single-stranded 3' overhangs.

[0027] Another object of the present invention is a nanoparticle comprising an antisense RNA as defined above, the antisense RNA preferably being conjugated to squalene or a derivative thereof, the nanoparticle comprising, for example, an siRNA, the sense strand of which is preferably conjugated to squalene or a derivative thereof.

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

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

[0030] Another object of the present invention is an antisense RNA targeted to the mRNA encoding the PMP22 protein, as defined above, for use in the treatment of Charcot-Marie-Tooth disease 1A (CMT-1A). Thus, the antisense RNA makes it possible to restore normal levels of PMP22 expression in patients suffering from Charcot-Marie-Tooth disease 1A.

[0031] The antisense RNA may be provided in the form of nanoparticles comprising the antisense RNA, such as the nanoparticles defined above.

[0032] The antisense RNA may be provided in the form of a pharmaceutical composition as defined above.

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

[0034] The antisense RNA may be used in combination with at least another drug useful in the treatment of Charcot-Marie-Tooth 1A.

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

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

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

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

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

[0040] The term "non-human mammal" includes, for example, a rat, mouse, pig, cat, dog, rabbit, or primate.

[0041] Charcot-Marie-Tooth 1A (CMT1A) disease results from a 1.5 Mb duplication on chromosome 17p11.2 that contains the PMP22-encoding 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] By "functional PMP22 protein" herein is meant a protein encoded by a gene that can increase PMP22 expression (when duplication occurs at the DNA level) at both the mRNA level (e.g., tested by real-time polymerase chain reaction after RNA extraction followed by reverse transcription (RT-qPCR)) and the protein level (e.g., tested by Western blot and by immunohistochemistry), leading to demyelination.

[0044] Those skilled in the art can easily determine whether a given PMP22 protein is functional by well-known methods, such as a proband with a family history combined with very slow nerve conduction velocity, or DNA testing, RT-qPCR, Western blot, and / or electrophysiological testing.

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

[0046] The PMP22 protein may comprise or consist of a sequence that is at least 80% identical to the sequence of SEQ ID NO: 10, 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: 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 may comprise or consist 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 to the sequence of SEQ ID NO: 9 in which the nucleotide "T" is replaced by "U".

[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 may comprise or consist 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 to the sequence of SEQ ID NO: 11 in which the nucleotide "T" is replaced by "U".

[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, compared to the reference sequence.

[0052] In the case of substitutions, the substitutions preferably correspond to conservative substitutions as shown in Table 1 below. In preferred embodiments, sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence only differ from the reference sequence 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 represent 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 another mammalian species than the reference sequence.

[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 by conservative substitutions, and / or represent a naturally occurring variant of the reference sequence, and / or represent a homologous sequence derived from another mammalian species than the reference sequence.

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

[0058] Alignment and determination of percentage identity can be performed manually or automatically, for example, using the Needle program, which is 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 nucleic 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, compared to the reference sequence.

[0060] In the case of nucleotide substitutions, the substitutions may represent silent substitutions or substitutions that lead to conservative substitutions in the translated amino acid sequence compared to the reference sequence, for example as shown in Table 1 above.

[0061] Nucleic sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may represent a naturally occurring variant of the reference sequence and / or represent a homologous sequence derived from another mammalian species 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 by substitutions that result in silent and / or conservative amino acid substitutions, and / or may represent a naturally occurring variant of the reference sequence, and / or may represent a homologous sequence derived from a mammalian species other than the reference sequence.

[0063] Antisense RNA targeting the mRNA encoding the PMP22 protein The present invention particularly relates to antisense RNAs that target the mRNA encoding the PMP22 protein.

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

[0065] Antisense RNAs that target the mRNA encoding the PMP22 protein are hereinafter referred to as "antisense RNAs."

[0066] The 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 Schwann cells, by 40% to 60%, preferably 40% to 55%, more preferably 40% to 50%, for example, 40% to 45% or 45% to 50%, and / or reduces the amount of PMP22 mRNA in cells, preferably Schwann cells, by 40% to 60%, preferably 40% to 55%, preferably 40% to 50%, for example, 40% to 45% or 45% to 50%.

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

[0069] Determining whether an antisense RNA can reduce the amount of PMP22 in a cell, and optionally quantifying the percentage of reduction, can be carried out, for example, by assessing the amount of PMP22 protein in a cell in the presence and absence of the antisense RNA being tested (see, e.g., the Examples section).

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

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

[0072] Thus, an antisense RNA is a nucleic acid that targets the mRNA encoding PMP22.

[0073] The term "nucleic acid targeting a given mRNA" as used herein refers to 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 allows the mRNA-targeting nucleic acid to specifically bind to the mRNA under intracellular conditions.

[0074] As used herein, the phrase "a sequence that is perfectly complementary to a second sequence" refers to the reverse complementary counterpart of the second sequence.

[0075] Antisense RNAs targeting 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 RNA. Because the sequences of SEQ ID NO: 9 and SEQ ID NO: 11 are cDNA sequences, the nucleotide "T" must be replaced with the nucleotide "U" to obtain the mRNA sequence.

[0076] Antisense RNA as defined above can be single-stranded or double-stranded RNA (ribonucleic acid), such as siRNA.

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

[0078] The antisense RNA as defined above may contain at least one non-standard nucleotide, such as a non-naturally occurring nucleotide or deoxyribonucleotide.

[0079] Antisense RNA as defined above may comprise or consist of an RNA portion and at least one additional portion, such as a deoxyribonucleotide portion.

[0080] The antisense RNA as defined above may 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, e.g., 19, 20, or 21 nucleotides.

[0081] The antisense RNA as defined above may, for example, comprise or consist of 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, e.g., 19, 20, or 21 nucleotides.

[0082] The antisense RNA defined above may comprise 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, 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 interfering agent.

[0084] The antisense RNA, in particular the RNA interfering agent, as defined above may 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" comprises 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 comprises an antisense strand complementary to the mRNA encoding the PMP22 protein, and a sense strand.

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

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

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

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

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

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

[0094] "MicroRNA" or "miRNA" refers to a short, non-coding RNA approximately 22 nucleotides in length. miRNAs are post-transcriptional regulators of target genes and are generally expressed in a highly tissue- or developmental stage-specific manner. Based on the properties of existing miRNA genes, artificial miRNAs can be designed and expressed.

[0095] "dsRNA" is RNA that has the same double strand as DNA, but with uracil instead of thymine. It forms the genetic material of many viruses and is a major component of the interferon system, which stimulates the immune system against viral infections.

[0096] The antisense RNA defined above may target a portion of the sequence of SEQ ID NO: 9, the sequence of SEQ ID NO: 11, or a naturally occurring variant thereof, in which 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 portion 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.

[0098] An antisense RNA complementary to a portion of a sequence that is 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 portion 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, wherein for purposes of 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 portion 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 as defined above is (i) - nucleotides 989 to 1007 of sequence SEQ ID NO: 11, - nucleotides 970 to 988 of sequence SEQ ID NO: 9, - nucleotides 1721 to 1739 of sequence SEQ ID NO: 11, - nucleotides 1726 to 1744 of the sequence SEQ ID NO: 9, - nucleotides 431 to 449 of sequence SEQ ID NO: 11, - nucleotides 429 to 447 of the sequence SEQ ID NO: 9, - nucleotides 1805 to 1823 of sequence SEQ ID NO: 11, - nucleotides 1809 to 1827 of sequence SEQ ID NO: 9, - nucleotides 921 to 939 of the sequence SEQ ID NO: 11, or nucleotides 903 to 921 of the sequence SEQ ID NO: 9 consisting of, contained in, or overlapping with any part of; or (ii) a portion that is homologous to a portion 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 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, e.g., 16, 17, 18, or 19 nucleotides, of a sequence selected from the group consisting of the sequences 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, the 3' or 5' overhangs consisting of, e.g., two dTs.

[0104] The antisense RNA, preferably siRNA, as defined above may comprise or consist of (i) a sequence selected from the group consisting of the sequences 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, the 3' and / or 5' overhangs consisting of, for example, two dTs.

[0105] The antisense RNA as defined above is preferably an siRNA comprising or consisting of (i) a sequence selected from the group consisting of the sequences 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 the 3' overhangs preferably consisting of two dTs.

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

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

[0108] The antisense RNAs defined above, in particular shRNAs, can be cloned into vectors and then delivered to cells.

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

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

[0111] The nanoparticles may be loaded with antisense RNA.

[0112] The use of nanoparticles makes it possible to increase the half-life of antisense RNA, especially in the case of siRNA.

[0113] Moreover, nanoparticles may allow for specific delivery to target cells, for example, by being intrinsically targeted to the target cells or by being bound to a ligand specific for 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-assemble into 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 a derivative thereof, which further anneals to the antisense strand of the siRNA, thereby forming nanoparticles, particularly after nanoprecipitation.

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

[0119] The siRNA-containing nanoparticles as defined above may be - adding a dibenzocyclooctyne residue to the 5' end of the sense strand of the siRNA to obtain a modified sense strand; - attaching squalene to said modified sense strand, in particular via bioconjugation of the azide functionality of squalene to a dibenzocyclooctyne residue, - adding the antisense strand of the siRNA to anneal the two strands of the siRNA; - optionally, adding acetone and water to precipitate the nanoparticles, in particular by slowly adding one phase (aqueous or organic) to the other, preferably under stirring, and - Optionally, evaporating the acetone, for example using a stream of nitrogen, to obtain an aqueous suspension of pure siRNA-SQ nanoparticles. It can be obtained by a method comprising:

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

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

[0122] For example, the nanoparticles have a size comprised between 100 and 200 nm, more preferably between 170 nm and 190 nm, for example 180 nm.

[0123] The nanoparticles preferably have a low polydispersity index, in particular a polydispersity of less than 0.3 nm, more preferably less 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 comprise (i) a sequence selected from the group consisting of the sequences SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and (ii) an siRNA comprising or consisting of two 3' overhangs, wherein the 3' overhangs preferably consist of two dTs.

[0127] The nanoparticles containing the antisense RNA may 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 defined above, in particular an siRNA targeting the mRNA encoding the PMP22 protein defined above, and a pharmaceutically acceptable carrier.

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

[0130] Thus, the antisense RNA may be provided in the form of exosomes comprising or conjugated to nucleic acid, liposomes comprising or conjugated to nucleic acid, and / or nanoparticles comprising or conjugated to nucleic acid, such as the nanoparticles defined above.

[0131] The language "pharmaceutically acceptable" is intended to encompass any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and which is preferably not deleterious 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 comprise a nucleic acid encoding the antisense RNA in the case of an siRNA, or a vector comprising said nucleic acid, particularly when the antisense RNA is part of an shRNA.

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

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

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

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

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

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

[0142] In one embodiment, pharmaceutical compositions are presented in unit dosage forms to facilitate accurate dosing.The term "unit dosage form" refers to a physically discrete unit suitable as a unit dosage for human subjects and other non-human mammals, each unit containing a predetermined amount of active substance calculated to produce a desired therapeutic effect in association with suitable pharmaceutical excipients.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 a pharmaceutical product as defined above and instructions for administration, which may indicate, for example, the medical indication, the route of administration, the dosage, and / or the group of patients to be treated.

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

[0145] The non-human mammal is, for example, a mouse, rat, cat, dog, rabbit, or primate.

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

[0147] Subjects can be of any age, eg, infants, children, adolescents, adults, elderly, and of any gender.

[0148] Treatment of Charcot-Marie-Tooth 1A disease As used herein, by "treating Charcot-Marie-Tooth 1A disease" is meant at least partially halting 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 of the lower limbs, weakness and / or loss of sensation in the hands, thereby normalizing gait and / or preventing or reducing foot drop; - halt, slow or reverse muscle weakness and / or atrophy of the lower limbs, weakness and / or loss of sensation in the hands, thereby normalizing gait and / or reducing foot drop; and / or - Normalizing nerve conduction velocity Includes:

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

[0152] Antisense RNA targeted to the mRNA encoding PMP22 is in particular as defined above.

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

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

[0155] Treatment of Charcot-Marie-Tooth 1A disease is specifically as defined above.

[0156] Administering antisense RNA, particularly siRNA, targeted to the mRNA encoding PMP22 can be carried out using a variety of techniques well known in the art, including naked administration and / or administration in a pharmaceutically acceptable carrier such as nanoparticles.

[0157] The nanoparticles comprising antisense RNA according to the invention are in particular as defined above.

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

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

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

[0161] Antisense RNA targeted to 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 efficacy of the antisense RNA and the nature of any carrier used. Determination of an appropriate amount for any given composition is within the skill of the art through standard series of tests designed to assess appropriate therapeutic levels.

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

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

[0164] Antisense RNA targeted to mRNA encoding PMP22 can be administered as a single injection or several injections, for example, once or twice per week, for at least three weeks and / or up to three months.

[0165] The antisense RNA may 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 invention will be further illustrated in light of the following examples and figures.

[0167] A brief description of arrays SEQ ID NO:1 is the sense strand sequence of an siRNA comprising the antisense strand of SEQ ID NO:12.

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

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

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

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

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

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

[0174] SEQ ID NO:8 is the sense strand sequence of an siRNA comprising 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, 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 a cDNA encoding mouse PMP22 protein, reference NM_008885.3 in the NCBI database, available on July 30, 2018.

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

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

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

[0181] SEQ ID NO: 15 is an RNA sequence that is fully complementary to nucleotides 989 to 1007 of the sequence of SEQ ID NO: 11 and partially complementary to nucleotides 970 to 988 of the sequence of SEQ ID NO: 9 (two mismatches).

[0182] SEQ ID NO: 16 is an RNA sequence that is completely complementary to nucleotides 1721 to 1739 of the sequence of SEQ ID NO: 11 and completely complementary to nucleotides 1726 to 1744 of the sequence of SEQ ID NO: 9.

[0183] SEQ ID NO:17 is an RNA sequence that is completely complementary to nucleotides 431 to 449 of the sequence of SEQ ID NO:11 and completely complementary to nucleotides 429 to 447 of the sequence of SEQ ID NO:9.

[0184] SEQ ID NO: 18 is an RNA sequence that is completely complementary to nucleotides 1805 to 1823 of the sequence of SEQ ID NO: 11 and completely complementary to nucleotides 1809 to 1827 of the sequence of SEQ ID NO: 9.

[0185] SEQ ID NO:19 is an RNA sequence that is completely complementary to nucleotides 921 to 939 of the sequence of SEQ ID NO:11 and completely complementary to nucleotides 903 to 921 of the sequence of SEQ ID NO:9. [Brief explanation of the drawings]

[0186] [Figure 1] Effect of siRNA PMP22 on PMP22 gene expression. Using Kruskal-Wallis analysis followed by Dunn's test, statistical differences were found between siRNA PMP22 (1-8) and the siRNA control (CT). Statistical differences were also found between siRNA PMP22 (1-8) and untreated cells (NT). [Figure 2] Effect of siRNA PMP22 on PMP22 protein levels. Protein levels were assessed by Western blot for siRNAs 5, 6, 7, and 8. Quantitation of relative PMP22 protein expression compared to GAPDH is represented by bars. [Figure 3]Effect of various concentrations of siRNA PMP22 on PMP22 and P0 mRNA expression. MSC-80 cells were transfected with 25 nM and 50 nM of siRNA control (siCt) and 25 nM, 50 nM, and 100 nM of siPMP22 #7 (si7) for 72 and 96 hours. PMP22 and P0 expression was assessed by real-time PCR and normalized to GAPDH expression. The 50 nM concentration was found to reduce PMP22 mRNA expression by 50% and had a long-lasting effect (up to 96 hours) without affecting P0 mRNA expression. [Figure 4] Effect of various concentrations of siRNA PMP22 on cell viability. Cell viability was assessed by using an MTT assay after 72 and 96 hours of transfection of siRNA control and siRNA PMP22 #7 at the same concentrations as in Figure 3. The 50 nM concentration has no significant effect on MSC-80 cell viability. [Figure 5] In vitro efficacy of siRNAPMP22-SQ nanoparticles and their effect on cell viability. MSC-80 cells were transfected with non-vectorized siRNACtrl (siCtrl), vectorized siRNACtrl with squalene (siCtrlSQNP), non-vectorized siPMP22 (siPMP22), or vectored siPMP22 with squalene (siPMP22-SQ NP) using Lipofectamine iMAX® for 48 and 72 hours. After 48 and 72 hours, cells were harvested, and mRNA was extracted and analyzed for PMP22 and PO gene expression (Figure 5) and cell viability (Figure 6). Nanoformulated PMP22-SQ inhibited PMP22 production over time to the same extent as non-vectorized siRNA PMP22, without affecting PO expression or cell viability. [Figure 6]In vitro efficacy of siRNAPMP22-SQ nanoparticles and their effect on cell viability. MSC-80 cells were transfected with non-vectorized siRNACtrl (siCtrl), vectorized siRNACtrl with squalene (siCtrlSQNP), non-vectorized siPMP22 (siPMP22), or vectored siPMP22 with squalene (siPMP22-SQ NP) using Lipofectamine iMAX® for 48 and 72 hours. After 48 and 72 hours, cells were harvested, and mRNA was extracted and analyzed for PMP22 and PO gene expression (Figure 5) and cell viability (Figure 6). Nanoformulated PMP22-SQ inhibited PMP22 production over time to the same extent as non-vectorized siRNA PMP22, without affecting PO expression or cell viability. [Figure 7] PMP22 gene expression (A, B) and behavioral tests (C, D, E) in B6 wild-type and transgenic PMP22 mice (n=5). PMP22 (A) and P0 (B) mRNA expression was assessed in the sciatic nerve of transgenic mice JP18 on a B6 background relative to their wild-type counterparts. Behavioral tests performed on the mice: beam walking test (C), locotronic (D), and paw withdrawal test (E). [Figure 8] PMP22 gene expression (A and B) and behavioral tests (C, D) in CBA wild-type and transgenic PMP22 mice (n=3). PMP22 (A) and P0 (B) mRNA expression was assessed in the sciatic nerve of transgenic mice JP18 on a CBA background relative to their wild-type counterparts. Behavioral tests performed on the mice: locotronic (C) and paw withdrawal test (D). [Figure 9]Effect of PMP22-SQ nanoparticles on behavior in a JP18 B6 background (n=5 / group). Mice were treated with vehicle (5% dextrose, siRNA control nanoparticles, siRNA PMP22 nanoparticles). Then, beam walking (A) and locotronic (B) tests were performed in addition to grip strength tests (forelimb force (C) and forelimb and hindlimb force (D)). [Figure 10] Effect of PMP22-SQ nanoparticles on behavior of mice in CBA background (n=3). Beam walking test was performed on mice before and after treatment with siRNA PMP22 nanoparticles. [Figure 11] Characterization of the siRNA PMP22-SQ bioconjugate. The recovered product was characterized by MALDI-TOF MS to have a molecular weight of 7628 (data not shown), and then after annealing with the antisense strand, the resulting nanoparticles were examined for their size by DLS (see Table 4 below) and for their morphology by Cryo-TEM (cryo-electron microscopy): the images show that the siRNA PMP22-SQ NPs have the same size as obtained by DLS. [Figure 12] Schematic diagram for the synthesis of the conjugated siRNA-squalene, SQ-5'DBCO(NHC6)-siRNA, via strain-promoted Huisgen cycloaddition of dibenzocyclooctyne. For simplicity, only one regioisomeric triazole is shown. A. Synthesis of siRNA PMP22-SQ bioconjugate: 1:50 siRNA:SQ molar ratio; solvent: DMSO, acetone, water; temperature: room temperature; incubation time: overnight with stirring; B. Evasion of acetone: under nitrogen flow. C. Purification by HPLC. D. Evasion of organic solvent by lyophilization; E. Bioconjugate annealing with the AS (antisense) strand; F. Bioconjugate solubilization in water:acetone (2:1). [Figure 13]Behavioral analysis of double transgenic JP18 / JY13 B6 CMT1A mice. Twelve-week-old double transgenic mice were divided into three groups (n = 6 each). They received the following treatments: 5% dextrose, siRNA Ct-SQ NP, and siPMP22-SQ NP, respectively. The wild-type B6 group served as a control. A beam-walking test was performed before and after treatment. The time taken by each mouse was recorded over three passes (mean ± SD). The JP18 / JY13 siPMP22-SQ NP group showed normalized time after treatment compared with the WT-B6 group and was significantly faster than the group receiving 5% dextrose and siRNA Ct-SQ NP. A locotronic test was performed after the end of treatment, which, like the beam-walking test, showed significant results. Three passes were recorded, and the data shown are the average (mean ± SD) of nine independent mice. (*) indicates significance between WT-B6 and other groups. (#) indicates significance between siRNA Ct NP group and other groups. ***, ###: p<0.001 (Anova followed by Bonferroni test). [Figure 14] Grip strength test analysis for double transgenic CMT1A mice. Grip strength tests were performed on both the forelimbs and the whole limbs at the end of treatment. JP18 / JY13 mice receiving siPMP22-SQ NP showed a significant improvement in their muscle strength compared with untreated mice (double transgenic 5% dextrose) and the JP18 / JY13 siRNA Ct-SQ NP group. Their strength became comparable to that of the WT-B6 group. (*) indicates significance between WT-B6 and other groups. (#) indicates significance between JP18 / JY13 5% dextrose and JP18 / JY13 siPMP22-SQ NP. ***, ###: p<0.001 (Anova followed by Bonferroni test). [Figure 15]Electrophysiological analysis of single-transgenic JP18 (A and B) and double-transgenic JP18 / JY13 CMT1A mice (C and D): compound muscle action potentials (A and C) and sensory nerve velocity (B and D). Data represent mean ± SD. (*) indicates significance between WT-B6 and other groups. (#) indicates significance between JP18 or JP18 / JY13 5% dextrose and JP18 or JP18 / JY13 siPMP22-SQ NP. *#: p<0.05; **: p<0.01; ***, ###: p<0.001 (Anova followed by Bonferroni test). [Figure 16] Fiber density and g-ratio analysis of small and large diameter fibers in toluidine-stained semi-ultrathin sections of the sciatic nerve of single transgenic JP18 CMT1A mice. No significant effect on myelination was detected among the different groups analyzed. [Figure 17] Fiber density and g-ratio analysis of small and large diameter fibers in toluidine-stained semi-ultrathin sections of the sciatic nerve of double transgenic JP18 / JY13 B6 CMT1A mice. No significant effect on myelination was detected among the different groups analyzed. [Figure 18] Quantitative analysis of the levels of transcription factors SOX10 (A) and KROX20 (B) in single transgenic JP18 mice. A. Data represent the mean ± SD, and each group consisted of three different mice. * represents significance between the WT-B6 group and other groups, and # represents significance between JP18 5% dextrose and JP18siPMP22-SQ NP. *: p<0.05; ***: p<0.001, ###: p<0.001 (Anova followed by Bonferroni test). B. Data represent the mean ± SD, and each group consisted of three different mice. * represents significance between the WT-B6 group and other groups, and # represents significance between JP18 5% dextrose and JP18siPMP22-SQ NP. **: p<0.01; ***: p<0.001, #: p<0.05; ###: p<0.001 (Anova followed by Bonferroni test). [Figure 19]Quantitative analysis of axon regeneration NF marker levels in single transgenic JP18 mice. Data represent mean ± SD, and each group consisted of three different mice. * indicates significance between the WT-B6 group and other groups, and # indicates significance between JP18 5% dextrose and JP18siPMP22-SQ NP. ***: p<0.001, ###: p<0.001 (Anova followed by Bonferroni test). [Figure 20] Quantitative analysis of Schwann cell markers SOX10 (A) and KROX20 (B) and axon regeneration NF marker (C) in double transgenic JP18 / JY13 CMT1A mice. Data represent mean ± SD. Each group consisted of three different mice. * indicates significance between the WT-B6 group and other groups, and # indicates significance between the JP18 / JY13 5% dextrose and JP18 / JY13 siPMP22-SQ NP. *: p<0.05; **: p<0.01; ***: p<0.001, ###: p<0.001 (Anova followed by Bonferroni test). [Figure 21] Long-term effects of beam walking and locotronic tests on double-transgenic JP18 / JY13 transgenic mice. JP18 / JY13 B6 mice received two cycles of treatment at 2.5 mg / kg per cycle. Treatment was stopped for 21 days between the two cycles to investigate relapse. Data from the beam walking test (left panel) represent the time spent crossing the bar (mean ± SD). Data from the locotronic test (right panel) represent the time recorded by a computerized program (mean ± SD). Mice were followed weekly for analysis of recovery and relapse periods. (*) indicates significance between WT-B6 and other groups. (#) indicates significance between JP18 / JY13 5% dextrose and JP18 / JY13 siPMP22-SQ NP. **, p<0.01; ***, p<0.001 (Anova followed by Bonferroni test). [Figure 22]Long-term effect analysis of the grip strength test on double-transgenic JP18 / JY13 transgenic mice. JP18 / JY13 B6 mice received two cycles of treatment at 2.5 mg / kg per cycle. Treatment was stopped for 21 days between the two cycles to investigate relapse. Grip strength test data (left panel) represent the force exerted by the mice in their forelimbs (mean ± SD). Grip strength test data (right panel) represent the force exerted by the mice in all limbs (mean ± SD). Mice were followed weekly for analysis of recovery and relapse periods. (*) represents significance between WT-B6 and other groups. (#) represents significance between JP18 / JY13 5% dextrose and JP18 / JY13 siPMP22-SQ NP. **, p<0.01, ***, p<0.001 (Anova followed by Bonferroni test). DETAILED DESCRIPTION OF THE INVENTION [Example]

[0187] Materials and Methods siRNA and chemical modifications Designed sequences of sense and antisense siRNA strands were purchased from Eurogentec, France. They were synthesized and 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 to provide nuclease stabilization. To preserve functionality, a dibenzocyclooctyne (DBCO) reactive group was introduced at the 5' end of the sense strand of each siRNA sequence via an N-(hexamethylenel)-6-oxohexanamide spacer (C6). To generate siRNA 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 Mg acetate, 100 mM K acetate] at 95°C for 3 minutes, then incubated at room temperature for 45 minutes before storage at -20°C.

[0188] Screening of siRNA against PMP22 To restore basal levels of PMP22 gene expression, eight siRNAs against PMP22 (see Tables 2 and 3) and a scrambled siRNA control (siRNA CTRL) were designed using three different methods (Tafer software, Thermofisher software, and the Reynold method). The siRNAs were transfected into a Schwann cell model (MSC 80) that endogenously expresses PMP22 using Lipofectamine iMAX®. The ability of the siRNAs to reduce PMP22 expression was tested at 24, 48, and 72 hours. The expression of both PMP22 and P0 genes was assessed by real-time PCR. The best siRNA sequences that could inhibit PMP22 expression by approximately 50% with a long-lasting effect without affecting P0 levels (PO deregulation has been described to be involved in CMT-1 disease) were then tested at various concentrations of 25 nM, 50 nM, and 100 nM to check their 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 siRNA to squalene and polyisoprenyl chains: Squalene was conjugated to siRNA via Huisgen cycloaddition of dibenzocyclooctyne (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). Briefly, the sense strand of the oligonucleotide was modified at the 5' end with a dibenzocyclooctyne residue, a commercially available modification for siRNA. Squalene carries an azide functional group. The conjugate was purified by HPLC and characterized by MALDI-TOFF mass spectrometry.

[0192] Annealing of both strands of siRNA PMP22 and siRNA CT was performed after bioconjugation of the sense strand to SQ, followed by precipitation in acetone / water. Under 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 nanoassemblies at a concentration of 10 μM. Control siRNA-SQ was prepared using the same protocol. The size of the nanoassemblies was determined by dynamic light scattering, and their zeta potential was determined by their electrophoretic mobility. The morphology of these squalene-based nanoassemblies was observed using cryo-transmission electron microscopy. Nanoassembly stability and drug release were tested in phosphate buffer (PBS) and cell culture medium.

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

[0194] (i) siRNA bioconjugation One nanomolar of the 5'-end-modified sense strand of siRNA PMP22, DBCO-C6 (1 mg / mL in DNAse / RNAse-free water), and 50 nmoles 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 under stirring at room temperature for 12 hours to obtain the siRNA PMP22-SQ bioconjugate. The next day, excess acetone was removed under nitrogen flow for 30 minutes, followed by a lyophilization step.

[0195] The bioconjugate was purified from excess unconjugated SQ by RP-HPLC on a polymer column as 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 the desired molar concentration of 10 μM. The same protocol was followed to obtain the siRNA Ct-SQ bioconjugate.

[0196] (ii) Purification of siRNAPMP22-SQ bioconjugate by HPLC HPLC purification was performed on a Thermoscientific high-performance liquid chromatography system (UltiMAte3000) 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 with a precolumn (Hamilton). Thermofisher Chromeleon software was used for data collection using 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 consisted of 5% acetonitrile, 90% water with 0.2 M TEAA (5%), pH 7.0, while mobile phase B consisted of 95% acetonitrile with 5% TEAA, 5% water. The gradients applied for purification were as follows: 0-8 min, linear gradient from 0% to 24% of phase B; 8-16 min, linear gradient from 24% to 90% of phase B; 16-18 min, linear gradient from 90% to 100% of phase B; 18-30 min, linear gradient from 100% of phase B; 30-32 min, linear gradient from 100% of phase B to 100% of phase A; and 32-42 min, re-equilibration at 100% of phase A. The siRNA-SQ bioconjugates were 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 A 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 acquire 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 described for the generation of siRNA from a single RNA strand, annealing of both siRNA PMP22 and siRNA Ct was performed after bioconjugation of the sense strand to SQ. Specifically, equimolar amounts of siPMP22C6-SQ bioconjugate and antisense siPMP22 were mixed in annealing buffer [30 mM HEPES-KOH (pH 7.4), 2 mM Mg acetate, 100 mM K acetate] and incubated at 95°C for 3 minutes, then incubated at room temperature for 45 minutes before storage at -80°C or direct precipitation. The same protocol was followed to obtain siRNA Ct-SQ bioconjugate.

[0199] (v) Preparation and Characterization of siRNAPMP22-SQ Nanoparticles The nanoparticles (NPs) siRNAPMP22-SQ and siRNA Ct-SQ were prepared by nanoprecipitation in acetone:water (1:2). Under stirring, one phase was slowly added to the other: 10 nmoles of siRNA-SQ was dissolved in 1 ml of DEPC-treated water and added dropwise to 500 ml 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 nanoassemblies at a concentration of 10 μM.

[0200] Hydrodynamic diameter (nm) was measured by dynamic light scattering (DLS) on a Malven Zeta Sizer NANO. Samples were analyzed at a concentration of 10 μM in HO. Three 5-minute measurements were performed per sample, and the mean diameter ± SD of three independent samples was calculated.

[0201] Cryo-transmission electron microscopy (cryo-TEM) was performed using a JEOL 2100 electron microscope at the electron microscopy platform (IBPS / Institut de Biologie Paris-Seine, Universite P. et M. Curie, Paris, France). A 4 μL droplet of siRNA PMP22-SQ NPs (2.2 mg / mL concentration) was deposited on a carbon-coated copper grid. Excess liquid was removed with blotting filter paper, and the samples were rapidly vitrified by placing them in liquid ethane using a guillotine. The samples were then transferred to a cryo-sample container. Observations were performed at an accelerating voltage of 200 kV under 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 study was first conducted 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 peripheral nerves, leading to the demyelination that is responsible for CMT-1A disease. Transgenic mice were purchased from TAAM CNRS after oocyte reviviscence in two genetic backgrounds, B6 and CBA. At 3 months of age, molecular and behavioral studies were performed in JP18 B6 and CBA mice to investigate 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 on the B6 or CBA background. For the JP18 mouse model, mouse pmp22 cDNA was transfected into PhCMV. * The gene was placed under the control of the -1 promoter, and therefore the mice overexpressed pmp22 throughout their lives.

[0203] This study was also carried out in a double transgenic mouse model (JP18 / JY13). This model was generated 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 using specific primers for PMP22 and tTA sequences, as previously described by Robertson et al.

[0204] Effect of siRNA administration against PMP22 on the progression of CMT-1A pathology JP18B6 mice were divided into three groups of five mice each, along with a wild-type group for comparison. One group received 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 via subocular IV injection twice a week, at an interval of 0.5 mg / kg per injection, for a cumulative dose of 2.5 mg / kg (a total of five treatments). At the end of treatment, the mice were sacrificed, and the sciatic nerves were harvested for further investigation. All animal experiments were approved by the Institutional Animal Experimentation Ethics Committee (CEEA) and the Research Council, registered with the French Ministry of Higher Education and Research (MESR, APAFIS#I 0131-2016112916404689 vl 6), and were performed in accordance with French laws and regulations under the conditions established by the European Community (Directive 2010 / 63 / EU). Experiments were performed in accordance with ethical standards and in accordance with the Declaration of Helsinki. Every effort was made to minimize animal suffering: treatment administration was performed under isoflurane anesthesia, and 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 cage. Food and water were provided ad libitum.

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

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

[0207] To investigate the long-term effects of siPMP22-SQ NPs, 12-week-old JP18 / JY13 B6 mice were used. In addition to a wild-type B6 control group, the mice were divided into three groups of six mice each: JP18 / JY13 vehicle, JP18 / JY13 siRNA Ct-SQ NPs, and JP18 / JY13 siPMP22-SQ NPs. Two cycles of treatment were administered. The first cycle consisted of a cumulative dose of 2.5 mg / kg of siPMP22-SQ NPs and siRNA Ct-SQ NPs, administered twice a week at an interval of 0.5 mg / kg per injection. Treatment was then stopped for three weeks to check for relapse. At the end of the first treatment cycle, three mice per group were sacrificed, and sciatic nerves were collected for further analysis. At week 4, a new cycle of treatment was initiated with another cumulative dose of 2.5 mg / kg siPMP22-SQ NPs and siRNA Ct-SQ NPs, administered twice weekly at 0.5 mg / kg injection intervals. Behavioral testing was performed before treatment, during the first treatment cycle at 1.5 mg / kg, during the first treatment cycle at 2.5 mg / kg, two weeks after the first treatment cycle was discontinued, three weeks after the first treatment cycle was discontinued, during the second treatment cycle at 1.5 mg / kg, and during the second treatment cycle at 2.5 mg / kg. Sciatic nerves were removed at the end of the second cycle for further analysis.

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

[0209] Locotronic: A locotronic device was used to test motor coordination during walking. Mice crossed a 75 x 5 x 20 cm flat ladder with bars (7 mm in diameter) set 2 cm apart. Infrared photocell sensors located above and below the bars monitored foot errors. The locotronic device was linked to software that automatically recorded the time taken by the mouse to cross the path as well as foot errors. Time and errors were assessed in three trials with a 15-minute rest between trials. Testing was performed at the end of treatment. Statistical analysis of the acquired data was performed by calculating the average of three trials per day over three days for each group. Data were presented as mean ± SD.

[0210] Grip strength test: Neuromuscular strength was assessed by using a grip strength test. This test was performed using a computerized grip strength measuring device. 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 mouse's forepaws, each mouse was gently supported by the base of its tail and the animal was allowed 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 its tail until the grip was released. This process was repeated three times, and the highest force was automatically recorded by the instrument in grams (g). To measure the force related to both limbs, each mouse was allowed to grasp a rectangular metal bar with its forepaws and hindpaws. It was then gently pulled 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: A hind paw withdrawal test was used to assess heat sensitivity: the platform on which the animals were placed was set at 52°C and the latency to sense the heat and withdraw their paw was recorded for analysis.

[0212] Electrophysiological investigations Testing was performed using standard EMG equipment (Natus / EMG) in accordance with the guidelines of the American Association of Neuromuscular and Electrodiagnostic Medicine. Anesthesia was achieved by isoflurane inhalation, and mice were placed in an induction chamber containing 1.5–2% isoflurane in pure oxygen. Anesthesia was maintained at the same level throughout the procedure via a face mask. Mice were placed on their front 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 anodal electrode was inserted at the superior base of the tail, while a receptor or recording needle was inserted into the medial part of the gastrocnemius muscle. An 8 mA supramaximal square-wave pulse was delivered through the stimulating needle and recorded as a magnitude through the muscle. For sensory nerve conduction velocity measurements, multiple stimulations of the caudal nerve were delivered through the stimulating needle, which was positioned 2 / 3 of the tail length and 2–2.5 cm from the receptor needle. A ground electrode was inserted midway between the stimulating and receptor electrodes. Sensory nerve conduction velocity was calculated from the stimulation latency and the distance between the stimulating and receptor electrodes.

[0213] Biological Testing The sciatic nerve histology of CMT-1A animals (treated or not with siRNA) was examined by electron microscopy, where the number of myelinated or unmyelinated axons and the thickness of the myelin sheath were assessed by determining the g ratio. The number of mitochondria per axon was then calculated to assess axonal pathology. 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 to control levels (WT animals or CMT-1A mice treated with siRNA PMP22). Neuromuscular junctions (NMJs) of CMT-1A mice treated or not with siRNA PMP22 were analyzed by immunohistochemistry. Muscle weakness in CMT-1A is attributed to axonal degeneration, leading to muscle denervation and atrophy.

[0214] In CMT-1A, neuromuscular junction loss 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 (innervation, denervation, reinnervation, etc.) will be performed.

[0215] Pharmacokinetics and biodistribution studies Radiolabeled free or SQ-conjugated siRNA PMP22 (siRNA PMP22-SQ 32 PMP22-SQ NPs) were intravenously injected into transgenic mice carrying CMT-1A. Organs and blood were collected at various time points, and the concentrations of free versus siRNA PMP22-SQ NPs were determined by counting radioactivity using a g counter. Radio-HPLC analysis also demonstrated that siRNA PMP22 was not degraded once accumulated in the desired tissues. Pharmacokinetic parameters were calculated (e.g., plasma half-life and clearance). Notably, greater than 50% of squalene is transported by LDL and HDL lipoproteins; therefore, possible interactions of siRNA PMP22-SQ NPs with blood components (i.e., LDL, HDL, VLDL, blood cells, etc.) were assessed as previously described by Sawle et al. (Journal of lipid research 43, pp. 335-343, 2002).

[0216] statistical analysis All data are presented as mean ± standard deviation (SD). Nonparametric Kruskal-Wallis analysis followed by Dunn's test or Anova followed by Bonferroni test were used to compare multiple treatments using GraphPad Prism. p < 0.05 was considered a statistically significant level.

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

[0218] Effect of various concentrations of siPMP22 on mRNA PMP22 and P0 and on cell viability We then investigated the effects of various concentrations of siPMP7 (25 nM, 50 nM, and 100 nM) on the gene expression of PMP22 and PO after transfection of MSC80 cells for 72 and 96 hours to determine the optimal concentration that could normalize PMP22 expression without affecting the expression of PO, a protein that is involved in myelin compaction and cell viability together with PMP22. Our results indicate that the optimal concentration is 50 nM. siPMP7 (50 nM) reduced PMP22 gene expression by 50% for 72 and 96 hours, respectively, while it did not affect PO gene expression (see Figure 3). Furthermore, cell viability testing using an MTT assay showed that 50 nM siPMP7 had no significant effect on MSC80 viability (see Figure 4). The siRNA control (siCt) did not alter the expression of the two genes investigated or cell viability.

[0219] siPMP7, subsequently named siPMP22, was the best candidate to continue investigation because it had the ability to reduce PMP22 levels by 50%, had no effect on P0 levels, and 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) is.

[0221] Binding of siRNA PMP22 and siRNA CTRL to squalene (SQ) and characterization of siRNA PMP22-SQ nanoparticles The bioconjugates siRNA PMP22-SQ and siRNA CTRL were obtained by Cu-free click chemistry with yields exceeding 90% thanks to the optimized reaction conditions detailed in Figure 12. For siRNA PMP22-SQ, the recovered bi-product, identified by HPLC and analyzed by MALDI-TOF MS, indicated that the bioconjugate had the expected 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 a Tyndall effect, suggesting the formation of nanoparticles. Dynamic light scattering (DLS) and cryoTEM images showed the formation of stable nanoparticles of approximately 180 nm with good polydispersity index (0.14–0.2) for 30 days, reflecting a homogeneous solution suitable for IV injection (see Figure 4). For siRNA CTRL, the molecular weight determined 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 days 0 and 30, respectively).

[0222] [Table 4]

[0223] These nanoparticles, siRNA PMP22-SQ and siRNA CTRL, were then tested in vitro over a time course (48 and 72 hours) for their efficacy and effect on cell viability. Previously, we demonstrated that squalene-based siRNA nanoparticles cannot spontaneously enter cells without any cationic compound. Therefore, SQ-based siRNA PMP22 nanoparticles and non-vectorized siRNA PMP22 were transfected into MSC-80 cells using Lipofectamine iMAX®. 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, neither nanoformulated nor non-vectorized siRNA controls affected PMP22 mRNA levels or cell viability when cells were transfected (see Figures 5 and 6 ).

[0224] In summary, this part of the study i) Squalenoylation of siRNA PMP22 resulted in approximately 100% yield after bioconjugation to SQ and approximately 85% yield after HPLC purification; ii) the resulting NPs are 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. showed.

[0225] In vivo experiments: PMP22 gene expression and behavioral testing in B6 and CBA wild-type and transgenic PMP22 mice First, we examined PMP22 expression in the sciatic nerve by comparing transgenic PMP22 mice on the B6 and CBA backgrounds with their wild-type counterparts. For both PMP22 mice on the B6 and CBA backgrounds, we found increased PMP22 mRNA expression compared with wild-type mice. P0 expression was found at similar levels in both strands (see Figures 7A and 7B, and Figures 8A and 8B).

[0226] Fine motor coordination and balance were assessed using beam walking and locotronic assays. The goal of these tests is for mice to remain upright and walk across an elevated, narrow beam to a safety platform. Interestingly, beam walking and locotronic assays showed increased time and number of errors for both PMP22 mice on the B6 and CBA backgrounds compared with wild-type mice, suggesting the development of CMT-1A disease. Indeed, as seen in Figures 7C, 7D, and 8C, a significant decrease in speed on the beam was observed in CMT-1A mice. In fact, CMT-1A mice walked twice as slower than control mice, and the number of foot slips (errors) in CMT-1A animals was increased. The paw withdrawal test, which detects unpleasant stimuli such as pain caused by stimulation of nociceptors (sensory neurons), was found to be identical for transgenic and wild-type mice, suggesting that the sensory neurons of these transgenic mice were not affected. Therefore, a useful model for our investigation represents CMT-1A pathology characterized by i) a less than two-fold increase in PMP22 expression due to a 1.5 Mb duplication on chromosome 17p11.2, ii) reduced motor neuron activity, and iii) sensory neurons were unaffected. Peripheral nerve injury tends to induce defects that can cause rodents to slip to one side.

[0227] Effect of PMP22-SQ nanoparticles on the behavior of JP18 B6 and CBA backgrounds Interestingly, a beam-walking test performed in JP18 on a B6 background showed that the time spent crossing the road by mice treated with siRNA PMP22-SQ nanoparticles was comparable to that of untreated wild-type mice (see Figure 8C; 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] Taken together, these results indicate that siRNAPMP22-SQ nanoparticles can restore the locomotor activity of CMT-1A transgenic mice.

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

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

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

[0232] TEM micrographs of ultrathin sections of sciatic nerves showed modulation of myelination in single and double transgenic mice treated with siPMP22-SQ NPs (data not shown). It was also shown that the nanoparticles localized in the cytoplasm of Schwann cells and could enter the nerve through the nodes of Ranvier (data not shown).

[0233] Next, the expression of proteins involved in myelination and axon regeneration was analyzed in the single-transgenic mouse model. The levels of the transcription factor SOX10, the myelinating factor KROX20, and axon regeneration NF markers were reduced in the JP18 5% dextrose and siRNA Ct groups; notably, siPMP22-SQ NP treatment significantly increased these levels compared to the JP18 5% dextrose group (see Figures 18A and 18B and 19). Similar results were obtained in the double-transgenic mouse model: siPMP22-SQ NP treatment increased myelinating transcription factors (SOX10 and KROX20) and enhanced axon 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 The long-term effects of siPMP22-SQ NPs on beam walking, locotronic, and grip strength tests were then analyzed. Double transgenic JP18 / JY13 B6 mice received two cycles of treatment at 2.5 mg / kg per cycle. Treatment was stopped for 21 days between cycles to investigate relapse. Mice were followed weekly to analyze recovery and relapse periods.

[0235] The motor activity of JP18 / JY13 mice recovered after 1.5 mg / kg administration (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 relapse 37 days after the first injection. From the same dose 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 original approach aims to develop a novel therapy for CMT-1A disease by specifically targeting the overexpression of peripheral myelin protein 22 (PMP22) with siRNA.

[0237] We successfully developed siRNA against PMP22, which could 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, knockdown of PMP22 did not affect MPZ (P0), which is involved in the myelination process together with PMP22 (dysregulation of MPZ can lead to another type of CMT, CMT1B). Furthermore, the mentioned siRNA did not affect cell viability.

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

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

[0240] In CMT-1A, the major form of CMT pathology, there are no comparable studies available, either in Europe or elsewhere in the world, describing the restoration of motor activity in mice by knocking down the expression of the gene by 50% by siRNA, thanks to its regulation in fine-tuning PMP22 expression.

Claims

1. A pharmaceutical composition comprising an antisense RNA targeting mRNA encoding PMP22 protein for use in treating Charcot-Marie-Tooth 1A (CMT-1A), the antisense RNA comprises 19 to 21 consecutive nucleotides of a sequence that is completely complementary to the mRNA encoding the PMP22 protein; the antisense RNA is an siRNA comprising (i) a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 19, and (ii) optionally one or two single-stranded overhangs; the antisense RNA reduces the amount of PMP22 protein in the cell; Pharmaceutical compositions.

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

3. 3. The pharmaceutical composition of claim 1, wherein the siRNA comprises one or two single-stranded overhangs, the single-stranded overhangs consisting of at least one deoxyribonucleotide T.

4. 4. The pharmaceutical composition of claim 1, wherein the antisense strand of the siRNA consists of a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 19, and the siRNA comprises one or two single-stranded overhangs.

5. 5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the antisense RNA is provided in the form of nanoparticles comprising the antisense RNA.

6. 6. The pharmaceutical composition for use according to any one of claims 1 to 5, wherein the antisense RNA is administered intravenously, intraperitoneally, subcutaneously or intraneurally, preferably to the sciatic nerve.

7. 7. The pharmaceutical composition for use according to any one of claims 1 to 6, wherein the antisense RNA is used in combination with at least another drug useful in the treatment of Charcot-Marie-Tooth 1A.

8. an antisense RNA targeting an mRNA encoding a PMP22 protein, the antisense RNA comprising 19 to 21 consecutive nucleotides of a sequence completely complementary to the mRNA encoding the PMP22 protein; the antisense RNA is an siRNA comprising (i) a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 18, and (ii) optionally one or two single-stranded overhangs; the antisense RNA reduces the amount of PMP22 protein in the cell; Antisense RNA.

9. 9. The antisense RNA targeting PMP22 of claim 8, wherein the siRNA comprises one or two single-stranded overhangs, each of the single-stranded overhangs consisting of at least one deoxyribonucleotide T.

10. An antisense RNA targeting PMP22 described in claim 8 or 9, wherein the antisense strand of the siRNA consists of a sequence selected from the group consisting of the sequences of SEQ ID NO: 16 and SEQ ID NO: 18, and the siRNA contains one or two single-stranded overhangs.

11. Nanoparticles comprising the antisense RNA of any one of claims 8 to 10.

12. The nanoparticle of claim 11 , wherein the antisense RNA is conjugated to squalene or a derivative thereof.

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