Allele-specific siRNA therapy for dynamin 2-related diseases

Abstract

The present invention relates to the treatment of myopathy. We report the identification of effective AS-siRNAs against two nucleotide versions of two non-pathogenic DNM2 SNPs that can be used to silence any mutations carried by the same mRNA. In addition, the first AS-siRNA was developed targeting a DNM2 mutation associated with a severe neonatal phenotype, namely the p.S619L mutation. They then developed other AS-siRNAs targeting the p.S619L and p.R465W mutations. They also report the functional benefits of this new set of siRNAs for several defects identified in patient-derived cell lines. The development of these new AS-siRNAs, in addition to the previous one targeting the p.R465W mutation, provides a large number of allele-specific molecules that can target the majority of AD-CNM patients. Interestingly, siRNAs against DNM2 SNPs are versatile molecules with greater potential applications in silencing DNM2 mutations in CMT and HSP, as well as in reducing DNM2 expression in a controlled manner in diseases associated with deleterious overexpression. Thus, the present invention relates to allele-specific siRNAs (AS-siRNAs) that can silence the expression of only one allele of a heterozygous DNM2 gene, where the target allele comprises a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rs12461992 (A or T), and / or a disease-causing mutation selected from the group consisting of c.1393C>T or c.1856C>T.

Description

[Technical Field]

[0001] The present invention relates to an allele-specific siRNA (AS-siRNA) that can silence the expression of only one allele of a heterozygous DNM2 gene, wherein the target allele contains a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rs12461992 (A or T), and / or a disease-causing mutation selected from the group consisting of c.1393C>T or c.1856C>T. [Background technology]

[0002] Autosomal dominant centronuclear myopathy (AD-CNM, MIM #160150) is a rare congenital myopathy associated with a wide clinical spectrum ranging from severe neonatal to mild adult forms (1). The classic late childhood or adult-onset form presents with delayed motor milestones and diffuse skeletal muscle weakness, primarily involving the facial and limb muscles, whereas pediatric patients affected with the severe neonatal form typically have generalized weakness, hypotonia, and facial weakness (2, 3). AD-CNM is caused by mutations in the DNM2 gene, which encodes dynamin 2 (DNM2) (4). Additionally, DNM2 mutations also cause rare forms of Charcot-Marie-Tooth disease (CMT) (5) and hereditary spastic paraplegia (HSP) (6). Deleterious DNM2 overexpression has been reported in several cancers (7-12) and X-linked recessive CNM (13), highlighting the significant involvement of DNM2 in human disease. DNM2 belongs to the large GTPase superfamily (14) and acts as a mechanochemical scaffolding molecule, oligomerizing and deforming biological membranes, leading to the formation and release of vesicles from the plasma membrane and intracellular membrane compartments. Furthermore, several studies have highlighted the role of DNM2 as a regulator of both the actin and microtubule cytoskeleton (15, 16). Over 30 DNM2 mutations have been reported in patients with AD-CNM (17), and when tested, mutant proteins are normally expressed (4, 18). The mutations are thought to mediate gain-of-function and / or dominant-negative effects through increased GTPase activity and the formation of abnormally stable DNM2 oligomers. (19, 20) Additionally, the lack of haploinsufficiency in AD-CNM is supported by data from patients and the lack of phenotypes produced by heterozygous knockout mice expressing 50% Dnm2. (13, 21)

[0003] We recently developed a therapeutic approach for DNM2-associated AD-CNM using allele-specific RNA interference (AS-RNAi), which specifically suppresses mutant protein expression from the mutant allele without reducing expression from the wild-type allele (22). AS-RNAi has been demonstrated as a powerful strategy in cells derived from patients and animal models of numerous dominantly inherited diseases (23), leading to two clinical trials targeting keratin 6a mutations that cause pachyonychia congenita (24) and a KRAS mutation implicated in pancreatic cancer (25). By applying this strategy, we reported functional rescue in a knock-in Dnm2R465W / + mouse model of AD-CNM and in patient-derived fibroblasts, both of which express the most common mutation encountered in patients (p.R465W, found in approximately 30% of patients) (22). Extending this strategy to the entire AD-CNM patient population will require either the development of personalized medicine through specific siRNAs for each reported mutation or the development of alternative approaches that allow targeting separate dominant DNM2 mutations using a limited number of allele-specific siRNAs. In the specific cases of triplet repeat diseases, such as Huntington's disease or spinocerebellar degeneration, where the sequence of gene mutations makes allele-specific silencing difficult, AS-siRNAs have been developed against disease-associated single nucleotide polymorphisms (SNPs) (23, 26-28). They developed a similar strategy for AD-CNM by exploiting the presence of two nonpathogenic SNPs in the DNM2 sequence that are frequently heterozygous in the general population. Summary of the Invention

[0004] Here, we report the identification of effective AS-siRNAs against two nucleotide versions of two nonpathogenic DNM2 SNPs that can be used to silence any mutations carried by the same mRNA. In addition, the first AS-siRNA was developed targeting a DNM2 mutation associated with a severe neonatal phenotype, namely the p.S619L mutation. They then developed other AS-siRNAs targeting the p.S619L and p.R465W mutations. They also report the functional benefits of this new set of siRNAs for several defects identified in patient-derived cell lines. The development of these new AS-siRNAs, in addition to the previous one targeting the p.R465W mutation, provides a large number of allele-specific molecules that can target the majority of AD-CNM patients. Interestingly, siRNAs against DNM2 SNPs are versatile molecules with even greater potential applications for silencing DNM2 mutations in CMT and HSP, as well as for reducing DNM2 expression in a controlled manner in diseases associated with deleterious overexpression.

[0005] Therefore, the present invention relates to an allele-specific siRNA (AS-siRNA) capable of silencing the expression of only one allele of a heterozygous DNM2 gene, wherein the target allele comprises a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rs12461992 (A or T), and / or a disease-causing mutation selected from the group consisting of c.1393C>T or c.1856C>T. In particular, the present invention is defined by its claims.

[0006] Detailed Description of the Invention The present inventors have investigated a therapeutic approach based on specifically suppressing the expression of only one allele of DNM2 while preserving the other DNM2 allele. On the one hand, this strategy aims to reduce DNM2 expression levels in a controlled manner in diseases associated with DNM2 overexpression. On the other hand, this strategy may be useful in autosomal dominant diseases caused by heterozygous mutations in the DNM2 gene by specifically inhibiting the expression of the mutant allele without reducing the level of the wild-type DNM2 allele required for normal cell function. To this end, the present inventors have discovered a highly efficient allele-specific siRNA that can inhibit only one allele of the heterozygous DNM2 gene in cells in a controlled manner. This strategy has been patented in International Patent Application Publication No. 2018100010. However, we have not designed and tested all possible siRNAs for the two most frequent non-pathogenic DNM2 SNPs, rs2229920 (C or T) or rs12461992 (A or T), and the two most disease-causing mutations, c.1393C>T or c.1856C>T. In this application, we designed these siRNAs and tested them. They showed that some were highly effective both in vitro and in vivo.

[0007] In a first aspect, the present invention relates to an allele-specific siRNA (AS-siRNA) capable of silencing the expression of only one allele of a heterozygous DNM2 gene, wherein the target allele comprises a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rs12461992 (A or T), and / or a disease-causing mutation selected from the group consisting of c.1393C>T or c.1856C>T.

[0008] Dynamin 2 is encoded by the DNM2 gene (gene ID 1785). More precisely, the DNM2 gene is located within the short arm of chromosome 19 at position 13.2 (19p13.2). The dynamin 2 gene or gene product is also known by other names, including, but not limited to, CMT2M, CMTDI1, CMTDIB, DI-CMTB, DYN2, DYN2_human, dynamin II, and DYNII. DNM2 plays an important role in endocytosis and in the structural framework of cells (cytoskeleton). The protein interacts with multiple parts of the cytoskeleton, including microtubules and actin, which organize into filaments and provide structure. These parts of the cytoskeleton are involved in the movement of molecules within the cell, cell shape, cell motility, and cell attachment to each other or to the extracellular matrix. Therefore, alterations in the DNM2 gene can disrupt endocytosis and interfere with cytoskeletal positioning or dynamics, leading to abnormal cellular function. As previously described, several dominantly inherited disorders, such as autosomal dominant centronuclear myopathy, Charcot-Marie-Tooth disease, and hereditary spastic paraplegia, are caused by heterozygous mutations in the DNM2 gene. Overexpression of DNM2 is also pathological and has been implicated in some of the pathophysiological mechanisms of other diseases, such as X-linked myotubular myopathy or cancers, such as prostate and pancreatic cancer.

[0009] RNA interference is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Therefore, interfering RNA is RNA that can downregulate the expression of target proteins. For example, it includes small interfering RNA (siRNA), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and short hairpin RNA (shRNA) molecules. RNA interference refers to the phenomenon in which dsRNA specifically suppresses the expression of target genes at the post-transcriptional level. Under normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) several thousand base pairs in length. In vivo, dsRNA introduced into cells is cleaved by an enzyme called DICER into a mixture of short dsRNA molecules called siRNAs. In mammalian cells, the siRNAs produced by Dicer are approximately 21 base pairs (bp) in length. The siRNAs then bind to the RNase complex RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. RNA interference is also a valuable research tool because double-stranded 19- to 23-bp siRNAs can be used to selectively and robustly induce the silencing of specific genes of interest. The main advantage of this approach is specificity, since siRNAs can discriminate between two sequences even when they differ by only a single nucleotide.

[0010] The present inventors have used the specificity of siRNA to specifically inhibit one allele of a heterozygous DNM2 gene, and as a result, in that particular case, the siRNA is referred to as an "allele-specific siRNA" (AS-siRNA).

[0011] AS-siRNA refers to any siRNA that can specifically silence only one allele of a target gene, where an allele is one of several alternative forms of a gene occupying a given locus on a chromosome. "Gene silencing" refers to the suppression or reduction of gene expression. Gene silencing can be mediated through processes that affect transcription and / or processes that affect post-transcriptional mechanisms. In some embodiments, gene silencing occurs when an siRNA triggers the degradation of a gene's mRNA in a sequence-specific manner via RNA interference. Thus, a gene includes coding sequences and / or regulatory sequences required for expression. For example, "gene" refers to a nucleic acid fragment that expresses mRNA, functional RNA, or a specific protein, including regulatory sequences. "Gene" also includes non-expressed DNA segments that may, for example, form recognition sequences for other proteins.

[0012] In the context of the present invention, the gene is the DNM2 gene, which encodes the dynamin 2 protein. Thus, the AS-siRNA of the present invention specifically silences one allele of the DNM2 gene, which is a variant form of the DNM2 gene.

[0013] In the context of the present invention, the DNM2 gene is a heterozygous DNM2 gene. A heterozygous DNM2 gene is a DNM2 gene that exists in a cell in a heterozygous state. "Heterozygous" means that a given chromosomal locus has two different alleles. Diploid organisms such as humans contain two copies of each chromosome (one maternal and one paternal chromosome), called homologous chromosomes. Therefore, each homologous chromosome has one allele of a given gene. If the two alleles of a given gene differ with respect to a given mutation or polymorphism, the diploid organism is heterozygous.

[0014] In the context of the present invention, a cell or organism is heterozygous for the DNM2 gene, the DNM2 gene being present in the heterozygous state, ie the two alleles of the DNM2 gene differ with respect to a given mutation or polymorphism.

[0015] In one embodiment of the present invention, heterozygosity refers to a genotype in which one allele has a wild-type DNM2 sequence and the other allele has a sequence encoding a DNM2 variant, particularly where the sequence encoding the DNM2 variant contains a mutation not present in the wild-type sequence.

[0016] In a specific embodiment, the DNM2 gene is heterozygous for the presence of a non-pathological polymorphism. In this second embodiment, the AS-siRNA of the present invention targets only one allele of the DNM2 gene, regardless of whether it contains the non-pathological polymorphism or not.

[0017] In a particular embodiment, the DNM2 gene is heterozygous for the presence of a disease-causing mutation. In this embodiment, the AS-siRNA of the invention targets an allele of the DNM2 gene that contains the disease-causing mutation.

[0018] As-siRNA targeting alleles of the DNM2 gene containing non-pathological polymorphisms In a particular aspect, the present invention relates to an AS-siRNA capable of silencing the expression of only one allele of a heterozygous DNM2 gene in a cell, wherein the DNM2 gene is heterozygous for the presence of a non-pathological polymorphism.

[0019] In certain embodiments, the DNM2 gene contains a common heterozygous non-pathological polymorphism. "Common heterozygous non-pathological polymorphism" refers to a polymorphism that has a high heterozygous frequency, i.e., that is frequently found in the heterozygous state in the population. "Frequent" refers to a polymorphism that is found in the heterozygous state in at least 20%, 30%, 40%, particularly at least 40% of the general population.

[0020] "Non-pathological polymorphism" refers to a mutation in the nucleic acid sequence of a gene that is not associated with disease. Therefore, according to the present invention, a non-pathological polymorphism corresponds to a sequence mutation in a gene that, when considered independently of other sequence variations, is not associated with pathology by itself. For clarity, when a non-pathological polymorphism is heterozygous in a cell, it means that both polymorphisms are considered non-pathological when considered independently of other sequence variations that may occur on the same gene. Non-pathological polymorphisms can include mutations in coding and non-coding regions. Furthermore, non-pathological polymorphisms include nucleotide substitutions, deletions, and / or additions, including those that result in missense and nonsense mutations that do not lead to pathology. In particular, non-pathological polymorphisms are single nucleotide substitutions.

[0021] In particular, when the overexpression of non-mutated DNM2 is associated with a pathological condition, the AS-siRNA of the present invention can be used to reduce the expression of DNM2 protein by targeting heterozygous common polymorphisms. For example, the overexpression of non-mutated DNM2 protein is correlated with X-linked myotubular myopathy or cancers such as prostate cancer and pancreatic cancer. Therefore, the present invention relates to AS-siRNA that targets DNM2 alleles containing non-pathological polymorphisms.

[0022] In another embodiment, the DNM2 allele containing the non-pathological polymorphism is located on the same allele as the heterozygous disease-causing mutation. By targeting heterozygous common polymorphisms rather than each specific disease-causing mutation, a single AS-siRNA can be used to inhibit the expression of more than one disease-causing mutation in more than one patient. Thus, in one embodiment, the targeted version of the heterozygous non-pathological polymorphism is located on the same allele as the disease-causing mutation and not on the wild-type allele that carries the other version of the polymorphism. In other words, targeting the heterozygous non-pathological polymorphism allows for differentiation between mutant and wild-type DNM2 alleles. The disease-causing mutation can be any heterozygous mutation in the DNM2 gene that is involved in or associated with a disease. For example, the disease-causing mutation in the DNM2 gene is involved in or correlates with a disease selected from autosomal dominant centronuclear myopathy (AD-CNM), T-cell acute lymphoblastic leukemia, Charcot-Marie-Tooth disease (CMT), or hereditary spastic paraplegia (HSP). In particular, disease-causing mutations in the DNM2 gene are involved in or correlate with autosomal dominant centronuclear myopathy (AD-CNM). In a specific embodiment, the DNM2 gene is heterozygous for the presence of a missense mutation selected from the group consisting of c.1393C>T; c.1105C>T; c.1106G>A; c.1565G>A; c.1856C>T; or c.1948G>A, which are involved in the following substitutions in the DNM2 protein sequence: p.R465W, p.R369W, p.R369Q, p.R522H, p.S619L, and p.E650K, respectively. In another specific embodiment, the DNM2 gene is heterozygous for the presence of a c.1393C>T mutation, which is involved in the p.R465W substitution in the DNM2 protein sequence.

[0023] Therefore, in a particular aspect, the present invention relates to an AS-siRNA capable of silencing the expression of only one allele of a heterozygous DNM2 gene in a cell, wherein the target allele comprises a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rs12461992 (A or T).

[0024] The present inventors have designed and tested many siRNAs that can target the most common non-pathological polymorphisms (rs2229920 (C or T), designated SNP1, or rs12461992 (A or T), designated SNP2) in in vitro and in vivo experiments. These sequences are listed in Tables 1 to 4.

[0025] Therefore, the present invention provides an allele-specific siRNA (AS-siRNA) that can silence the expression of only one allele of a heterozygous DNM2 gene, wherein the AS-siRNA is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99 , 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, and 151.

[0026] In particular, the present invention relates to an allele-specific siRNA (AS-siRNA) capable of silencing the expression of only one allele of a heterozygous DNM2 gene, wherein the AS-siRNA comprises a sense strand selected from the group consisting of SEQ ID NOs: 15, 17, 19, 33, 51, 53, 55, 57, 61, 67, 69, 71, 93, 95, 97, 135, 137, 141, 143, 145, and 147.

[0027] [Table 1] Table 1: siRNA for SNP1 human (rs2229920) with polymorphism T. The upper sequence corresponds to the sense strand (or sense sequence) (5'-3') and is called the "passenger sequence," and the lower sequence corresponds to the antisense strand (or antisense sequence) (3'-5') and is called the "guide sequence" (see, for example, Si1-SNP1-T).

[0028] [Table 2] Table 2: siRNA for SNP1 human (rs2229920) with polymorphism C. The upper sequence corresponds to the sense strand (or sense sequence) (5'-3') and is called the "passenger sequence," and the lower sequence corresponds to the antisense strand (or antisense sequence) (3'-5') and is called the "guide sequence" (see, for example, Si1-SNP1-C).

[0029] [Table 3] Table 3: siRNA for SNP2 human (rs12461992) with polymorphism A. The upper sequence corresponds to the sense strand (or sense sequence) (5'-3') and is called the "passenger sequence," and the lower sequence corresponds to the antisense strand (or antisense sequence) (3'-5') and is called the "guide sequence" (see, for example, Sil-SNP2-A).

[0030] [Table 4] Table 4: siRNA for SNP2 human (rs12461992) with polymorphism T. The upper sequence corresponds to the sense strand (or sense sequence) (5'-3') and is called the "passenger sequence," and the lower sequence corresponds to the antisense strand (or antisense sequence) (3'-5') and is called the "guide sequence" (see, for example, Sil-SNP2-T).

[0031] The AS-siRNA of the present invention can silence only one allele of the DNM2 gene containing a heterozygous non-pathological polymorphism by specifically hybridizing to the gene transcript (messenger RNA or mRNA) derived from the allele of the DNM2 gene. Therefore, the AS-siRNA of the present invention is complementary to the mRNA derived from the allele of DNM2 and binds to the mRNA through base pairing. The term "complementary" refers to the ability of a polynucleotide to form base pairs with another polynucleotide molecule. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. In particular, the degree of complementarity between the AS-siRNA of the present invention and the target mRNA is approximately 100%.

[0032] In a specific embodiment, the AS-siRNA of the present invention targets the region of the DNM2 gene transcript that contains the non-pathological polymorphism.Therefore, the AS-siRNA of the present invention is complementary to the sequence of the mRNA that contains the non-pathological polymorphism.The specificity of siRNA allows it to distinguish between two sequences even if they differ by only one nucleotide.This property allows the AS-siRNA of the present invention to target polymorphisms that result in single nucleotide substitutions.

[0033] In one specific embodiment, in the absence of a disease-causing mutation, the target allele (and, consequently, the target mRNA) can be arbitrarily selected to reduce the level of DNM2 mRNA and / or protein. In particular, when the goal is only to reduce the overall level of DNM2 mRNA or DNM2 protein, the AS-siRNA of the present invention can target mRNA with or without the non-pathological polymorphism. For example, the AS-siRNA of the present invention can target either of the DNM2 alleles carrying one of two versions of a heterozygous non-pathological polymorphism in which DNM2 is overexpressed in cells, for example, in X-linked myotubular myopathy or in cancers such as prostate cancer and pancreatic cancer.

[0034] In another specific embodiment, the target version of the non-pathological polymorphism is present on the same allele as the disease-causing mutation. Therefore, the AS-siRNA of the present invention targets and silences only the allele that carries the target version of the polymorphism and the disease-causing mutation. This specific embodiment requires prior identification of the location of the disease-causing mutation.

[0035] AS-siRNA targeting the allele of the DNM2 gene containing the disease-causing mutation In one embodiment of the present invention, the DNM2 gene is heterozygous for the presence of a disease-causing mutation. Therefore, the DNM2 gene exists in two different forms corresponding to two alleles: one DNM2 allele is a "wild-type allele," while the other is a "mutant allele." In this embodiment, the AS-siRNA of the present invention specifically targets and silences the allele of the DNM2 gene containing the disease-causing mutation without targeting the wild-type allele. Thus, the AS-siRNA can silence the expression of dynamin 2 mRNA and dynamin 2 protein derived from the mutant allele without affecting the expression of mRNA and protein derived from the wild-type allele.

[0036] In particular, the disease-causing mutation may be any deletion, insertion, or substitution of nucleotides in the DNM2 gene that is involved in or correlates with pathology. In a preferred embodiment, the disease-causing mutation is a dominant mutation. A dominant mutation refers to any mutation that leads to a dominant allele. A "dominant allele" refers to an allele that exerts its effect on a phenotype greater than the presence of a recessive allele of the same gene. The terms dominant and recessive allele are defined relative to each other and are not absolute. In other words, the phenotypic consequences of a dominant mutation are observed in heterozygous individuals with one mutant allele and one wild-type allele. Recessive alleles only show their effects when an individual has two copies of the mutant allele (also known as homozygosity) or two different mutant alleles (also known as compound heterozygosity).

[0037] In certain embodiments, the dominant mutation is a gain-of-function mutation. A gain-of-function mutation is defined as a mutation that confers a new or enhanced activity to a protein. A gain-of-function mutation is a type of mutation in which the altered gene product possesses a new molecular function or a new pattern of gene expression. Consequently, disease-causing mutations in DNM2 lead to a gain of function of the dynamin 2 protein.

[0038] In certain embodiments, the dominant mutation is a loss-of-function mutation with a dominant-negative effect. A loss-of-function mutation is defined as a mutation that results in the loss or reduction of the normal activity of a protein. A dominant-negative effect is defined as a product of a mutant allele that alters the function of the product from a wild-type allele. This occurs, for example, when oligomerization is required for normal function of the protein and the mutant protein can oligomerize with the wild-type protein. Consequently, disease-causing mutations in DNM2 lead to loss of function of the wild-type dynamin 2 protein due to the presence of the mutated dynamin 2 protein.

[0039] In another specific embodiment, the DNM2 gene that is heterozygous for the disease-causing mutation is not haploinsufficient.Haploinsufficiency occurs when one copy of the gene is inactivated or deleted, and the remaining functional copies of the gene are insufficient to produce a sufficient amount of gene product to maintain normal function.In other words, after silencing the mutant allele by the AS-siRNA of the present invention, the wild-type DNM2 allele of the present invention can maintain normal function.Therefore, the present invention preferably relates to an autosomal dominant disease that does not have haploinsufficiency.

[0040] In certain embodiments, dominant mutations in the DNM2 gene lead to autosomal dominant disorders. Autosomal dominant disorders are disorders in which an individual has one copy of a mutant gene and one normal gene on a pair of autosomes (autosomes are any chromosomes that are not sex chromosomes). Individuals with autosomal dominant disorders have a 50% chance of passing on the mutant gene, and therefore the disorder, to each of their children.

[0041] In one preferred embodiment, the autosomal dominant disease is selected from autosomal dominant centronuclear myopathy (AD-CNM), T-cell acute lymphoblastic leukemia, Charcot-Marie-Tooth disease (CMT), or hereditary spastic paraplegia (HSP). Preferably, the autosomal dominant disease is autosomal dominant centronuclear myopathy (AD-CNM).

[0042] In another embodiment, the disease-causing mutation in the DNM2 gene is associated with or correlates with a disease selected from autosomal dominant centronuclear myopathy (AD-CNM), T-cell acute lymphoblastic leukemia, Charcot-Marie-Tooth disease (CMT), or hereditary spastic paraplegia (HSP). Preferably, the disease-causing mutation in the DNM2 gene is associated with or correlates with autosomal dominant centronuclear myopathy (AD-CNM).

[0043] According to the present invention, the disease-causing mutations c.1393C>T and c.1856C>T are associated with the following substitutions in the DNM2 protein sequence: p.R465W and p.S619L, respectively.

[0044] Thus, in a particular aspect, the present invention relates to an AS-siRNA capable of silencing the expression of only one allele of a heterozygous DNM2 gene in a cell, wherein the target allele comprises a disease-causing mutation selected from the group consisting of c.1393C>T or c.1856C>T.

[0045] We have designed and tested many siRNAs that can target the most common disease-causing mutations (c.1856C>T, also known as S619L, and c.1393C>T, also known as p.R465W) in vitro and in vivo experiments. These sequences are listed in Tables 5 and 6.

[0046] Therefore, the present invention also relates to an allele-specific siRNA (AS-siRNA) capable of silencing the expression of only one allele of a heterozygous DNM2 gene, wherein the AS-siRNA comprises a sense strand selected from the group consisting of SEQ ID NOs: 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 215, 217, 223, 225, and 227.

[0047] In particular, the present invention relates to an allele-specific siRNA (AS-siRNA) capable of silencing the expression of only one allele of a heterozygous DNM2 gene, wherein the AS-siRNA comprises a sense strand selected from the group consisting of SEQ ID NOs: 157, 163, 167, 171, 175, 177, 179, 181, and 185.

[0048] [Table 5] Table 5: siRNAs for the S619L mutation. The top sequence corresponds to the sense strand (or sense sequence) (5'-3') and is called the "passenger sequence," and the bottom sequence corresponds to the antisense strand (or antisense sequence) (3'-5') and is called the "guide sequence" (see, for example, Sil-S619L).

[0049] [Table 6] Table 6: siRNA for mutation R465W. The upper sequence corresponds to the sense strand (or sense sequence) (5'-3') and is called the "passenger sequence", and the lower sequence corresponds to the antisense strand (or antisense sequence) (3'-5') and is called the "guide sequence" (see, for example, Sil-R465W).

[0050] The AS-siRNA of the present invention can silence the mutant allele of the DNM2 gene by specifically hybridizing to the gene transcript (messenger RNA or mRNA) derived from the mutant allele of the DNM2 gene. Therefore, the AS-siRNA of the present invention is complementary to the mRNA derived from the mutant allele of DNM2 and binds to the mRNA through base pairing. The term "complementary" refers to the ability of a polynucleotide to form base pairs with another polynucleotide molecule. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. In particular, the degree of complementarity between the AS-siRNA of the present invention and the target mRNA is approximately 100%.

[0051] In a specific embodiment, the AS-siRNA of the present invention targets the region of the DNM2 gene transcript containing the disease-causing mutation. Thus, the AS-siRNA of the present invention is complementary to the sequence of the mRNA containing the disease-causing mutation. The specificity of the siRNA makes it possible to distinguish between two sequences even if they differ by only one nucleotide. This property allows the AS-siRNA of the present invention to target disease-causing mutations, such as mutations resulting in single nucleotide substitutions.

[0052] General Information Regarding All siRNAs of the Invention In a specific embodiment, the AS-siRNA of the present invention contains a nucleotide overhang at the 3'-end of each strand. In a more specific embodiment, the AS-siRNA of the present invention contains a dinucleotide overhang consisting of two deoxythymidines (dTdT) at the 3'-end of each strand (sense and antisense).

[0053] Another aspect of the present invention relates to a vector encoding the AS-siRNA of the present invention, wherein the vector is in particular a plasmid or a viral vector such as an AAV vector.

[0054] "Sense strand" refers to the strand of the AS-siRNA that has the same sequence as the target allele containing a non-pathological polymorphism or disease-causing mutation. The other strand of the AS-siRNA is therefore called "antisense" because its sequence is complementary to the target DNM2 mRNA, called the "sense" sequence (so that the sense segment of the mRNA "5'-AAGGUC-3'" will be blocked by the antisense mRNA segment "3'-UUCCAG-5'").

[0055] In a specific embodiment, the AS-siRNA of the present invention is used to reduce DNM2 mRNA and / or DNM2 protein expression by 20-60%, such as 20, 30, 40, 50, or 60%. In particular, the AS-siRNA of the present invention is used to reduce DNM2 mRNA and / or DNM2 protein expression by about 50%. "About" refers to a value of + or -10%, particularly + or -5%. For example, about 50% refers to 45-55%, particularly 47.5-52.5%.

[0056] The present invention contemplates various ways to deliver target mRNAs using the AS-siRNAs of the present invention: AS-siRNAs can be administered to cells directly or as isolated oligonucleotides using transfection reagents such as lipid derivatives, liposomes, calcium phosphate, nanoparticles, microinjection, or electroporation.

[0057] In another embodiment, the present invention contemplates introducing the AS-siRNA into cells in the form of a vector. Accordingly, another aspect of the present invention relates to a vector encoding the AS-siRNA of the present invention. The vector may be, in particular, a plasmid or a viral vector. Representative viral vectors useful in practicing the present invention include, but are not limited to, vectors derived from adenovirus, retrovirus, in particular lentivirus, poxvirus, herpes simplex virus I, and adeno-associated virus (AAV).

[0058] In certain embodiments, the AAV vector is AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, Rh10, Rh74, or any other serotype of AAV capable of infecting humans, monkeys, or other species.

[0059] The selection of an appropriate viral vector will naturally depend on the target cell and viral tropism. In certain embodiments, the target cell is a muscle cell, although viral vectors with a broad range of tropism, including muscle tropism in particular, can also be implemented. In certain embodiments, for example, for use in intramuscular injection, an AAV1 vector is implemented. In another embodiment, the vector is to be administered via a systemic route (e.g., via an intravascular or intraarterial route), and the vector is an AAV8 or AAV9 vector.

[0060] In certain embodiments, the present invention also relates to shRNAs (short hairpin RNAs) corresponding to the AS-siRNAs of the present invention, which have an even sharper hairpin turn. The shRNA hairpin structure is then cleaved into siRNAs by the cellular machinery. A further aspect of the present invention relates to vectors encoding shRNAs corresponding to the AS-siRNAs of the present invention.

[0061] In another aspect, the present invention also relates to target cells comprising the AS-siRNA of the present invention or transfected or transduced with the vector of the present invention. For example, the target cells can be selected from muscle cells (or cells of muscle lineage), such as myoblasts, e.g., patient-derived myoblasts, or fibroblasts, such as patient-derived fibroblasts.

[0062] Uses of the AS-siRNA of the present invention In another aspect, the present invention relates to an in vitro method for silencing expression of a mutant allele of the DNM2 gene without silencing expression of a wild-type allele of the DNM2 gene in target cells, such as muscle target cells (e.g., muscle cells such as myoblasts, particularly patient-derived myoblasts), comprising introducing an AS-siRNA or vector of the present invention into said target cells.

[0063] In another aspect, the present invention relates to an in vitro method for silencing expression of one allele of a DNM2 gene carrying a heterozygous non-pathological polymorphism without silencing expression of the other allele of the DNM2 gene in a target cell, the method comprising introducing an AS-siRNA or vector of the present invention into the target cell.

[0064] In a further aspect, the present invention relates to an AS-siRNA, vector, or cell of the present invention for use in a method for treating a disease caused by a disease-causing mutation in the DNM2 gene in a subject in need thereof. In particular, the AS-siRNA, vector, or cell of the present invention is used in a method for treating centronuclear myopathy (such as autosomal dominant centronuclear myopathy), T-cell acute lymphoblastic leukemia, Charcot-Marie-Tooth disease (CMT), or hereditary spastic paraplegia (HSP) in a subject in need thereof. More preferably, the AS-siRNA, vector, or cell of the present invention is used in a method for treating autosomal dominant centronuclear myopathy.

[0065] In another aspect, the present invention relates to an AS-siRNA, vector, or cell of the invention for use in a method for treating a muscular dystrophy, such as Duchenne muscular dystrophy, in a subject in need thereof.

[0066] In another aspect, the present invention relates to an AS-siRNA, vector, or cell of the present invention for use in a method for treating a disease associated with overexpression of dynamin 2 in a subject in need thereof, preferably for treating X-linked myotubular myopathy, or cancer such as prostate cancer and pancreatic cancer.

[0067] In certain embodiments, the present invention relates to an AS-siRNA, vector, or cell of the present invention for use in treating centronuclear myopathy (such as autosomal dominant centronuclear myopathy), T-cell acute lymphoblastic leukemia, Charcot-Marie-Tooth disease (CMT), or hereditary spastic paraplegia (HSP) in a subject in need thereof.

[0068] In certain embodiments, the present invention relates to a method for treating muscular dystrophy, such as Duchenne muscular dystrophy, in a subject in need thereof, comprising administering to the subject an effective amount of an AS-siRNA, vector, or cell of the present invention.

[0069] As used herein, the term "subject" refers to mammals such as rodents, felines, canines, and primates. In particular, the subject according to the present invention is a human.

[0070] As used herein, the term "treatment" or "treating" refers to both preventative or preventative treatment and curative or disease-modifying treatment, including treatment of subjects at risk of or suspected of having a disease, as well as subjects who have been diagnosed with a disease or medical condition, including the suppression of clinical recurrence. Treatment can be administered to a subject who has a medical disorder or who may ultimately suffer from a disorder to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disorder or relapsing disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of disease treatment, e.g., a pattern of medication used during therapy. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used in the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the subject during the initial period of the treatment regimen. The induction regimen may employ (in part or in whole) a "loading regimen," which may involve administering a higher dose of drug than the physician would employ during the maintenance regimen, administering the drug more frequently than the physician would administer the drug during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used to maintain a subject during disease treatment, for example, to keep the subject in remission for an extended period of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, or yearly) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain predetermined criteria (e.g., disease symptoms, etc.)).

[0071] The AS-siRNA of the present invention, vectors or cells according to the present invention may be formulated and administered to treat any disease caused by heterozygous mutations in the DNM2 gene or caused by overexpression of DNM2, preferably to treat autosomal dominant centronuclear myopathy, T-cell acute lymphoblastic leukemia, Charcot-Marie-Tooth disease, hereditary spastic paraplegia, X-linked myotubular myopathy, or cancers such as prostate cancer and pancreatic cancer. The AS-siRNA of the present invention, vectors or cells according to the present invention may be formulated by any means that brings the AS-siRNA into contact with its site of action in a subject in need thereof.

[0072] The present invention also provides pharmaceutical compositions comprising the AS-siRNA of the present invention, vectors, or cells according to the present invention. Such compositions comprise a therapeutically effective amount of a therapeutic agent (the AS-siRNA, vector, or cells of the present invention) and a pharmaceutically acceptable carrier. In specific embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals and humans, or listed in the United States or European Pharmacopoeia, or other generally recognized pharmacopoeias. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline, water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0073] The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the subject.

[0074] The pharmaceutical composition is adapted for any type of administration to mammals, particularly humans, and is formulated according to routine procedures.The composition is formulated by using suitable conventional pharmaceutical carriers, diluents, and / or excipients.The administration of the composition can be via any common route, as long as the target tissue is available via that route.

[0075] The amount of the therapeutic agent of the present invention that will be effective in treating nucleotide repeat expansion can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays can be optionally employed to help predict the optimal dosage range. The exact dosage to be employed in the formulation will also depend on the route of administration and the severity of the disease, and should be determined according to the judgment of the practitioner and the circumstances of each patient. The dosage of AS-siRNA, vector, or cell administered to a subject in need thereof will vary based on several factors, including, but not limited to, the route of administration, the age of the subject, or the expression level required to achieve the required therapeutic effect. Those skilled in the art can easily determine the required dosage range based on these and other factors based on their knowledge in this field.

[0076] The present invention will be further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0077] [Figure 1] Identification of allele-specific siRNA for the C version of SNP1. A. DNM2 mRNA expression 48 hours after 30 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n≧4 per condition). B. Quantification of C / T ratio after transfection with 30 nM siRNA (n≧4 per condition). C. DNM2 mRNA expression 48 hours after 100 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n≧4 per condition). D. Quantification of C / T ratio after transfection with 100 nM siRNA (n≧4 per condition). E. Quantification of densitometric signal after 100 nM siRNA transfection. GAPDH was used as a loading control (n≧4). Data information: In scatter plots, bars are mean values ​​and error bars indicate SEM. Relative to scrambled siRNA (Sc), ****P<0.0001, ***P<0.001, **P<0.01, and *P<0.5 using a two-tailed Mann-Whitney U test. [Figure 2]Identification of allele-specific siRNA for the T version of SNP1. A. DNM2 mRNA expression 48 hours after 30 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n ≥ 4 per condition). B. Quantification of the T / C ratio after transfection with 30 nM siRNA (n ≥ 4 per condition). C. DNM2 mRNA expression 48 hours after 100 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n ≥ 4 per condition). D. Quantification of the T / C ratio after transfection with 100 nM siRNA (n ≥ 4 per condition). E. Quantification of densitometric signal after 100 nM siRNA transfection. GAPDH was used as a loading control (n ≥ 4). Data information: In scatter plots, bars are mean values, and error bars indicate SEM. *P < 0.5 versus scrambled siRNA (Sc) using a two-tailed Mann-Whitney U test. [Figure 3] Identification of allele-specific siRNA for the T version of SNP2. A. DNM2 mRNA expression 48 hours after 30 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n≧4 per condition). B. Quantification of the T / A ratio after transfection with 30 nM siRNA (n≧4 per condition). C. DNM2 mRNA expression 48 hours after 100 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n≧4 per condition). D. Quantification of the T / A ratio after transfection with 100 nM siRNA (n≧4 per condition). E. Quantification of densitometric signal after 100 nM siRNA transfection. GAPDH was used as a loading control (n≧4). Data information: In scatter plots, bars are mean values ​​and error bars indicate SEM. Relative to scrambled siRNA (Sc), ****P<0.0001, ***P<0.001, **P<0.01, and *P<0.5 using a two-tailed Mann-Whitney U test. [Figure 4]Identification of allele-specific siRNA for the A version of SNP2. A. DNM2 mRNA expression 48 hours after 30 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n ≥ 4 per condition). B. Quantification of the A / T ratio after transfection with 30 nM siRNA (n ≥ 4 per condition). C. DNM2 mRNA expression 48 hours after 100 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n ≥ 4 per condition). D. Quantification of the A / T ratio after transfection with 100 nM siRNA (n ≥ 4 per condition). E. Quantification of densitometric signal after 100 nM siRNA transfection. GAPDH was used as a loading control (n ≥ 4). Data information: In scatter plots, bars are mean values, and error bars indicate SEM. **P < 0.01 and *P < 0.5 relative to scrambled siRNA (Sc) using a two-tailed Mann-Whitney U test. [Figure 5] Identification of allele-specific siRNA for the S619L DNM2 mutation. A. DNM2 mRNA expression 48 hours after 30 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n ≥ 4 per condition). B. Quantification of mutant / WT ratio after transfection with 30 nM siRNA (n ≥ 4 per condition). The mutant allele harbors the C version of SNP1. C. DNM2 mRNA expression 48 hours after 100 nM siRNA transfection. Quantification of DNM2 expression normalized to HPRT (n ≥ 4 per condition). D. Quantification of mutant / WT ratio after transfection with 100 nM siRNA (n ≥ 4 per condition). E. Quantification of densitometric signal after 100 nM siRNA transfection. GAPDH was used as a loading control (n ≥ 4). Data information: In scatter plots, bars represent mean values ​​and error bars indicate SEM. Vs. scrambled siRNA (Sc), ***P<0.001 and *P<0.1 using a two-tailed Mann-Whitney U test. [Figure 6]Effect of AS-siRNA on transferrin uptake and cell surface of patient-derived fibroblasts. A. Transferrin uptake under basal conditions (n ​​= 500–700 cells from three independent experiments). CTCF: corrected total cell fluorescence. B. Transferrin uptake after 48 hours of transfection with 30 nM scrambled siRNA (sc) or allele-specific siRNA against the S619L mutation or two SNPs (n = 200–300 cells from two independent experiments). C. Transferrin uptake after 48 hours of transfection with 100 nM scrambled siRNA (sc) or allele-specific siRNA against the S619L mutation or two SNPs (n = 200–300 cells from two independent experiments). D. Cell surface (μm2) under basal conditions (n ​​= 500–700 cells from three independent experiments). E. Cell surface after 48 hours of transfection with 30 nM scrambled siRNA (sc) or allele-specific siRNA against the S619L mutation or two SNPs (n = 200-300 cells from two independent experiments). F. Cell surface after 48 hours of transfection with 100 nM scrambled siRNA (sc) or allele-specific siRNA against the S619L mutation or two SNPs (n = 200-300 cells from two independent experiments). Data information: Bars represent the mean, error bars indicate SEM. Statistical analysis was performed using Kruskal-Wallis for A–F (****p<0.0001 for all histograms) followed by Dunn's test for control cells in A and D (****P<0.0001, ***P<0.001, adjusted p-values), control cells transfected with scrambled siRNA in B, C, E, and F (****P<0.0001, adjusted p-values), or patient cell lines transfected with scrambled siRNA in B, C, E, and F (†P<0.05, ††P<0.01, and ††††P<0.0001, adjusted p-values). [Figure 7]Migration and adhesion assays in patient-derived cells. A. Mean velocity (μm / min) of CNM- and control-fibroblasts under basal conditions (n=50 cells tracked). B. Mean velocity of cells after 48 hours of transfection with 30 nM scrambled siRNA (sc) or allele-specific siRNA against the S619L mutation or two SNPs (n=50-70 cells tracked). C. Mean velocity of cells after 48 hours of transfection with 100 nM scrambled siRNA (sc) or allele-specific siRNA against SNP1 (n=50-70 cells tracked). D. Adhesion under basal conditions (n=8 independent assays). E. Adhesion assay after 48 hours of transfection with 30 nM scrambled siRNA (sc) or allele-specific siRNA against the S619L mutation or SNP1 (n=5 or 6 independent transfections). F. Adhesion assays (n = 6 independent transfections) 48 hours after transfection with 100 nM scrambled siRNA (sc) or allele-specific siRNA against the S619L mutation or SNP1. Data information: Bars represent mean values, error bars indicate SEM. Statistical analysis was performed using Kruskal-Wallis for migration assays A-C (all histograms, ****p<0.0001), followed by Dunn's test for A vs. control cell 1 (***p<0.001) or control cell 2 (φφp<0.01), or B and C vs. scrambled siRNA-transfected control cells (****p<0.0001), or B and C vs. scrambled siRNA-transfected patient cell lines (††††p<0.0001). Anova tests were performed on the adhesion assay in A (**P<0.01), B, and C (****P<0.0001), followed by post hoc tests against control cells in A (**P<0.01), and against control cells transfected with scrambled siRNA in B and C (****P<0.0001, ***P<0.001, **P<0.01), and against patient cell lines transfected with scrambled siRNA (††p<0.01, ††p<0.05).

[0078] Working Example: Materials & Methods Cell culture and transfection Healthy control and CNM patient-derived fibroblast cell lines were obtained from the MyoLine platform (Institute of Myology, Paris, France) for the immortalization of human cells in accordance with European recommendations and French legislation. Cell lines were cultured at 37°C (5% CO) in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, France) containing 10% fetal calf serum (FCS) supplemented with penicillin (100 units / ml) and streptomycin (100 μg / ml). Patient and control fibroblasts were cultured as previously described. 45 The cells were immortalized using a lentiviral vector containing a sequence encoding the catalytic subunit of human telomerase (hTERT). For transfection, cells were grown to 70% confluency and transfected with siRNA using RNAimax transfection reagent (Life Technologies, France) according to the manufacturer's protocol. The siRNA concentration for each experiment is indicated in the corresponding figure. Allele-specific and scrambled siRNA were purchased from Eurogentec (Belgium), and sequences are available upon request. Cells were used for functional assessment or harvested for RNA and protein extraction 48 hours after transfection.

[0079] Total RNA extraction and cDNA analysis Total RNA was isolated from cells using NucleoSpin RNA (Macherey-Nagel, France) according to the manufacturer's protocol. Cells were disrupted in lysis buffer by pipetting up and down several times. Total RNA (500 ng) was subjected to reverse transcription using an oligo-dT primer in a final volume of 20 μl using the Superscript III Reverse Transcriptase Kit (Life Technologies, France). Reverse transcription was performed at 50°C for 50 minutes, with a final step of 85°C for 5 minutes. To determine the allelic version of each SNP present on the mutant allele, PCR encompassing the SNP and mutation was performed and cloned using the pGEMT vector system (Promega), and approximately 10 single clones were sequenced (Eurofins, France). DNM2 expression was quantified by semiquantitative RT-PCR relative to HPRT housekeeping gene expression. The RT product (1 μl) was subjected to PCR performed at 96°C for 3 minutes, followed by 27 cycles including denaturation at 96°C for 25 seconds, annealing at 58°C for 25 seconds, and polymerization at 72°C for 40 seconds, with a final step of 72°C for 5 minutes. The number of 27 PCR cycles was selected to have amplification in the exponential range for DNM2 and HPRT. To quantify the allele specificity of the assessed siRNAs, assays for SNP1 and SNP2 were developed using restriction enzymes that allow discrimination between the two alleles after digestion of the RT-PCR products. PCR was designed to amplify the region of the DNM2 transcript encompassing the SNP. The RT product (1 μl) was subjected to SNP1 or SNP2 PCR performed at 96°C for 3 minutes, followed by 40 cycles including denaturation at 96°C for 25 seconds, annealing at 58°C for 25 seconds, and polymerization at 72°C for 40 seconds, with a final step of 72°C for 5 minutes. The number of cycles is chosen to be at the end of the exponential phase of the amplification.For SNP1, 10 μl of the 20 μl PCR product was digested overnight at 37°C with 7 units of BglI (New England Biolabs, France), and for SNP2, 15 μl of the 30 μl PCR product was digested overnight at 37°C with 7 units of Psp5II (New England Biolabs, France). Imaging of PCR products after agarose gel electrophoresis was performed using the Geni2 gel imaging system (Ozyme, France), and the associated signals were quantified using ImageJ software (NIH; http: / / rsbweb.nih.gov / ij). All PCR primers used in this study were from Eurogentec (Belgium), and sequences are available upon request.

[0080] Protein extraction and Western blot Cell pellets were homogenized in lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, and 1% protease inhibitor cocktail (Sigma-Aldrich, France) and incubated for 20 minutes at 4°C on a rotating rotor. After cell lysate detachment, samples were lysed by sonication twice for 10 seconds at 30% of maximum power in a VCX 130 Vibra-cell sonicator (Sonics, USA). After centrifugation (12,000 g, 4°C, 20 minutes), the protein concentration in the supernatant was determined using a BCA Protein Assay Kit (Thermo Scientific Pierce, France). Five milligrams of protein was mixed with loading buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, and bromophenol blue) and denatured at 90°C for 5 minutes. Protein samples were separated on 10% SDS-PAGE prestained gels and transferred to PVDF membranes (0.45 μm pore size, Life Technologies, France) overnight at 100 mA at 4°C or to nitrocellulose membranes (BioRad Turbo Transfer System) by transblotting at 25 V, 2.5 mA for 8 min. The membranes were blocked in PBS containing 5% nonfat dry milk and 0.1% Tween-20 for 2 h at room temperature and then exposed to rabbit polyclonal anti-dynamin 2 antibody (ab3457 or ab65556, Abcam, France), rabbit polyclonal anti-transferrin receptor antibody (ab84036, Abcam, France), or rabbit polyclonal anti-GAPDH antibody (sc-25778, Santa Cruz, France) in PBS-Tween-20 0.1% and nonfat dry milk 1% overnight at 4°C. The membrane was rinsed in PBS-Tween-20 0.1% and incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit, Jackson ImmunoResearch, United Kingdom) in PBS-Tween-20 0.1% for 2 hours.Chemiluminescence was detected using an ECL detection kit (Merck‐Millipore, Germany) in a BioRad ChemidocMP imaging system, and signal quantification was performed using ImageJ software.

[0081] Transferrin uptake assay and cell surface Transfected cells were cultured in DMEM at 37°C for 45 minutes. Transferrin-AlexaFluor488 (Life Technologies, France) was added at 20 μg / ml for 15 minutes at 37°C. Cells were then washed in DMEM pH 2 and PBS and fixed in 4% paraformaldehyde for 15 minutes. Z-stack images were acquired using an Axio Observer Apotome.2 microscope (Zeiss, Germany) using a 20× Plan Apochromat Zeiss objective. Individual cells were manually outlined on summed projection images of the confocal stacks to determine the cell surface (μm 2 ) was measured and transferrin uptake was calculated for each cell using ImageJ software according to the formula: corrected total cell fluorescence (CTCF) = integrated density of cells - (mean background fluorescence × cell area).

[0082] Adhesion assay Cells were harvested by trypsinization 48 hours after transfection or under basal conditions, and 35,000 cells were seeded onto 12 mm-diameter glass coverslips in 24-well plates (three technical replicates for each transfection). After incubation in DMEM-10% FCS supplemented with penicillin (100 units / ml) and streptomycin (100 μg / ml) for 1 hour at 37°C, unattached cells were removed by PBS washing, and cells attached to the coverslips were fixed in 4% paraformaldehyde for 15 minutes. Glass coverslips were mounted on slides using Vectashield medium (Vector Laboratories) containing DAPI to stain nuclei. Images were acquired using an Axio Observer Apotome.2 microscope (Zeiss, Germany) with a 2.5× Plan NeoFluar Zeiss objective, and the number of attached cells per μm2 of coverslip was counted by nuclei counting using ImageJ software. Three independent experiments were performed for each condition.

[0083] Cell Tracking and Analysis Cells were seeded at low density in 24-well plastic plates under basal conditions or 48 h after transfection. Bright-field images were acquired every 20 or 30 min at 10x magnification for 65 h (for transfected cells and basal conditions, respectively) using an inverted video microscope equipped with a thermal and CO2-controlled chamber (Nikon Ti2, Oko-Lab) driven by NIS (Nikon). After reconstruction of the 24-h video, 50–70 cells per condition were randomly selected and manually tracked by tracking the nuclear position using ImageJ software and the MTrackJ plugin to determine the mean and maximum velocities. 46 Other motility parameters (mean velocity of movement, number and duration of pauses) were obtained using the Skypad Microsoft Excel add-in, which automatically analyzes particles in 2D trajectories. 47A threshold of 10 μm was set to account for cells migrating between the two time frames, and a velocity threshold of 0.1 μm / min was set to account for the associated displacement.

[0084] Data Analysis and Statistics Graphs and statistical analyses were performed using GraphPad Prism software version 6 or 9 (GraphPad Software, LaJolla, California, USA). Values ​​were expressed as mean ± SEM. The number of samples (n) represents the number of independent biological replicates, as indicated in the legend. The number, mean, and SEM of analyzed values ​​for all graphs presented in the figures are listed in Supplementary Table 1.

[0085] We analyzed our data using nonparametric statistical tests when normality could not be assumed (Shapiro-Wilk test) or tested (n too small). In this case, statistical comparisons between groups were performed using the unpaired, two-tailed Mann-Whitney U test for siRNA screening, or the Kruskal-Wallis test, followed by Dunn's if significant, for transferrin uptake, cell size, and migration assays. Adhesion assays were analyzed using an ANOVA followed by a Bonferroni post-hoc test or Welch's ANOVA to correct for unequal variances, followed by Dunnett's as a post-hoc test. p values ​​shown for post-hoc tests are adjusted p values. P < 0.05 was considered statistically significant.

[0086] result Identification of targetable SNPs and heterozygous cells We conducted an in silico analysis specifically aimed at identifying DNM2 SNPs exhibiting the highest frequency of heterozygosity in humans. We searched the Ensembl Genome Browser (https: / / www.ensembl.org - human genome assembly GRCh38.p13) for synonymous variants in the DNM2 open reading frame or variants in the 5'- and 3'-untranslated transcribed regions (UTRs) exhibiting the second most common allele frequency of 0.1-0.5 in the general population. Using these criteria, we identified two SNPs (data not shown). The first SNP was a T / C mutation (rs2229920, hereafter referred to as SNP1), identified as a synonymous variant at alanine 713 of the DNM2 protein (c.2139T>C, NM_001005361.3). The allele frequencies for SNP1 were determined to be 0.688 for the T allele and 0.321 for the C allele in 250,390 counts (gnomAD exome r2.1.1), yielding a theoretical heterozygosity (HTZ) frequency of 0.43 (calculated as 2 × T allele frequency × C allele frequency). Consistent with this, PCR and Sanger sequencing in a cohort of 52 CNM patients identified 42.3% SNP1 heterozygosity. The second SNP was an A / T mutation (rs12461992, hereafter referred to as SNP2), identified as a 3'-UTR variant (*268A>T, NM_001005361.3) located 268 nucleotides after the stop codon. The allele frequencies for SNP2 were determined to be 0.824 for allele A and 0.176 for allele T, yielding a theoretical HTZ frequency of 0.29 in 143,008 counts (gnomAD exome r3.0); 23% SNP2 heterozygosity was found in our patient cohort. No clinical symptoms were associated with SNP1 and SNP2.

[0087] We next aimed to identify cells carrying the SNPs in the HTZ state, which was necessary for screening allele-specific siRNAs against the SNPs. RT-PCR products encompassing the SNPs were amplified from fibroblast cell lines derived from two healthy controls and two CNM patients. Sanger sequencing of these RT-PCR products led to the identification of one healthy control cell line heterozygous for the two SNPs, one CNM cell line carrying both SNPs in the HTZ state (p.R522H DNM2 mutation), and one CNM cell line carrying only SNP1 in the HTZ state (p.S619L DNM2 mutation) (data not shown). Through single-allele RT-PCR, cloning, and Sanger sequencing, we determined that in the R522H cell line, the mutant mRNA also carried the C version of SNP1 and the T version of SNP2, and in the S619L cell line, the mutant mRNA carried the C version of SNP1 (data not shown). Overall, we identified two SNPs targetable by AS-RNAi, one healthy control cell line to perform siRNA screening, and two patient-derived cell lines for functional evaluation of the identified AS-siRNAs.

[0088] Allele-specific siRNA for the C allele of SNP1 For all screens of allele-specific siRNAs for SNP1 and SNP2 reported in this study, we used one healthy control cell line and searched for allele-specific siRNAs that led to approximately 50% expression of DNM2 mRNA and protein due to specific silencing of the target allele. We first aimed to identify AS-siRNAs against the C version of SNP1 (Figure 1). Twelve 19-nucleotide siRNAs (referred to as si5–si17) with a single mismatch at positions 5–17 with the nontargeting T version were assessed. At low concentrations (30 nM), si6 and si11 failed to significantly reduce DNM2 mRNA expression compared to cells transfected with scrambled siRNA (Figure 1A). In addition, si5, si13, and si14 led to an excessive reduction in DNM2 transcripts compared to the expected 50%, while seven siRNAs (si7, si8, si9, si10, si12, si15, and si17) reduced DNM2 expression within the expected range (Figure 1A). RT-PCR and BglI restriction enzyme digestion assays were used to distinguish between the C (digested) and T (undigested) alleles of SNP1 (data not shown). The seven siRNAs reduced the C / T ratio compared to scrambled siRNA (Figure 1B), with lower ratios achieved by si8 and si9. Quantification of each allele relative to HPRT mRNA showed that si8, si9, and si10 reduced the expression of the C allele without affecting the T allele (data not shown). At the same concentrations, si8, si9, and si10 also reduced DNM2 protein expression by approximately 50% compared to scrambled siRNA in Western blots (data not shown). Maintenance of allele specificity at higher concentrations (100 nM) was then assessed for si8, si9, and si10. The three siRNAs reduced total DNM2 mRNA content, approaching the expected 50% reduction for si8 and si9 (Figure 1C). Allele specificity of si8 and si9 for the target C allele was maintained, as evidenced by the reduced C / T ratio (Figure 1D), and supported by quantification of each allele relative to HPRT expression (data not shown).The maintenance of potency and allele specificity of si8 and si9 at higher concentrations was also demonstrated for DNM2 protein by Western blot, which showed a significant reduction in DNM2 protein content that did not exceed 50% (Figure 1E). Overall, these data validate si8 and si9 as the best allele-specific siRNAs for the C version of SNP1.

[0089] Allele-specific siRNA for the T allele of SNP1 To identify AS-siRNAs against the T version of SNP1, we assessed siRNAs with mismatches at positions 8, 9, and 10 relative to the non-targeting C allele, following our previous screening. At low concentrations (30 nM), si8 and si9 significantly reduced DNM2 expression within the expected range (Figure 2A). RT-PCR assays and BglI digestion, used to distinguish between the targeting T allele and the non-targeting C allele, showed that the three siRNAs significantly reduced the T / C ratio compared to scrambled siRNA (Figure 2B). Notably, an increase in the non-targeting C allele was observed with si9 (data not shown), suggesting gene regulation promoting the expression of the non-targeting allele. At this concentration, the three siRNAs did not alter DNM2 protein content (data not shown). At higher concentrations (100 nM), total DNM2 mRNA levels were reduced for the three siRNAs, remaining close to the 50% threshold for si8 and si9 (Figure 2C), and the allele specificity of the three siRNAs for the target T allele was maintained, as demonstrated by the reduced T / C ratio (Figure 2D) and quantification of each allele relative to HPRT expression (data not shown). A significant increase in non-target alleles was still observed for si9 and si10 (data not shown). At this concentration, Western blots showed a significant decrease in DNM2 protein content for si8, si9, and si10 compared to scrambled siRNA (Figure 2E). Overall, these data validate si8 and si9 as allele-specific siRNAs for the T version of SNP1.

[0090] Allele-specific siRNA for the T allele of SNP2 To screen AS-siRNAs against the T version of SNP2, we assessed 12 siRNAs with mismatches to the non-targeting A allele at positions 6–17 (Figure 3). When transfected at 30 nM for 48 h, si11, si12, si13, si14, si15, si16, and si17 reduced DNM2 mRNA expression compared to cells transfected with scrambled siRNA, with all of them leading to a nearly 50% reduction, except for si14 and si15 (Figure 3A). RT-PCR and Psp5II restriction enzyme digestion assays were used to distinguish between the T-digested and A-undigested alleles (Figure 3B). Using this assay, we demonstrated that lower T / A ratios were achieved by si11, si16, and si17 compared to scrambled siRNA (Figure 3B). Quantification of each allele relative to HPRT mRNA demonstrated that the decrease in the T / A ratio was due to a reduction in expression of the T allele, without a reduction in the A allele (data not shown). For si17, a significant increase in the A allele was also observed (data not shown). At this low concentration, only si11 and si16 reduced DNM2 protein expression by the expected approximately 50% compared to scrambled siRNA (data not shown). We next assessed the maintenance of allele specificity for si11, si16, and si17 at higher concentrations (100 nM). Semiquantitative RT-PCR demonstrated a significant reduction of total DNM2 mRNA by approximately 50% for each siRNA (Figure 3C). The three siRNAs reduced the T / A ratio compared to scrambled siRNA, with si11 and si16 specifically affecting the T allele (data not shown) (Figure 3D). At this higher concentration, DNM2 protein content did not exceed 50% (Figure 3E). Overall, these data validate si11 and si16 as efficient allele-specific siRNAs against the T version of SNP2.

[0091] Allele-specific siRNA for the A allele of SNP2 Previous results for the T allele of SNP2 indicated a large number of mismatch positions in siRNAs that would be effective for developing AS-siRNAs. Consequently, we began screening for AS-siRNAs against the A allele by introducing a single mismatch in the central region of the siRNA (positions 9, 10, and 11), which frequently showed the highest specificity in previously reported studies (Figure 4) (23). At low concentrations (30 nM), si9, si10, and si11 significantly reduced DNM2 expression in the expected 50% range (Figure 4A). RT-PCR assays and Psp5II digestion, used to distinguish between the targeting A allele and the non-targeting T allele, showed that the three siRNAs significantly reduced the A / T ratio compared to scrambled siRNA, with si9 and si11 having the greatest effect on the A allele (Figure 4B). At this concentration, DNM2 protein content was significantly reduced with si10 and si11 (data not shown). At higher concentrations (100 nM), significant reductions in total DNM2 mRNA were observed for the three siRNAs (Figure 4C), and Psp5II digestion of RT-PCR products demonstrated maintenance of allele specificity (Figure 4D). Quantification of each allele relative to HPRT expression demonstrated reduction of the A allele using si9 and si11, and upregulation of the non-targeting T allele with the three siRNAs (data not shown). At this concentration, DNM2 protein content, as quantified by Western blot, was reduced by si10 and si11 (Figure 4E), but not by si9, likely due to upregulation of the non-targeting T allele (data not shown). Overall, these data validate si11 as the most efficient allele-specific siRNA for the A version of SNP2.

[0092] Allele-specific siRNA for the p.S619L DNM2 mutation To compare the functional benefits of siRNAs directed against the SNP or mutation, we screened AS-siRNAs against p.S619L (c.C1856T) (Figure 5). Using S619L patient-derived fibroblasts, we screened 15 siRNAs (si3–si17), named according to the position of the mismatch with the wild-type (WT) sequence of DNM2 mRNA. At low concentrations (30 nM), si9 and si11 failed to significantly reduce DNM2 mRNA expression compared to scrambled siRNA, while si4, si5, si7, and si16 led to an excessive reduction in DNM2 transcripts compared to the expected 50% (Figure 5A). Nine siRNAs (si3, si6, si8, si10, si12, si13, si14, si15, and si17) significantly reduced DNM2 expression, with si6, si8, si10, si13, si15, and si17 reducing mRNA levels by approximately 50%, as expected (Figure 5A). Given that the S619L mutation does not introduce or remove a restriction site compared to the WT sequence, we used the presence of SNP1 in a heterozygous state in this patient cell line to quantify the allele specificity of the nine siRNAs using BglI digestion of the SNP1 sequence (the C allele of SNP1 and the DNM2 mutation are carried by the same mRNA; data not shown). The nine siRNAs significantly reduced the C / T (i.e., mutant / WT) ratio compared to the scrambled siRNA (Figure 5B), with a lower ratio achieved by si10. Quantification of each allele relative to HPRT mRNA showed that nine siRNAs reduced expression of the mutant allele, some of which induced an increase in the WT allele (Supplementary Figure 6A). We assessed the effect of four of the 15 siRNAs (si6, si8, si10, and si13) on DNM2 protein content and found that four siRNAs showed significant reductions that did not exceed 50% (data not shown). At higher concentrations (100 nM), significant reductions in total DNM2 mRNA were observed for the four siRNAs (Figure 5C), maintaining allele specificity (Figure 5D), with the greatest effect on mutant mRNA being achieved with si10.At this concentration, DNM2 protein content, as quantified by Western blot, was significantly reduced by the four siRNAs, with the reduction not exceeding 50% (Figure 5E). Overall, these data validate the four allele-specific siRNAs (si6, si8, si10, and si13) against the S619L DNM2 mutant allele.

[0093] Rescue of clathrin-mediated endocytosis and cell surface by AS-siRNA DNM2 is well recognized for its role in endocytosis, and defective clathrin-mediated endocytosis (CME) has previously been demonstrated in fibroblasts derived from AD-CNM patients (18, 29). We assessed CME through fluorescent transferrin uptake measurements in healthy controls and two DNM2-CNM fibroblast cell lines. Under basal conditions, CME was decreased in fibroblasts carrying the R522H mutation (R522H-fibroblasts) and increased in fibroblasts carrying the S619L mutation (S619L-fibroblasts) compared with control fibroblasts (Figure 6A). Western blot analysis demonstrated comparable expression of the transferrin receptor between control and mutant cells (data not shown). Among the AS-siRNAs identified by our in vitro screening, we used si8 against the C version of SNP1 (SNP1-si8-C, hereafter referred to as siSNP1), si11 against the T version of SNP2 (SNP2-si11-T, hereafter referred to as siSNP2), and si10 against the p.S619L mutation (S619L-si10, hereafter referred to as siS619) for functional evaluation (data not shown). The decrease in transferrin uptake observed in R522H-fibroblasts was not altered by siSNP1 and siSNP2 when transfected at 30 nM for 48 h (Figure 6B). At 100 nM, transferrin uptake values ​​increased by siSNP1 and siSNP2 and approached the control value for siSNP1 after 48 h (Figure 6C). For S619L-fibroblasts, the increase in transferrin was significantly reduced by siS619L and siSNP1 transfected at 30 nM, which was maintained at 100 nM (Fig. 6B and C).

[0094] Measuring cell surface area for transferrin uptake quantification revealed defects, with significantly smaller cell size for R522H-fibroblasts and larger cell size for S619L-fibroblasts compared to control cell lines (Figure 6D). The effects of transfection of three AS-siRNAs of interest were then assessed on cell surface changes in patient-derived cells (Figures 6E and 6F). Among the AS-siRNAs tested, siSNP2 improved cell size in R522H-fibroblasts when transfected at 100 nM. For S619L-fibroblasts, siSNP1 improved cell size from 30 nM, while siS619L was only able to restore cell size when transfected at 100 nM. Overall, AS-siRNAs directed against nonpathogenic SNPs were found to be as effective at restoring CME and cell surface area in patient-derived cells harboring two distinct DNM2 mutations as AS-siRNAs directly targeting the mutant nucleotide.

[0095] Rescue of cell migration by AS-siRNA DNM2 dysfunction due to its overexpression is known to promote cell migration, invasion, and metastasis in cancer. 12 Therefore, we investigated the migration defects in two fibroblast cell lines harboring the p.R522H and p.S619L CNM mutations. We tracked single cells for 24 hours and compared the motility behavior in two patient-derived cell lines and two healthy control cell lines. While both control cells exhibited similar motility behavior (Figure 7A), mutant fibroblasts exhibited cell motility defects, including a decreased average speed (Figure 7A), increased pause duration, and a decreased speed when migrating only for S619L-fibroblasts (data not shown).

[0096] To assess the benefit of allele-specific siRNA, we performed the same experiment 48 hours after cell transfection with either scrambled or allele-specific siRNA (data not shown). At a concentration of 30 nM, all measured parameters were reduced in mutant fibroblasts transfected with scrambled siRNA compared with the scrambled control cell line. At this concentration, siSNP1 improved all reduced motility parameters (i.e., average speed, number and duration of pauses, and speed of motility) in R522H-fibroblasts. siSNP2 completely rescued all parameters to control values ​​(Figure 7B). For S619L fibroblasts, siSNP1 and siS619L were effective in rescuing or nearly rescuing motility parameters to control values ​​(Figure 7B). A second series of experiments was performed at a final concentration of 100 nM to assess the possibility of complete restoration of motility parameters with siSNP1 in R522H cells. As shown in Figure 7C, 100 nM siSNP1 rescued all motility parameters. Overall, these results highlight the reduced cell motility associated with DNM2-CNM mutations, which is restored using allele-specific siRNA.

[0097] Rescue of cell adhesion by AS-siRNA The defects in both plasma membrane turnover and cell migration due to impaired endocytosis revealed in the DNM2 mutant cell lines (Figures 6 and 7) may also suggest an effect of the DNM2 mutation on cell adhesion. We next assessed the adhesive capacity of the two CNM fibroblast cell lines by quantifying the number of adherent cells per μm2 1 h after seeding on glass coverslips. Compared to controls, an approximately 60% decrease in cell density was measured in S619L-fibroblasts, while no change occurred in R522H-fibroblasts (Figure 7D). Consequently, we evaluated the effect of AS-siRNA transfected for 48 h only in S619L-fibroblasts. At 30 nM, siS619L and siSNP1 failed to reverse the adhesion defect (Figure 7E), whereas partial rescue was achieved at 100 nM, when values ​​intermediate between those of patient-derived cells and healthy control cells were measured (Figure 7F). Overall, these results indicated that an adhesion defect may exist in cells derived from CNM patients, and that selected AS-siRNAs against a single SNP or mutation ameliorated this phenotype to a similar extent.

[0098] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated herein by reference into the present disclosure.

[0099] [Table 7] TIFF2025530647000008.tif244169 TIFF2025530647000009.tif229169 TIFF2025530647000010.tif243169 TIFF2025530647000011.tif224169

Claims

1. An allele-specific siRNA (AS-siRNA) capable of silencing the expression of only one allele of a heterozygous DNM2 gene, wherein the target allele contains a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rs12461992 (A or T), and / or a disease-causing mutation selected from the group consisting of c. 1393C>T or c. 1856C>T.

2. 2. The AS-siRNA capable of silencing the expression of only one allele of a heterozygous DNM2 gene in a cell according to claim 1, wherein the target allele comprises a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rs12461992 (A or T).

3. AS-siRNA is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 3. The AS-siRNA capable of silencing the expression of only one allele of a heterozygous DNM2 gene in a cell according to claim 2, comprising a sense strand selected from the group consisting of 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, and 151.

4. 4. The AS-siRNA capable of silencing the expression of only one allele of a heterozygous DNM2 gene in a cell according to claim 3, wherein the AS-siRNA comprises a sense strand selected from the group consisting of SEQ ID NOs: 15, 17, 19, 33, 51, 53, 55, 57, 61, 67, 69, 71, 93, 95, 97, 135, 137, 141, 143, 145, and 147.

5. 2. The AS-siRNA capable of silencing expression of only one allele of a heterozygous DNM2 gene in a cell according to claim 1, wherein the target allele comprises a disease-causing mutation selected from the group consisting of c. 1393C>T or c. 1856C>T.

6. 6. The AS-siRNA capable of silencing expression of only one allele of a heterozygous DNM2 gene in a cell according to claim 5, wherein the AS-siRNA comprises a sense strand selected from the group consisting of SEQ ID NOs: 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 215, 217, 223, 225, and 227.

7. 7. The AS-siRNA capable of silencing the expression of only one allele of a heterozygous DNM2 gene in a cell according to claim 6, wherein the AS-siRNA comprises a sense strand selected from the group consisting of SEQ ID NOs: 157, 163, 167, 171, 175, 177, 179, 181, and 185.

8. 8. The AS-siRNA capable of silencing the expression of only one allele of a heterozygous DNM2 gene in a cell according to any one of claims 1 to 7, wherein the AS-siRNA contains a dinucleotide overhang consisting of two deoxythymidines (dTdT) at the 3' end of each strand (sense and antisense).

9. A vector encoding the AS-siRNA according to any one of claims 1 to 8.

10. An in vitro method for silencing expression of a mutant allele of the DNM2 gene without silencing expression of a wild-type allele of the DNM2 gene in a target cell, such as a muscle target cell (e.g., a muscle cell such as a myoblast, particularly a patient-derived myoblast), the method comprising introducing into the target cell an AS-siRNA or vector according to any one of claims 1 to 9.

11. 11. An in vitro method for silencing expression of a mutant allele of the DNM2 gene without silencing expression of a wild-type allele of the DNM2 gene in a target cell, as described in claim 10, wherein the mutant allele comprises a non-pathological polymorphism and a disease-causing mutation in said mutant allele.

12. 10. The AS-siRNA or vector according to any one of claims 1 to 9 for use in a method for treating muscular dystrophy, such as Duchenne muscular dystrophy, in a subject in need thereof.

13. 10. The AS-siRNA or vector according to any one of claims 1 to 9 for use in a method for treating a disease associated with overexpression of dynamin 2 in a subject in need thereof, preferably for treating X-linked myotubular myopathy, or cancers such as prostate cancer and pancreatic cancer.

14. 10. The AS-siRNA or vector of any one of claims 1 to 9 for use in the treatment of centronuclear myopathy (such as autosomal dominant centronuclear myopathy), T-cell acute lymphoblastic leukemia, Charcot-Marie-Tooth disease (CMT), or hereditary spastic paraplegia (HSP) in a subject in need thereof.

15. 10. A method for treating muscular dystrophy, such as Duchenne muscular dystrophy, in a subject in need thereof, comprising administering to the subject an effective amount of the AS-siRNA or vector of any one of claims 1 to 9.

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