Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/34—Allele or polymorphism specific uses

Definitions

• the present invention relates to an allele specific siRNA (AS-siRNA) able to silence the expression of only one allele of a heterozygous DNM2 gene wherein the targeted allele comprises a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rsl2461992 (A or T) and/or a disease-causing mutation selected from the group consisting of c,1393C>T or c, 1856C>T.
• AS-siRNA allele specific siRNA
• AD-CNM Autosomal dominant centronuclear myopathy
• MIM #160150 Autosomal dominant centronuclear myopathy
• the classical late-childhood or adult-onset form exhibits delayed motor milestones and diffuse skeletal muscle weakness mainly involving facial and limb muscles whereas paediatric patients affected by the severe neonatal form usually have generalized weakness, hypotonia, and facial weakness (2,3).
• AD-CNM results from mutations in the DNM2 gene which encodes dynamin 2 (DNM2) (4).
• DNM2 mutations also cause rare forms of Charcot-Mari e-Tooth disease (CMT) (5) and hereditary spastic paraplegia (HSP) (6) and a deleterious DNM2 overexpression was reported in several cancers (7-12) and the X- linked recessive CNM (13), highlighting a large DNM2 involvement in human diseases.
• CMT Charcot-Mari e-Tooth disease
• HSP hereditary spastic paraplegia
• DNM2 belongs to the superfamily of large GTPases (14) acting as a mechanochemical scaffolding molecule that oligomerizes and deforms biological membranes leading to the formation and release of vesicles from the plasma membrane and intracellular membrane compartments.
• DNM2 DNM2
• AD- CNM patients More than 30 DNM2 mutations have been reported in AD- CNM patients (17) and, when tested, the mutant protein is normally expressed (4,18). Mutations are thought to be responsible for a gain of function and/or a dominant-negative effect through an increased GTPase activity and formation of abnormal stable DNM2 oligomers (19,20).
• absence of haploinsufficiency in AD-CNM is supported by data from patients and absence of phenotype developed by heterozygous knockout mice expressing 50% Dnm2 (13,21).
• AS-RNAi allele-specific RNA interference
• AS- siRNA have been developed against disease-associated single nucleotide polymorphisms (SNP) (23,26-28). They took advantage of the presence in the DNM2 sequence of two non-pathogenic SNPs frequently heterozygous in the general population to develop a similar strategy for the AD-CNM.
• SNP disease-associated single nucleotide polymorphisms
• the inventors report the identification of effective AS-siRNA against the two nucleotide versions of the two non-pathogenic DNM2 SNPs which may be used to silence any mutation carried by the same mRNA.
• the first AS-siRNAs targeting a DNM2 mutation associated with severe neonatal phenotype, i.e. the p.S619L mutation have been developed and in a second time, they developed other AS-siRNA targeting the mutation p.S619L and the mutation p.R465W. They also report the functional benefits of this new set of siRNAs on several defects identified in patient-derived cell lines.
• siRNAs against the DNM2 SNPs represent versatile molecules with larger potential applications to silence DNM2 mutations in CMT and HSP and to reduce DNM2 expression in a controlled manner in diseases associated with deleterious overexpression.
• the present invention relates to an allele specific siRNA (AS-siRNA) able to silence the expression of only one allele of a heterozygous DNM2 gene wherein the targeted allele comprises a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rsl2461992 (A or T) and/or a disease-causing mutation selected from the group consisting of C.1393OT or C.1856OT.
• AS-siRNA allele specific siRNA
• the inventors have investigated a therapeutic approach based on the specific suppression of the expression of only one allele of DNM2, preserving the other DNM2 allele.
• this strategy aims to reduce in a controlled way the DNM2 expression level, in the case of diseases related to overexpression of DNM2.
• this strategy would be useful in autosomal dominant inherited diseases due to heterozygous mutation in the DNM2 gene, by specifically inhibiting the expression of a mutant allele without reducing the level of the wild type DNM2 allele which is required for a normal cellular function.
• the present inventors discovered very efficient allele specific siRNAs able to inhibit, in a controlled way, only one allele of a heterozygous DNM2 gene in a cell.
• the invention relates to an allele specific siRNA (AS-siRNA) able to silence the expression of only one allele of a heterozygous DNM2 gene wherein the targeted allele comprises a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rsl2461992 (A or T) and/or a disease-causing mutation selected from the group consisting of C.1393OT or C.1856OT.
• AS-siRNA allele specific siRNA
• the 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 (19p 13.2).
• the dynamin 2 gene or gene products are also known by other names, including but not limited to CMT2M, CMTDI1, CMTDIB, DI-CMTB, DYN2, DYN2 HUMAN, dynamin II, DYNII.
• DNM2 has an important role in endocytosis and in the cell's structural framework (cytoskeleton). The protein interacts with multiple parts of the cytoskeleton, including microtubules and actin, which organize into filaments to provide structure.
• DNM2 cytoskeletons
• An alteration in the DNM2 gene may thus disrupt endocytosis and interfere with the arrangement or dynamics of cytoskeletons leading to abnormal cellular function.
• DNM2 several dominant genetic diseases are caused by heterozygous mutations of the DNM2 gene such as autosomal dominant centronuclear myopathy, Charcot-Mari e-Tooth disease and Hereditary Spastic Paraplegia.
• Overexpression of DNM2 is also pathological and involved in some pathophysiological mechanisms of other diseases such as X-linked myotubular myopathy or cancers, for example prostate cancer and pancreatic cancer.
• RNA interference is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules.
• An interfering RNA is therefore an RNA which is capable of down-regulating the expression of the targeted protein.
• siRNA small interfering RNA
• dsRNA double-stranded RNA
• ssRNA single-stranded RNA
• shRNA short hairpin RNA
• RNA interference designates a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-transcriptional level. In normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) of several thousands of base pair length.
• a dsRNA introduced into a cell is cleaved by an enzyme called DICER into a mixture of short dsRNA molecules called siRNA.
• the siRNAs produced by Dicer are about 21 base-pairs (bp) in length.
• RISC RNA-induced silencing complex
• RNA interference is also a valuable research tool, as double strand siRNA of 19 to 23 bp may be used to selectively and robustly induce suppression of specific genes of interest.
• the major interest of this approach is the specificity, as an siRNA is able to discriminate two sequences even when differing by only a single nucleotide.
• the present inventors used said specificity of siRNA to specifically inhibit one allele of a heterozygous DNM2 gene. Consequently, in that particular case, the siRNA is called an “allele specific siRNA” (AS-siRNA).
• AS-siRNA allele specific siRNA
• AS-siRNA any siRNA able to specifically silence only one allele of a targeted gene, an allele being 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 may be mediated through processes that affect transcription and/or through processes that affect post-transcriptional mechanisms. In some embodiments, gene silencing occurs when siRNA initiates the degradation of the mRNA of the gene in a sequence-specific manner via RNA interference.
• a gene includes coding sequences and/or the regulatory sequences required for expression.
• Gene refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences. "Genes” also include nonexpressed DNA segments that may, for example, form recognition sequences for other proteins.
• the gene is the DNM2 gene, coding for Dynamin 2 protein.
• an AS-siRNA of the invention specifically silences one allele of the DNM2 gene, which is a variant form of the DNM2 gene.
• the DNM2 gene is a heterozygous DNM2 gene.
• a heterozygous DNM2 gene is a DNM2 gene present in a heterozygous state in a cell.
• heterozygous is meant 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), that are called homologous chromosomes. Therefore, each homologous chromosome carries one allele of a given gene.
• a diploid organism is heterozygous when said two alleles of a given gene are different in respect to a given variation or polymorphism.
• a cell or an organism is heterozygous in respect to the DNM2 gene wherein the DNM2 gene is present in a heterozygous state, that is to say wherein the two alleles of DNM2 gene are different in respect to a given variation or polymorphism.
• heterozygous 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.
• the sequence encoding a DNM2 variant comprises a mutation that is not present in the wild-type sequence.
• the DNM2 gene is heterozygous for the presence of a non- pathological polymorphism.
• the AS-siRNA of the invention targets only one of the allele of the DNM2 gene, comprising or not said non-pathological polymorphism.
• the DNM2 gene is heterozygous for the presence of a disease-causing mutation.
• the AS-siRNA of the invention targets the allele of the DNM2 gene comprising said disease-causing mutation.
• the invention relates to an AS-siRNA able to silence 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.
• the DNM2 gene comprises a common heterozygous non- pathological polymorphism.
• “Common heterozygous non pathological polymorphism” refers to a polymorphism with high heterozygous frequency that is to say that is frequent in the population at heterozygous state.
• “frequent” is meant a polymorphism which is found at heterozygous state in at least 20%, 30%, 40% of general population, particularly at least 40%.
• non-pathological polymorphism is meant a variation in the nucleic acid sequence of a gene that is not associated with a disease.
• a non-pathological polymorphism corresponds to a sequence variation in a gene that, when considered independently of other sequence modifications, is not by itself associated to a pathology.
• a non-pathological polymorphism is heterozygous in a cell, it means that both polymorphisms are considered non-pathological, if considered independently of other sequence variations that might occur on the same gene.
• Non-pathological polymorphisms may include variations in coding and non-coding regions.
• non- pathological polymorphisms include nucleotide substitutions, deletions, and/or additions, including those that result in missense and nonsense mutations which do not lead to a pathology.
• the non-pathological polymorphism is a single nucleotide substitution.
• the AS-siRNA of the invention can be used to reduce the expression of DNM2 protein, especially when overexpression of DNM2 in absence of mutation is associated with a pathological condition.
• overexpression of DNM2 protein, in absence of mutation is correlated to X-linked myotubular myopathy or cancer such as prostate cancer and pancreatic cancer.
• the invention relates to an AS- siRNA wherein the AS-siRNA targets a DNM2 allele comprising a non-pathological polymorphism.
• the DNM2 allele comprising a non-pathological polymorphism is on the same allele as a heterozygous disease-causing mutation.
• a single AS- siRNA can be used to inhibit expression of more than one disease-causing mutation in more than one patient.
• the targeted version of the heterozygous non-pathological polymorphism is present on the same allele as said disease-causing mutation and is absent on the wild type allele which harbors the other version of the polymorphism. In other words, targeting a heterozygous non-pathological polymorphism allows differentiating mutant and wild-type DNM2 alleles.
• Said disease-causing mutation can be any heterozygous mutation within the DNM2 gene responsible for or associated to a disease.
• the disease-causing mutation within the DNM2 gene is responsible for or correlated to 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).
• AD-CNM Autosomal Dominant Centronuclear Myopathy
• CMT Charcot-Marie-Tooth disease
• HSP Hereditary Spastic Paraplegia
• AD-CNM Autosomal Dominant Centronuclear Myopathy
• the DNM2 gene is heterozygous for the presence of a missense mutation selected in the group consisting of: c, 1393C>T; c. H05C>T; c. H06G>A; c,1565G>A; c, 1856C>T or c, 1948G>A, respectively responsible for the following substitution in the DNM2 protein sequence: p.R465W, p.R369W, p.R369Q, p.R522H, p.S619L, and p.E650K.
• the DNM2 gene is heterozygous for the presence of the c, 1393C>T mutation, responsible for the p.R465W substitution in the DNM2 protein sequence.
• the invention relates to an AS-siRNA able to silence the expression of only one allele of a heterozygous DNM2 gene in a cell, wherein the targeted allele comprises a non-pathological polymorphism selected from the group consisting of rs2229920 (C or T) or rsl2461992 (A or T).
• the inventors have indeed designed many siRNA able to target the most frequent non- pathological polymorphisms (rs2229920 (C or T) called SNP1 or rsl2461992 (A or T) called SNP2) and tested in in-vitro and in-vivo experiments. These sequences are listed in the tables 1 to 4.
• the invention also relates to an allele specific siRNA (AS-siRNA) able to silence the expression of only one allele of a heterozygous DNM2 gene
• AS-siRNA comprises a sense strand selected in the group consisting of SEQ ID NO: 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.
• the invention relates to an allele specific siRNA (AS-siRNA) able to silence the expression of only one allele of a heterozygous DNM2 gene wherein the AS-siRNA comprises a sense strand selected in the group consisting of SEQ ID NO: 15, 17, 19, 33, 51, 53, 55, 57, 61, 67, 69, 71, 93, 95, 97, 135, 137, 141, 143, 145 and 147.
• AS-siRNA allele specific siRNA
• Table 1 siRNA against the SNP1 dyna (rs2229920) with a polymorphism T.
• the sequence at the top corresponds to the sense strand (or the sense sequence) (5 ’-3’) and is call “passenger sequence” and the at the bottom corresponds to the antisense strand (or the antisense sequence) (3 ’-5’) and is call “guide sequence” (see the Sil-SNPl-T for example).
• Table 2 siRNA against the SNP1 practise (rs2229920) with a polymorphism C.
• the sequence at the top corresponds to the sense strand (or the sense sequence) (5 ’-3’) and is call “passenger sequence” and the at the bottom corresponds to the antisense strand (or the antisense sequence) (3 ’-5’) and is call “guide sequence” (see the Sil-SNPl-C for example).
• Table 3 siRNA against the SNP2 dyna (rs 12461992) with a polymorphism A.
• the sequence at the top corresponds to the sense strand (or the sense sequence) (5 ’-3’) and is call “passenger sequence” and the at the bottom corresponds to the antisense strand (or the antisense sequence) (3 ’-5’) and is call “guide sequence” (see the SH-SNP2-A for example).
• Table 4 siRNA against the SNP2 dyna (rs 12461992) with a polymorphism T.
• the sequence at the top corresponds to the sense strand (or the sense sequence) (5 ’-3’) and is call “passenger sequence” and the at the bottom corresponds to the antisense strand (or the antisense sequence) (3 ’-5’) and is call “guide sequence” (see the SH-SNP2-T for example).
• the AS-siRNA of the invention is able to silence only one allele of DNM2 gene comprising a heterozygous non-pathological polymorphism, by hybridizing specifically to the gene transcript (messenger RNA or mRNA) derived from said allele of DNM2 gene.
• AS-siRNA of the invention is therefore complementary to mRNA derived from said allele of DNM2 and binds to said mRNA by base pairing.
• the term "complementary" refers to the ability of polynucleotides to form base pairs with another polynucleotide molecule. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands.
• the degree of complementarity between the AS-siRNA according to the invention and the target mRNA is equal to about 100%.
• AS-siRNA of the invention targets a region of the DNM2 gene transcript comprising said non-pathological polymorphism.
• AS-siRNA of the invention is complementary to a sequence of the mRNA comprising said non- pathological polymorphism.
• the specificity of siRNA allows discriminating two sequences, even when differing by a single nucleotide. This property allows the AS-siRNA of the invention targeting polymorphism resulting to single nucleotide substitution.
• the target allele (and consequently the target mRNA) could be arbitrarily chosen, in absence of disease-causing mutation, in order to reduce the level of DNM2 mRNA and/or protein.
• the AS-siRNA of the invention could target the mRNA carrying or not said non-pathological polymorphism, when the goal is only to reduce the overall level of DNM2 mRNA or DNM2 protein.
• the AS-siRNA of the invention could target anyone of the DNM2 allele carrying one of the two version of the heterozygous non-pathological polymorphism, wherein DNM2 is overexpressed in a cell, for example in X-linked myotubular myopathy or cancer such as prostate cancer and pancreatic cancer.
• the targeted version of the non-pathological polymorphism is present on the same allele as a disease-causing mutation. Therefore, the AS- siRNA of the invention targets and silences only the allele carrying said targeted version of the polymorphism and said disease-causing mutation. This particular embodiment requires prior confirming the location of the disease-causing mutation.
• the DNM2 gene is heterozygous for the presence of a disease-causing mutation. Therefore, the DNM2 gene is present in two different forms corresponding to the two alleles: one DNM2 allele is a “wild type allele” whereas the other is a “mutant allele”.
• the AS-siRNA of the invention specifically targets and silences the allele of the DNM2 gene comprising said disease-causing mutation without targeting the wild type allele. The AS-siRNA is thus able to 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.
• the disease-causing mutation could be any deletion, insertion or substitution of nucleotide(s) within the DNM2 gene which is responsible for a pathology or which is correlated to a pathology.
• the disease-causing mutation is a dominant mutation.
• dominant mutation is meant any mutation that leads to a dominant allele.
• dominant allele is meant an allele that exerts its effect on phenotype over the presence of a recessive allele of the same gene.
• the terms dominant and recessive alleles are defined relative to one another and are not absolute. In other words, the phenotypic consequences of a dominant mutation are observed in a heterozygous individual carrying one mutant allele and one wild type allele. Recessive alleles only show their effect if the individual has two copies of the mutated allele (also known as being homozygous) or two different mutated alleles (also known as composite heterozygosity).
• the dominant mutation is a gain-of-function mutation.
• a gain-of-function mutation is defined as a mutation that confers new or enhanced activity on 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, the disease-causing mutation within the DNM2 leads to a gain-of-function of Dynamin 2 protein.
• the dominant mutation is a loss-of-function mutation by 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.
• Dominant-negative effect is defined as the product of a mutated allele alters the function of the product from the wild-type allele. This occurs, for example, when oligomerization is required for normal function on a protein and when the mutated protein is able to oligomerize with the wild-type protein. Consequently, the disease-causing mutation within the DNM2 leads to a loss-of-function of the wild-type Dynamin 2 protein due to the presence of the mutated Dynamin 2 protein.
• the DNM2 gene which is heterozygous for a diseasecausing mutation is not haploinsufficient.
• Haploinsufficiency occurs when one copy of a gene is inactivated or deleted and the remaining functional copy of the gene is not adequate to produce sufficient amount of the gene product to preserve normal function.
• the wild type DNM2 allele of the invention is able to preserve normal function, following the silencing of the mutant allele by AS-siRNA of the invention. Therefore, the present invention preferably relates to autosomal dominant disease in which there is no haploinsufficiency.
• the dominant mutation within the DNM2 gene leads to an autosomal dominant disease.
• An autosomal dominant disease is a disease wherein the individual has one copy of a mutant gene and one normal gene on a pair of autosomal chromosomes (autosomal chromosome being any chromosome which is not a sex chromosome).
• An individual with autosomal dominant diseases has 50% chance of passing the mutant gene and therefore the disorder on to each of its children.
• the autosomal dominant disease is selected from Autosomal Dominant Centronuclear Myopathy (AD-CNM), T-cell acute lymphoblastic leukemia, Charcot-Mari e-Tooth disease (CMT) or Hereditary Spastic Paraplegia (HSP).
• AD-CNM Autosomal Dominant Centronuclear Myopathy
• CMT Charcot-Mari e-Tooth disease
• HSP Hereditary Spastic Paraplegia
• the autosomal dominant disease is Autosomal Dominant Centronuclear Myopathy (AD-CNM).
• the disease-causing mutation within the DNM2 gene is responsible for or correlated to a disease selected from Autosomal Dominant Centronuclear Myopathy (AD-CNM), T-cell acute lymphoblastic leukemia, Charcot-Mari e-Tooth disease (CMT) or Hereditary Spastic Paraplegia (HSP).
• AD-CNM Autosomal Dominant Centronuclear Myopathy
• CMT Charcot-Mari e-Tooth disease
• HSP Hereditary Spastic Paraplegia
• the disease-causing mutation within the DNM2 gene is responsible for or correlated to Autosomal Dominant Centronuclear Myopathy (AD-CNM).
• the disease-causing mutations c, 1393C>T and c,1856C>T are respectively responsible for the following substitution in the DNM2 protein sequence: IND.R465W and p.S619L.
• the invention relates to an AS-siRNA able to silence the expression of only one allele of a heterozygous DNM2 gene in a cell, wherein the targeted allele comprises a disease-causing mutation selected from the group consisting of c, 1393C>T or Georgia, 1856C>T.
• the inventors have indeed designed many siRNA able to target the most frequent disease-causing mutation (c,1856OT also called S619L and C.1393OT also called p.R465W) and tested in in-vitro and in-vivo experiments. These sequences are listed in the tables 5 to 6.
• the invention also relates to an allele specific siRNA (AS-siRNA) able to silence the expression of only one allele of a heterozygous DNM2 gene
• AS-siRNA comprises a sense strand selected in the group consisting of SEQ ID NO: 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.
• the invention relates to an allele specific siRNA (AS-siRNA) able to silence the expression of only one allele of a heterozygous DNM2 gene wherein the AS-siRNA comprises a sense strand selected in the group consisting of SEQ ID NO: 157, 163, 167, 171, 175, 177, 179, 181 and 185.
• AS-siRNA allele specific siRNA
• Table 5 siRNA against the mutation S619L.
• the sequence at the top corresponds to the sense strand (or the sense sequence) (5 ’-3’) and is call “passenger sequence” and the at the bottom corresponds to the antisense strand (or the antisense sequence) (3 ’-5’) and is call “guide sequence” (see the SH-S619L for example).
• Table 6 siRNA against the mutation R465W.
• the sequence at the top corresponds to the sense strand (or the antisense sequence) (5 3 ’) and is call “passenger sequence” and the at the bottom corresponds to the antisense strand (or the antisense sequence) (3 ’-5’) and is call “guide sequence” (see the SH-R465W for example).
• the AS-siRNA of the invention is able to silence the mutant allele of DNM2 gene, by hybridizing specifically to the gene transcript (messenger RNA or mRNA) derived from said mutant allele of DNM2 gene.
• the AS-siRNA of the invention is therefore complementary to a mRNA derived from said mutant allele of DNM2 and binds to said mRNA by base pairing.
• the term "complementary" refers to the ability of polynucleotides to form base pairs with another polynucleotide molecule. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands.
• the degree of complementarity between the AS-siRNA according to the invention and the target mRNA is equal to about 100%.
• the AS-siRNA of the invention targets a region of the DNM2 gene transcript comprising said disease-causing mutation.
• the AS-siRNA of the invention is complementary to a sequence of the mRNA comprising said disease-causing mutation.
• the specificity of siRNA allows discriminating two sequences, even when differing by a single nucleotide. This property allows the AS-siRNA of the invention targeting diseasecausing mutations, such as mutations resulting to single nucleotide substitution.
• the AS-siRNA of the invention contains nucleotide overhangs on 3' end of each strand. In a more particular embodiment, the AS-siRNA of the invention contains dinucleotide overhangs made of two deoxythymidines (dTdT) on 3' end of each strand (sense and antisense).
• dTdT deoxythymidines
• Another aspect of the invention relates to a vector encoding the AS-siRNA of the invention, the vector being particularly a plasmid or a viral vector, such as an AAV vector.
• sense strand is meant the strand of the AS-siRNA which has the same sequence as the targeted allele comprising the non-pathological polymorphism or the disease-causing mutation. Therefore, the other strand of AS-siRNA is called “anti-sense” because its sequence is complementary to the targeted DNM2 mRNA, which is called the “sense” sequence (so that a sense segment of mRNA " 5'-AAGGUC-3' " would be blocked by the anti-sense mRNA segment " 3'-UUCCAG-5' ").
• the AS-siRNA of the invention is used to reduce expression of DNM2 mRNA and/or DNM2 protein by 20-60%, such as 20, 30, 40, 50 or 60%.
• the AS-siRNA of the invention is used to reduce expression of DNM2 mRNA and/or DNM2 protein by about 50%.
• « about » is meant a value of + or - 10 %, Particularly + or - 5 %.
• about 50% means from 45 to 55%, Particularly from 47.5 to 52.5%.
• the present invention contemplates various ways of reaching the target mRNA with AS-siRNA of the invention.
• the AS-siRNA may be administered to the cell as isolated oligonucleotide, either directly or using transfection reagents such as lipidic derivatives, liposomes, calcium phosphate, nanoparticles, microinjection or electroporation.
• the present invention contemplates introducing the AS-siRNA into the cell in the form of a vector.
• a vector encoding the AS-siRNA of the invention.
• the vector may in particular be a plasmid or a viral vector.
• Representative viral vectors useful in the practice of the invention include, without limitation, a vector derived from adenovirus, retrovirus, in particular lentivirus, poxviruses, herpes simplex virus I and adeno-associated virus (AAV).
• the AAV vector is AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9 AAV10, AAV3B, AAV-2i8, RhlO, Rh74 or any other serotypes of AAV that can infect human, monkeys or other species.
• targeted cells are muscle cells, but viral vectors with broad tropism, including in particular the muscle tropism, may also be implemented.
• an AAV1 vector is implemented, for example for use in intramuscular injections.
• the vector is to be administered via the systemic route (for example via the intravascular or intraarterial route), and the vector is an AAV8 or AAV9 vector.
• the invention also relates to shRNA (short hairpin RNA) corresponding to the AS-siRNA of the invention, with a further tight hairpin turn. The shRNA hairpin structure is then cleaved by the cellular machinery into siRNA.
• a further aspect of the invention relates to a vector encoding shRNA corresponding to AS-siRNA of the invention.
• the invention also relates to a target cell comprising an AS-siRNA of the invention or which is transfected or transduced with a vector of the invention.
• the target cell may be selected from: a muscle cell (or a cell of the muscle lineage), such as myoblast, for example a patient-derived myoblast, or a fibroblast such as a patient-derived fibroblast.
• the present invention relates to an in vitro method for silencing the expression of the mutated allele of DNM2 gene without silencing the expression of the wild type allele of the DNM2 gene in a target cell, such as a muscle target cell (for example a muscle cell, such as a myoblast, in particular a patient-derived myoblast), comprising introducing in said target cell an AS-siRNA or a vector of the invention.
• a target cell such as a muscle target cell (for example a muscle cell, such as a myoblast, in particular a patient-derived myoblast)
• introducing in said target cell an AS-siRNA or a vector of the invention.
• the present invention relates to an in vitro method for silencing the expression of one allele of DNM2 gene carrying a heterozygous non-pathological polymorphism without silencing the expression of the other allele of the DNM2 gene in a target cell, comprising introducing in said target cell an AS-siRNA or a vector of the invention.
• the present invention relates to an AS-siRNA, a vector or a cell of the invention for use in a method for treating, in a subject in need thereof, a disease induced by a disease-causing mutation in the DNM2 gene.
• an AS-siRNA, a vector or a cell of the invention are used in a method for treating, in a subject in need thereof, a centronuclear myopathy (such as autosomal dominant centronuclear myopathy), T-cell acute lymphoblastic leukemia, Charcot-Marie-Tooth disease (CMT) or Hereditary Spastic Paraplegia (HSP).
• a centronuclear myopathy such as autosomal dominant centronuclear myopathy
• T-cell acute lymphoblastic leukemia Charcot-Marie-Tooth disease (CMT) or Hereditary Spastic Paraplegia (HSP).
• HSP Hereditary Spastic Paraplegia
• an AS-siRNA, a vector or a cell of the invention are used
• the present invention relates to an AS-siRNA, a vector or a cell of the invention for use in a method for treating, in a subject in need thereof a muscular dystrophy such as Duchenne muscular dystrophy.
• the present invention relates to an AS-siRNA, a vector or a cell of the invention for use in a method for treating, in subject in need thereof, a disease associated with overexpression of dynamin 2, preferably for treating X-linked myotubular myopathy, or cancer such as prostate cancer and pancreatic cancer.
• the invention relates to an AS-siRNA, a vector or a cell of the invention for use in the treatment of a centronuclear myopathy (such as autosomal dominant centronuclear myopathy), T-cell acute lymphoblastic leukemia, Charcot-Mari e-Tooth disease (CMT) or Hereditary Spastic Paraplegia (HSP) in a subject in need thereof.
• a centronuclear myopathy such as autosomal dominant centronuclear myopathy
• T-cell acute lymphoblastic leukemia Charcot-Mari e-Tooth disease (CMT) or Hereditary Spastic Paraplegia (HSP)
• CMT Charcot-Mari e-Tooth disease
• HSP Hereditary Spastic Paraplegia
• the invention relates to a method for treating muscular dystrophy such as Duchenne muscular dystrophy in a subject in need thereof comprising administrating to said subject an effective amount of an AS-siRNA, vector or cell of the invention.
• the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human.
• treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
• the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
• therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
• a therapeutic regimen may include an induction regimen and a maintenance regimen.
• the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
• the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
• An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
• maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
• a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
• the AS-siRNA of the invention, the vector or the cell according to the invention can be formulated and administered to treat any disease caused by a heterozygous mutation in the DNM2 gene or caused by overexpression of DNM2, preferably to treat autosomal dominant centronuclear myopathy, T-cell acute lymphoblastic leukemia, Charcot-Mari e-Tooth disease, Hereditary Spastic Paraplegia, X-linked myotubular myopathy, or cancer such as prostate cancer and pancreatic cancer.
• AS-siRNA of the invention, the vector or the cell according to the invention are formulated by any means that produces contact of the AS-siRNA with its site of action in the subject in need thereof.
• compositions comprising the AS- siRNA of the invention, the vector or the cell according to the invention.
• Such compositions comprise a therapeutically effective amount of the therapeutic (the AS-siRNA, vector or cell of the invention), and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. or European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and humans.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as saline solution, water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• Physiological saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.
• Saline solutions 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.
• compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
• These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
• Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. 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, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
• the pharmaceutical composition is adapted for any type of administration to a mammal, in particular a human being and is formulated in accordance with routine procedures.
• the composition is formulated by using suitable conventional pharmaceutical carrier, diluent and/or excipient. Administration of the composition may be via any common route so long as the target tissue is available via that route.
• the amount of the therapeutic of the invention which will be effective in the treatment of a nucleotide repeat expansion can be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be employed to help predict optimal dosage ranges.
• the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances.
• the dosage of the AS-siRNA, the vector or the cell administered to the subject in need thereof will vary based on several factors including, without limitation, the route of administration, the subject's age or the level of expression necessary to obtain the required therapeutic effect. One skilled in the art can readily determine, based on its knowledge in this field, the dosage range required based on these factors and others.
• FIGURES are a diagrammatic representation of FIGURES.
• Figure 1 Identification of allele-specific siRNA against the C version of SNP1.
• A Expression of the DNM2 mRNA 48 hours after siRNA transfection at 30 nM. Quantification of DNM2 expression normalized to HPRT (n >4 per condition).
• B Quantification of the C/T ratio (n >4 per condition) after transfection with siRNA at 30 nM.
• C Expression of the DNM2 mRNA 48 hours after siRNA transfection at 100 nM. Quantification of DNM2 expression normalized to HPRT (n >4 per condition).
• D Quantification of the C/T ratio (n >4 per condition) after transfection with siRNA at 100 nM.
• E Quantification of the signal by densitometry after siRNA transfection at 100 nM concentration.
• GAPDH was used as a loading control (n >4).
• Figure 2 Identification of allele-specific siRNA against the T version of SNP1.
• A Expression of the DNM2 mRNA 48 hours after siRNA transfection at 30 nM. Quantification of DNM2 expression normalized to HPRT (n >4 per condition).
• B Quantification of the T/C ratio (n >4 per condition) after transfection with siRNA at 30 nM.
• C Expression of the DNM2 mRNA 48 hours after siRNA transfection at 100 nM. Quantification of DNM2 expression normalized to HPRT (n >4 per condition).
• D Quantification of the T/C ratio (n >4 per condition) after transfection with siRNA at 100 nM.
• E Quantification of the signal by densitometry after siRNA transfection at 100 nM concentration.
• GAPDH was used as a loading control (n >4).
• Figure 3 Identification of allele-specific siRNA against the T version of SNP2.
• A Expression of the DNM2 mRNA 48 hours after siRNA transfection at 30 nM. Quantification of DNM2 expression normalized to HPRT (n >4 per condition).
• B Quantification of the T/A ratio (n >4 per condition) after transfection with siRNA at 30 nM.
• C Expression of the DNM2 mRNA 48 hours after siRNA transfection at 100 nM. Quantification of DNM2 expression normalized to HPRT (n >4 per condition).
• D Quantification of the T/A ratio (n >4 per condition) after transfection with siRNA at 100 nM.
• E Quantification of the signal by densitometry after siRNA transfection at 100 nM concentration.
• GAPDH was used as a loading control (n >4).
• Figure 4 Identification of allele-specific siRNA against the A version of SNP2.
• A Expression of the DNM2 mRNA 48 hours after siRNA transfection at 30 nM. Quantification of DNM2 expression normalized to HPRT (n >4 per condition).
• B Quantification of the A/T ratio (n >4 per condition) after transfection with siRNA at 30 nM.
• C Expression of the DNM2 mRNA 48 hours after siRNA transfection at 100 nM. Quantification of DNM2 expression normalized to HPRT (n >4 per condition).
• D Quantification of the A/T ratio (n >4 per condition) after transfection with siRNA at 100 nM.
• E Quantification of the signal by densitometry after siRNA transfection at 100 nM concentration.
• GAPDH was used as a loading control (n >4).
• Figure 5 Identification of allele-specific siRNA against the S619L DNM2 mutation.
• A Expression of the DNM2 mRNA 48 hours after siRNA transfection at 30 nM. Quantification of DNM2 expression normalized to HPRT (n >4 per condition).
• B Quantification of the mutant/WT ratio (n >4 per condition) after transfection with siRNA at 30 nM. The mutant allele harbors the C version of SNP1.
• C Expression of the DNM2 mRNA 48 hours after siRNA transfection at 100 nM. Auantification of DNM2 expression normalized to HPRT (n >4 per condition).
• D Quantification of the mutant/WT ratio (n >4 per condition) after transfection with siRNA at 100 nM.
• Figure 6 Impact of AS-siRNA on transferrin uptake and cell surface of patient- derived fibroblasts.
• A. Transferrin uptake under basal conditions (n 500-700 cells from 3 independent experiments).
• B. Transferrin uptake after 48 hours transfection with 30 nM scramble siRNA (sc) or allele-specific siRNA against the S619L mutation or the 2 SNPs (n 200-300 cells from 2 independent experiments).
• Figure 7 Migration and adhesion assays in patient-derived cells.
• Adhesion assay after 48 hours transfection with 30 nM scramble siRNA (sc) or allele-specific siRNA against the S619L mutation or the SNP1 (n 5 or 6 independent transfections).
• Healthy controls and CNM patient-derived fibroblast cell lines were obtained from the MyoLine platform for the immortalization of human cells (Institute of Myology, Paris, France) in accordance with European recommendations and French legislation.
• Cell lines were cultured at 37°C (5% CO2) 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 pg/ml).
• DMEM Dulbecco's modified Eagle's medium
• FCS fetal calf serum
• Penicillin 100 Units/ml
• Streptomycin 100 pg/ml
• RNAimax transfection reagent Life Technologies, France
• concentration of siRNAs for each experiment was indicated in corresponding figure legends. Allele-specific siRNA and scramble siRNA were purchased from Eurogentec (Belgium) and the sequences are available on request. Cells were used for functional evaluation or harvested for RNA and protein extraction 48h after transfection.
• RNAs were isolated from cells using NucleoSpin RNA (Macherey -Nagel, France) according to the manufacturer's protocol. Cells were passed through a pipetting up-down several times for disruption in the lysis buffer. Total RNAs (500 ng) were submitted to reverse transcription using the Superscript III reverse transcriptase kit (Life Technologies, France) using oligo-dT primers in a final volume of 20 pl. Reverse transcription was performed at 50°C for 50 minutes, and a final step of 85°C for 5 minutes was added.
• PCR encompassing SNPs and the mutation was performed and cloned using the pGEMT vector system (Promega) and about ten single clones were sequenced (Eurofins, France).
• the DNM2 expression was quantified by semi- quantitative RT-PCR relative to the HPRT housekeeping gene expression.
• RT product (1 pl) was submitted 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 and a final step at 72°C for 5 minutes.
• PCR The number of 27 PCR cycles has been selected to have the amplification in the exponential range for DNM2 and HPRT.
• assays were developed for SNP1 and SNP2 using restriction enzymes allowing discrimination between the 2 alleles after digestion of the RT-PCR products.
• PCR was designed to amplify regions of the DNM2 transcript encompassing the SNPs.
• RT product (1 pl) was submitted 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 and a final step at 72°C for 5 minutes.
• the number of cycles has been selected to be at the end of the exponential phase of amplification.
• Ten pl out of the 20 pl PCR products were digested overnight at 37°C using 7 units of Bgll (New England Biolabs, France) for SNP1, and 15 pl out of the 30 pl PCR products were digested overnight at 37°C using 7 units of Psp5II (New England Biolabs, France) for SNP2.
• Image acquisition of the PCR products after agarose gel electrophoresis was performed using a Geni2 gel imaging system (Ozyme, France), and the associated signal was quantified using Imaged Software (NIH; http://rsbweb.nih.gov/ij). All the PCR primers used in this study were from Eurogentec (Belgium) and sequences are available on request.
• Cell pellets were homogenized in lysis buffer containing 50 mM of Tris-HCl pH 7.5, 150 mM NaCl, 1% IGEPAL, 0.5% Deoxycholate Sodium and protease inhibitor cocktail 1% (Sigma-Aldrich, France) and kept on rotator for 20 minutes at 4°C. After cell lysates scraping, samples are lysed by sonication 2 times for 10 seconds at 30% of a maximum power of VCX 130 Vibra-cell ultrasonic processors (Sonics, USA). After centrifugation (12,000 g, 4°C, 20 minutes), protein concentration in the supernatant was determined with the BCA Protein Assay Kit (Thermo Scientific Pierce, France).
• Membranes were blocked for 2 hours at room temperature in PBS containing non-fat dry milk 5% and Tween-200.1% and then exposed to rabbit polyclonal anti-Dynamin 2 antibody (ab3457 or ab65556, Abeam, France) or rabbit polyclonal anti-Transferrin receptor antibody (ab84036, Abeam, France) or rabbit polyclonal anti-GAPDH antibody (sc-25778 Santa Cruz, France) in PBS-Tween-200.1%, non-fat dry milk 1% overnight at 4°C.
• Transfected cells were cultured in DMEM at 37°C for 45 minutes. Transferrin- AlexaFluor488 (Life Technologies, France) was added at 20 pg/ml at 37°C for 15 minutes. Cells were then washed in DMEM pH 2 and PBS and fixed in paraformaldehyde 4% for 15 minutes. Images z-stacks were acquired using an Axio Observer Apotome.2 microscope (Zeiss, Germany) using a 20x Plan Apochromat Zeiss objective.
• CTCF total corrected cell fluorescence
• Cells were harvested by trypsinization 48 hours after transfection or under basal conditions and 35000 cells were seeded on a 12-millimetre diameter glass coverslip in 24-well- plates (3 technical replicates for each transfection). After 1 hour incubation at 37°C in DMEM- 10% FCS supplemented with Penicillin (100 Units/ml) and Streptomycin (100 pg/ml), the non- attached cells were removed by PBS washing and cells attached on coverslips were fixed in paraformaldehyde 4% for 15 minutes. Glass coverslips were mounted on slides with Vectashield medium (Vector Laboratories) containing DAPI to stain nuclei.
• Vectashield medium Vector Laboratories
• the first SNP was a T/C variation (rs2229920, thereafter called SNP1) identified as a synonymous variant of the Alanine 713 (c.2139T>C, NM_001005361.3) of the DNM2 protein. Allele frequencies for SNP1 were determined at 0.688 for the allele T and 0.321 for the allele C on 250 390 counts (gnomAD exomes r2.1.1) resulting in a theorical heterozygous (HTZ) frequency of 0.43 (calculated as 2 x frequency of T allele x frequency of C allele). In agreement, PCR and Sanger sequencing in a cohort of 52 CNM patients identified 42.3% of SNP1 heterozygosity.
• the second SNP was an A/T variation (rs 12461992, thereafter called SNP2) identified as a 3 ’-UTR variant located 268 nucleotides after the stop codon (*268 A>T, NM_001005361.3). Allele frequencies for SNP2 were determined at 0.824 for the allele A and 0.176 for the allele T resulting in a theorical HTZ frequency of 0.29 on 143 008 counts (gnomAD exomes r3.0) and 23% of SNP2 heterozygosity was found in our cohort of patient. No clinical sign was associated with SNP1 and SNP2.
• RT-PCR products encompassing the SNPs were amplified from fibroblast cell lines from 2 healthy controls and 2 CNM patients. Sanger sequencing of these RT-PCR products led to identifying one healthy control cell line heterozygous for the 2 SNPs, one CNM cell line (p.R522H DNM2 mutation) harbouring the 2 SNPs at HTZ state, and one CNM cell line (p.S619L DNM2 mutation) harbouring only the SNP1 at HTZ state (data not shown).
• the 7 siRNA reduced the C/T ratio compared to scramble siRNA ( Figure IB) with the lower ratio reached by si8 and si9. Quantification of each allele relative to HPRT mRNA showed that si8, si9, and si 10 reduced expression of the allele C without affecting the T (data not shown).
• si8, si9, and si 10 also reduced expression of the DNM2 protein around the expected 50% compared to scramble siRNA in western blot (data not shown).
• the maintenance of allele-specificity at higher concentration (lOOnM) was then assessed for si8, si9, and silO.
• the 3 siRNA reduced total DNM2 mRNA content, and close to the expected 50% decrease for si 8 and si9 (Figure 1C). Allele-specificity of si8 and si9 against the targeted C allele was maintained as demonstrated by the C/T ratio reduction ( Figure ID) and confirmed by quantification of each allele relative to HPRT expression (data not shown).
• siRNAs si3, si6, si8, silO, si 12, si 13, si 14, si 15, and si 17 significantly reduced the DNM2 expression, and among them, the si6, si8, silO, sil3, sil5, sil7 reduced mRNA amount in the expected range around 50% (Figure 5 A).
• the si6, si8, silO, sil3, sil5, sil7 reduced mRNA amount in the expected range around 50% ( Figure 5 A).
• the S619L mutation does not introduce or remove a restriction site relative to the WT sequence
• the 9 siRNA significantly reduced the C/T (i.e.
• DNM2 is well recognized for its role in endocytosis and the defect of clathrin-mediated endocytosis (CME) was previously demonstrated in AD-CNM patient-derived fibroblasts (18,29).
• CME clathrin-mediated endocytosis
• CME was decreased in fibroblasts harbouring the R522H mutation (R522H-fibroblasts) and increased in fibroblasts harbouring the S619L mutation (S619L-fibroblasts) compared to control fibroblasts ( Figure 6A) and western blot showed similar expression of transferrin receptor between control and mutant cells (data not shown).
• siSNPl sil l against the T version of the SNP2
• siSNP2 sil 1-T
• siS619 silO against the p.S619L mutation
• DNM2 dysfunction through its overexpression is known to promote cell migration, invasion and metastasis in cancers 12. Therefore, we looked for migration defects in the 2 fibroblast cell lines carrying the p.R522H and the p.S619L CNM mutations.
• RNAi therapy targeting KRAS in combination with chemotherapy for locally advanced pancreatic cancer patients Oncotarget 6, 24560-70.
• Dynamin 2 interacts with a-actinin 4 to drive tumor cell invasion.
• Dynamin 2 potentiates invasive migration of pancreatic tumor cells through stabilization of the Rael GEF Vavl. Dev Cell 24, 573-85.
• N z -[4-(dipropylamino)benzylidene]-2- hydroxybenzohydrazide is a dynamin GTPase inhibitor that suppresses cancer cell migration and invasion by inhibiting actin polymerization. Biochemical and Biophysical Research Communications 443, 511-517.

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