FR2894967A1 - New interfering RNA, useful for treatment and prevention of diseases caused by morbilliviruses, are directed against the mRNA from the viral nucleoprotein gene - Google Patents

Abstract

Interfering RNA (A) directed against a region (R) of the mRNA of the N gene, which encodes the nucleoprotein of a morbillivirus, is new. (R) contains the motif defined by Seq. (1) RRWYNNRHUGGUUHGARA (1) where R = A or G; Y = C or U; W = A or U; H = A, C or U; N = A, C, G or U. An independent claim is included for an expression vector that contains a DNA sequence encoding (I), transcribable to (A), under control of a promoter. ACTIVITY : Virucide. MECHANISM OF ACTION : Vaccine; inhibition of viral replication by RNA interference.

Description

The present invention relates to the obtaining of interfering RNAs

  capable of inhibiting the replication of morbilliviruses, and their use for the prophylaxis or therapy of morbillivirus infections, especially for obtaining vaccines. The genus Morbillivirus belongs to the family Paramyxoviridae (Order Mononegavirales) and includes the PPRV (peste des petits ruminants virus), the RPV (Rinderpest virus or rinderpest virus), the MV (or MeV) ( Measles virus or measles virus), CDV (canine distemper virus or Carre's disease virus), DMV (dolphin morbillivirus or dolphin morbillivirus) and PDV (phocine distemper virus or marine seal virus).

  The genome of morbilliviruses is a single-stranded, non-segmented, negative polarity RNA. Its structure is schematised in FIG. 1. This single-stranded RNA comprises 6 genes: N, P, M, F, H and L. The products of the N (Nucleoprotein), P (Phosphoprotein) and L (proLein Large) genes RNA polymerase RNA dependent) associate with viral genomic RNA to form the nucleocapsid, which protects the viral genome, and provides a polymerase complex for replication and transcription of the virus.

  The products of the F genes (Fusion protein) and H (Hemagglutinin) are part of the viral envelope. The glycoprotein H allows the attachment of the virus to the target cell, and the glycoprotein F is involved in the fusion of the viral envelope and the cell membrane.

  The M gene product (Matrix Protein) provides the interface between the nucleocapsid and the virus envelope. RNA interference is a biological mechanism conserved during evolution, which induces the specific extinction of genes by specific degradation of the messenger RNAs and / or stopping their translation. Elie was first observed in Caenorhabditis elegans: Injection into this double-stranded RNA nematode inhibits the expression of the gene which contains the sequence of the injected molecule. It was then shown (FIRE et al., Nature, 391, 806, 1998) that it was the double-stranded RNA that was responsible for this mechanism. In the cell, this double-stranded RNA is rapidly segmented by an RNase III type endonuclease, called DICER, into small 21 to 28 nucleotide RNAs: the small siRNA siRNA interfering agents (ZAMORE et al., Cell, 101, 25, 2000). SiRNAs are incorporated into an enzyme complex called RISC for "RNA-Induced Silencing Complex". The RISC complex dissociates the strands of siRNAs, and guides the pairing of the antisense strands with their complementary target sequences. The messenger RNAs containing these target sequences are then cleaved at the level of the duplex thus formed or their translation by the ribosomes is blocked.

  In mammals, the presence of double-stranded RNA larger than 30 pb in a cell also induces an interferon-mediated response, which results in non-sequence-specific messenger RNA degradation. It has been shown (ELBASHIR et al., Nature, 411, 494, 2001) that the use of siRNAs in mammalian cells does not induce this interferon response, and allows specific inhibition of gene expression. containing the complementary sequences of these siRNAs.

  RNA interference has been extensively used against viruses of various families, to target different genes for the purpose of studying their function, and / or for anti-viral therapy purposes. For non-segmented, single-stranded RNA viruses, the first experiments were performed in pneumovirus, the synovial respiratory virus (RSV) (BITKO & BARIK, BMC Microbiol, 1, 34, 2001). A siRNA targeting the P-protein gene (subunit of the RNA dependent RNA polymerase), resulted in a 90-fold reduction in the expression of this protein, accompanied by a drastic reduction in the size of the protein. expression of all the viral proteins, and a reduction of the viral titre; a siRNA targeting the F protein gene induces a specific reduction in the synthesis of this protein, without affecting that of the other viral proteins, and inhibits the formation of syncitia. PCT Application WO2005 / 056021 describes the use of siRNA to target and inactivate the gene coding for a non-structural pro-Lein of RSV, pro-Lein NS1, and thereby increase the interferon response to RSV. A recent publication by S. BARIK (BARIK, Virus Res, 102, 27, 2004) reviews the different approaches used to control the replication of unsegmented, single-stranded siRNA RNA viruses. Targeting the genes encoding the P or L proteins, which are essential subunits of the viral polymerase complex, leads to an almost complete disappearance of viral RNA synthesis; Targeting non-essential genes to the synthesis of viral RNA but implicated in the virus's interactions with the host cell produces more variable results. RNA interference potentially represents a particularly interdening tool for the prophylaxis and / or treatment of viral infections. However, in spite of all the theoretical interest of this approach, putting it into practice to obtain effective antiviral activity poses various problems, particularly with regard to the choice of target sequences used in siRNAs. The term "target gene" is used herein to designate a gene which is sought to be extinguished, and that of "target sequence" to designate a portion of the mRNA of a gene. -recognized by a particular siRNA. One of the problems in choosing target sequences is the frequency of mutations, which is usually very high among viruses. A mutation occurring in the siRNA target sequence may allow the mutant virus to escape recognition. It is therefore desirable to choose a target sequence kept between different viruses, or mutations are less likely to appear.

  On the other hand, although it is relatively easy, for a given target gene, to define siRNAs allowing to obtain a certain attenuation of the expression of this gene, it is much more problematic to obtain siRNAs allowing achieve a target gene silencing level sufficient to inhibit viral replication. It is indeed known that the efficiency of the RNA interference can vary considerably from one siRNA to the other. Numerous factors appear to be involved in this variability, relating in particular to the sequencecible itself (for example the G / C content, the presence of certain bases at certain positions), to the position of this target sequence in the target gene. and the presence of secondary structures of mRNA that may decrease the accessibility of the target sequence for siRNA. Different methods have been proposed to try to predict the efficacy of siRNAs (GILMORE et al., J Drug Target, 12, 315, 2004; UI-TEI & SAIGO, Tanpakushitsu Kakusan Koso, 49, 2662, 2004; AMARZGUIOUI & PRYDZ, Biochem Biophys Res Commun, 316, 1050, 2004, HEALE et al., Nucleic Acids Res, 33, e30, 2005, REYNOLDS et al., Nat Biotechnol, 22, 326, 2004, ARZIMAN et al., Nucleic Acids Res, 33 , W582, 2005, HUESKEN et al., Nat Biotechnol, 23, 995, 2005). However, despite the progress made in rationalizing the selection criteria of optimal target sequences, this choice remains largely empirical, and its results are random. The inventors have hypothesized that the extinction of the gene coding for the N protein of morbilliviruses could make it possible to obtain the inhibition of viral replication, and began to investigate whether this extinction could be obtained using siRNAs. targeting regions of this gene conserved between morbilliviruses.

  They have succeeded in defining, in one of these conserved regions, a locus containing a target sequence making it possible to define siRNAs capable of inhibiting the expression of the N protein, this inhibition inducing an inhibition of the replication of the morbilliviruses.

  Within the meaning of the present invention, the term "inhibition of the expression of a gene" is understood to mean a reduction of at least 85%, preferably at least 90%, of the level of expression of a gene relative to its normal level. Inhibition of viral replication is understood to mean a decrease of at least 95%, preferably at least 98%, of the amount of virus compared to that produced under normal replication conditions. The subject of the present invention is therefore a method for inhibiting the replication of a morbillivirus characterized in that it comprises inhibiting the N gene of a morbillivirus with the aid of an interfering RNA targeting the MRNA of said gene containing the motif defined by the following general sequence: embedded image in which: A = Adenine C = Cytosine G = Guanine U = Uracil R = A or GY = C or UW = A or UH = A or C or UN = A or C or G or U. The general sequence SEQ ID NO: 1 was established from the alignment of the N gene sequences of the PPRV, RPV, MV, CDV, DMV morbilliviruses, and PDV, shown in Figure 2.

  The subject of the present invention is also interferent RNAs that can be used for carrying out the method according to the invention, namely interferent RNAs directed against the region of the N gene of a morbillivirus containing the motif defined by the general sequence SEQ ID. NO: 1. For example, to inhibit the expression of the N gene of measles virus, an interfering RNA targeting the mRNA region of said gene containing the following sequence will be selected: GGUUCGGAUGGUUCGAGA (SEQ ID NO: 2); to inhibit the expression of the N gene of the RPV, an interfering RNA targeting the mRNA region of said gene containing the following sequence will be selected: AGUCUUACUGGUUUGAGA (SEQ ID NO: 3); to inhibit the expression of the N gene of PPRV, an interfering RNA targeting the mRNA region of said gene containing the following sequence will be selected: GGAUCAACUGGUUUGAGA (SEQ ID NO: 4); to inhibit the expression of the CDV N gene, an interfering RNA targeting the mRNA region of said gene containing the following sequence will be selected: AAUUAGGCUGGUUAGAAA (SEQ ID NO: 5); to inhibit the expression of the N-gene of PDV, an interfering RNA targeting the mRNA region of said gene containing the following sequence will be selected: AAAUGGGCUGGUUAGAAA (SEQ ID NO: 6); to inhibit the expression of the DMV N gene, an interfering RNA targeting the mRNA region of said gene containing the following sequence will be selected: GAACCCAUUGGUUUGAGA (SEQ ID NO: 7). Typically, an interfering RNA according to the invention comprises a portion of 19 to 29 bp, preferably 19 to 23 bp, of an antisense sequence of the N gene of a morbillivirus, said portion containing a motif defined by the general sequence UYUCDAACCADYNNRWYY (SEQ ID NO: 8), wherein A, C, G, U, R, Y, W, and N are as defined above and D = G, A or U.

  The sequence SEQ ID NO: 8 represents the antisense sequence of the target sequence SEQ ID NO: 1 defined above. For example, in the case of measles virus, an interfering RNA according to the invention contains at least the following sequence: UCUCGAACCAUCCGAACC (SEQ ID NO: 9); in the case of RPV, an interfering RNA according to the invention contains at least the following sequence: UCUCAAACCAGUAAGACU (SEQ ID NO: 10); in the case of PPRV, an interfering RNA according to the invention contains at least the following sequence: UCUCAAACCAGUUGAUCC (SEQ ID NO: 11); in the case of CDV, an interfering RNA according to the invention contains at least the following sequence: UCUCUAACCAGCCUAAUU (SEQ ID NO: 12); in the case of PDV, an interfering RNA according to the invention contains at least the following sequence: UUUCUAACCAGCCCAUUU (SEQ ID NO: 13); in the case of DMV, an interfering RNA according to the invention contains at least the following sequence: UCUCAAACCAAUGGGUUC (SEQ ID NO: 14).

  If necessary, an interfering RNA according to the invention may be used in combination with one or more other interfering RNAs targeting other regions of the morbillivirus genome, and in particular other regions of the N. gene.

  In this context, it is possible in particular to use, in combination with an interfering RNA according to the invention: an interfering RNA directed against a region of the mRNA of the N gene containing a motif defined by the following general sequence: GSMGRUUYAUGGUVKCDYU (SEQ ID NO: 15), wherein A, C, G, U, R, Y, and D are as defined above and M = A or C, K = G or U, S = G or C, V = G, A or C, and / or an interfering RNA directed against a region of the mRNA of the N gene containing a motif defined by the following general sequence: GCHYUDGGNYUDCAYGARU (SEQ ID NO: 16) wherein A, C, G, U, R, Y, D, H, and N are as defined above. The general sequences SEQ ID NO: 15 and SEQ ID NO: 16 were established from the alignment of the N-gene sequences of the PPRV, RPV, MV, CDV, DMV, and PDV morbilliviruses, shown in FIG. For example: to inhibit the expression of the N gene of the measles virus, an RNA may be used in combination with an interfering RNA targeting the mRNA region of said gene containing the sequence GGUUCGGAUGGUUCGAGA (SEQ ID NO: 2). interferent targeting the mRNA region of said gene containing the following sequence: GCCGAUUCAUGGUCGCUCU (SEQ ID NO: 17) and / or an interfering RNA targeting the mRNA region of said gene containing the following sequence: GCUCUUGGACUGCAUGAAU (SEQ ID NO: 18 ); to inhibit the expression of the N gene of the RPV, it is possible to use, in combination with an interfering RNA targeting the mRNA region of said gene containing the sequence AGUCUUACUGGUUUGAGA (SEQ ID NO: 3), an interfering RNA targeting the region of 1 MRNA of said gene containing the following sequence: GCAGAUUUAUGGUGGCAUU (SEQ ID NO: 19) and / or an interfering RNA targeting the mRNA region of said gene containing the following sequence: GCACUGGGCCUGCAUGAAU (SEQ ID NO. 20) - to inhibit the expression of the N gene of the PPRV, in combination with an interfering RNA targeting the mRNA region of said gene containing the GGAUCAACUGGUUUGAGA sequence (SEQ ID NO: 4), an interfering RNA targeting the mRNA region of said gene containing the following sequence: GGCGGUUCAUGGUAUCUCU (SEQ ID NO: 21) and / or interfering RNA targeting the mRNA region of said gene containing the following sequence: GCAUUAGGCCUUCACGAGU (SEQ ID NO: 22); to inhibit the expression of the N gene of CDV, it is possible to use, in combination with an interfering RNA targeting the mRNA region of said gene containing the AAUUAGGCUGGUUAGAGA sequence (SEQ ID NO: 5), an interfering RNA targeting the region of 1 MRNA of said gene containing the following sequence: GGCGAUUCAUGGUGGCGCU (SEQ ID NO: 23) and / or interfering RNA targeting the mRNA region of said gene containing the following sequence: GCUCUUGGGUUGCAUGAGU (SEQ ID NO. 24) - to inhibit the expression of the N gene of the PDV, it is possible to use, in combination with an interfering RNA targeting the mRNA region of said gene containing the sequence AAAUGGGCUGGUUAGAAA (SEQ ID NO: 6), an interfering RNA targeting the mRNA region of said gene containing the next sequence: GGCGAUUUAUGGUGGCAUU (SEQ ID NO: 25) and / or interfering RNA targeting the mRNA region of said gene containing the following sequence: GCACUUGGUCUACAUGAGU (SEQ ID NO. 26) - to inhibit the expression of the DMV N gene, one can use, in combination with a Interfering RNA targeting the mRNA region of said gene containing the sequence GAACCCAUUGGUUUGAGA (SEQ ID NO: 7) an interfering RNA targeting the mRNA region of said gene containing the following sequence: GGAGAUUCAUGGUGGCAUU (SEQ ID NO: 27) and / or an interfering RNA targeting the mRNA region of said gene containing the following sequence: GCCUUAGGGUUGCAUGAAU (SEQ ID NO: 28). Interferent RNA directed against a region of the mRNA of the N gene containing a motif defined by the sequence SEQ ID NO: 15 comprises a portion of 19 to 29 bp, preferably 19 to 23 bp, of an antisense sequence of the gene N of a morbillivirus, said portion containing the general sequence ARHGMBACCAURAAYCKSC (SEQ ID NO: 29), wherein A, C, G, U, R, Y, M, H, K and S are as defined above, and B = G, U or C. An interfering RNA directed against a region of the mRNA of the N gene containing a motif defined by the sequence SEQ ID NO: 16 comprises a portion of 19 to 29 bp, preferably 19 to 23 pb, of an antisense sequence of the N gene of a morbillivirus, said portion containing the general sequence AYUCRUGHARNCCHARDGC (SEQ ID NO: 30), wherein A, C, G, U, R, Y, H, and N are such as defined above. The sequences SEQ ID NO: 29 and SEQ ID NO: 30 respectively represent the antisense sequences of the target sequences SEQ ID NO: 15 and SEQ ID NO: 16 defined above. For example, in the case of measles virus, an interfering RNA according to the invention containing at least the sequence UCUCGAACCAUCCGAACC (SEQ ID NO: 9) may be used in combination with an interfering RNA containing at least the following sequence: AGAGCGACCAUGAAUCGGC (SEQ ID NO: 31) and / or with interfering RNA containing at least the following sequence: AUUCAUGCAGUCCAAGAGC (SEQ ID NO: 32); in the case of RPV, an interfering RNA according to the invention containing at least the UCUCAAACCAGUAAGACU sequence (SEQ ID NO: 10) may be used in combination with an interfering RNA containing at least the following sequence: AAUGCCACCAUAAAUCUGC (SEQ ID NO: 33) ) and / or with an interfering RNA containing at least the following sequence: AUUCAUGCAGGCCCAGUGC (SEQ ID NO: 34); in the case of PPRV, an interfering RNA according to the invention containing at least the sequence UCUCAAACCAGUUGAUCC (SEQ ID NO: 11) may be used in combination with an interfering RNA containing at least the following sequence: AGAGAUACCAUGAACCGCC (SEQ ID NO: 35 ) and / or with interfering RNA containing at least the following sequence: ACUCGUGAAGGCCUAAUGC (SEQ ID NO: 36); in the case of CDV, an interfering RNA according to the invention containing at least the sequence UCUCUAACCAGCCUAAUU (SEQ ID NO: 12) may be used in combination with an interfering RNA containing at least the following sequence: AGCGCCACCAUGAAUCGCC (SEQ ID NO: 37 and / or with an interfering RNA containing at least the following sequence: ACUCAUGCAACCCAAGAGC (SEQ ID NO: 38); in the case of PDV, an interfering RNA according to the invention containing at least the sequence UUUCUAACCAGCCCAUUU (SEQ ID NO: 13) can be used in combination with an interfering RNA containing at least the following sequence: AAUGCCACCAUAAAUCGCC (SEQ ID NO: 39) and / or with an interfering RNA containing at least the following sequence: ACUCAUGUAGACCAAGUGC (SEQ ID NO: 40); in the case of DMV, an interfering RNA according to the invention containing at least the sequence UCUCAAACCAAUGGGUUC (SEQ ID NO: 14) may be used in combination with an interfering RNA containing at least the following sequence: AAUGCCACCAUGAAUCUCC (SEQ ID NO: 41 ) and / or with an interfering RNA containing at least the following sequence: AUUCAUGCAACCCAAAAGC (SEQ ID NO: 42). The different interfering RNAs defined above can be in various forms. It can be a single-stranded antisense RNA, capable of integrating into the RISC complex, and pairing with the target sequence, inducing its cleavage (TIJSTERMAN et al., Science, 295, 694, 2002 MARTINEZ et al., Cell, 110, 563, 2002, BARIK, Virus Res, 102, 27, 2004). However, it will preferably be an RNA containing both the target sequence and the corresponding antisense sequence, in the form of siRNA, or the case of sh (<< short hairpin >>) RNAs (YU et al. Proc Natl Acad Sci USA, 99, 6047, 2002, PADDISON et al., Genes Dev, 16, 948, 2002, SIOLAS et al., Nat Biotechnol, 23, 227, 2005), which is transformed into the cell. siRNA.

  SiRNAs are generally 21 to 25 nucleotides in length; they contain a double-stranded portion, usually 19 to 21 nucleotides, consisting of the target sequence and the corresponding antisense sequence; Either strand, or both, generally has at the end 3 a single-stranded extension of 2 or 3 nucleotides; most often (but not necessarily) it is a dinucleotide, of sequence TT. The sRNAs consist of a single strand, 50 to 70 nucleotides in length, which can fold to form a hairpin structure, containing a double-stranded portion of 19 to 29 nucleotides in length. by the target sequence and the corresponding antisense sequence, and a 5 to 10 nucleotide loop. They can also include, at the 3 'end, a single-stranded extension of 2 or 3 nucleotides. Interfering RNAs can also be used as microRNA precursors (pre-miRNAs). MicroRNAs (miRNAs) are about 22 single-stranded nucleotide RNAs that bind to the 3 'non-coding end of mRNA, thereby repressing the expression of this mRNA. without degrading it (unlike siRNAs or shRNAs). MiRNAs are important molecules for the natural regulation of gene expression in eukaryotic cells. The miRNAs are produced in the cytoplasm of cells from nuclear precursors of about 70 hairpin-like nucleotides, called pre-miRNAs, olives in miRNAs by the DICER complex. The sequence of a pre-miRNA, identified under natural conditions in a eukaryotic cell, can be modified to introduce an siRNA sequence, which, released by the DICER will then degrade another mRNA. This modified pre-miRNA then serves as a "vehicle" for the siRNA of interest, which results in increasing the efficiency of the interference. The possibility of using modified pre-miRNAs to inhibit viral replication has been demonstrated by BODEN et al. (Nucleic Acids Res., 13, 1154, 2004) for HIV-1. In this case, the efficiency of the interference may be greater than 80o that provided by the equivalent siRNA.

  These interfering RNAs can be obtained by conventional methods for the preparation of nucleic acids (for review see, for example, AMARZGUIOUI et al., FEBS Lett, 579, 5974, 2005).

  They can thus be prepared, for example by chemical synthesis, or by genetic engineering. In the latter case, an expression vector will be used containing a DNA sequence that can be transcribed into an interfering RNA according to the invention, placed under the control of an appropriate promoter. Generally, said promoter is a viral promoter, for example the T3 promoter, T7, SP6, pCMV or a polymerase III recognized promoter, for example the small U6 RNA promoter, or that of the HI RNA (MIYAGISHI & TAIRA, Nucleic Acids Res Suppl, 113, 2002); however, promoters recognized by polymerase II are also useful. The expression vectors defined above are also part of the subject of the present invention. Many methods for introducing interfering RNAs into cells or organisms in which it is desired to terminate the expression of a target gene are known per se. In the case where it is desired to temporarily extinguish the expression of the target gene, the interfering RNA can be directly administered, preferably associated with a suitable vehicle, to facilitate its entry into the cell and / or to Pro-Leger of degradation. Examples of vehicles that may be used include liposomes or nanoparticles.

  To achieve a longer-term extinction of the expression of the target gene, particularly in the case of mammalian cells, a vector is used to express the interfering RNA in the cell. Many vectors usable for this purpose are known in themselves. These include vectors derived from retroviruses, lentiviruses, or adenoviruses (BARTON & MEDZHITOV, Proc Natl Acad Sci USA, 99, 14943, 2002, TISCORNIA et al., Proc Natl Acad Sci USA, 100, 1844, 2003; XIA et al., Nat Biotechnol, 20, 1006, 2002, SHEN et al., FEBS Lett, 539, 111, 2003). The present invention also relates to the use of an interfering RNA or an expression vector according to the invention for the purpose of obtaining a medicament for the treatment or prevention of a morbillivirus infection. and in particular the treatment or prevention of measles, rinderpest, peste des petits ruminants, Carre's disease, seals' plague or DMV infection. The present invention also relates to a pharmaceutical composition comprising an interfering RNA or an expression vector according to the invention. Advantageously, said pharmaceutical composition is a vaccine. The present invention will be better understood with the aid of the following description, which refers to examples illustrating the obtaining of an interfering RNA according to the invention, and its use to block the replication of PPRV morbilliviruses. VPN. LEGEND OF FIGURES Figure 1: Schematic representation of the genome of morbilliviruses. Figure 2: Multiple alignment of the cDNA sequences of the N gene of different morbilliviruses, PPRV (X74443), RPV (X98291), MV (Z66517), CDV (NC001921), DMV (AJ608288) and PDV (X75717) by the Vector software NTI (Informax Inc.). The nucleotides identical to those of the consensus sequence indicated at the bottom of the alignment are represented by a dot; nucleotides that differ from the consensus sequence are indicated. Framed regions, or all nucleotides are indicated, represent the target sequences of siRNAs NRP1 and 2 on the sequence of RPV, and siRNAs NPPR1 and 3 and siRNAs NPPR5 to 10 on the sequence of PPRV. The codons << start >> and << stop >> are in bold and underlined characters. Figure 3: Effect of siRNAs on cytopathic effect (CPE) induced by peste des petits ruminants virus on VERO cells. Scale order of percent inhibition of CEP. Abscisses: concentration of siRNAs in nM. - • -: siRNA NPPR1; where. siRNA NPPR2; : siRNA NPPR3; where siRNA NPPR10; where: siRNA GAPDH; - - non-infected cells; - -. infected cells not transfected. Figure 4: Effect of siRNAs on expression of PPRV virus N protein in VERO cells. (a) Abscisses fluorescence due to the expression of the N protein; Ordinates: number of fluorescent cells; siRNA1 siRNA NPPR1; siRNA2: siRNA NPPR2; uninfected cells: negative control; infected cells positive control. (b) Abscisses: concentration (in nM) of siRNAs. Ordinates: percentage of fluorescent cells; ^: siRNA NPPR1; : siARN GAPDH. Figure 5: Effect of siRNAs NPPR1, 2 and 3 on the synthesis of viral RNA measured by quantitative RT-PCR in real time (QRT-PCR). Representation of two independent tests. Figure 6: Effect of siRNAs NPPR1 (vertical hatching), NPPR2 (white), NPPR3 (gray), NPPR10 (black) and GAPDH (diagonal hatching) on viral replication measured by viral titer in infected cell mats. EXAMPLES: MATERIALS AND METHODS 1) Viruses and sequences of interest The PPRV virus uses is the strain Nigeria 75/1 (DIALLO et al Rev Elev Med Vet Pays Trop 42, 311, 1989), attenuated by serial passages on cells ( SK / 1, BK / 1 and Vero / 55). This is a vaccine strain. The complete genome sequence of this strain is available on Genbank (accession number X74443). The RPV virus uses is the RBOK strain (PLOWRIGHT and FERRIS, Res Vet Sci, 3, 172, 1962), virus vaccine strain. attenuated by serial passages on cells (BK / 98 and Vero / 2). The complete genome sequence of this strain is available from Genbank (accession number Z30697). The PPRV and RPV viruses are propagated on VERO (ATCC) cells maintained in a monolayer in the presence of complete medium, ie Eagle MEM essential medium with salts. of Earle (Eurobio, Courtaboeuf, France), 10% of fetal bovine serum (Eurobio, Courtaboeuf, France) and 2 mM of L-glutamine (Gibco, Mife Technologies, UK). 2) Cell culture, transfection and viral infection Adjacent VERO cells were removed with a solution containing trypsin and EDTA (Sigma-Aldrich, Lyon, France) and then resuspended in MEM medium (Minimum Eagle Medium) complete at 105 cells / ml and distributed in 24-well plate wells. The plates are incubated at 37 ° C and 5% CO2. Subsequently, all cell incubations take place at 37 C in the presence of 5% CO2. When the cell layer reaches 70-80% confluence, the medium is eliminated, and then the cells are incubated for 30 minutes in MEM medium devoid of serum fetal bovine. For transfection, the medium is eliminated and replaced with LIPOFECTAMINETM 2000 (Invitrogen) at the rate of 500 ng in 200 μl of OPTI-MEM I Invitrogen transfection medium containing the siRNAs at different concentrations (from 6.5 to 100 nM). final in the transfection mixture). The following incubation takes place for three hours. For viral infection, the transfection medium is removed and replaced with MEM and 5% fetal bovine serum. The cells are incubated for 24 hours and then multiplied in cytopathic cells over 50% of the contacts. The MEM medium, bovine, is observed infected with the PPRV or RPV virus at an infection level of 0.1 50% cytopathic dose per medium without fetal serum. of bovine, the 50o dose being the amount of virus inducing an ECP-infecting cellular carpets. After one hour of cellular mat is rinsed twice with then MEM medium with 5% fetal serum added on the cells. The cells are daily and the evolution of the cytopathic effect (CPE) of the virus is evaluated according to a grid of percentages (scale ranging from 0%, not 100% CPE, maximum CPE). The effect of siRNAs is expressed as percent inhibition of ECP.

  Four to five days after infection, the cell culture supernatant on the one hand and the cells on the other hand are collected for viral titration and expression of viral antigens by flow cytometry. Controls consisting of untransfected and non-infected cells and infected and non-transfected cells are included in each series of siRNAs assays. 4) Viral Titration The cell culture supernatants or the cells themselves are stored at -70 ° C until used for titration. The viral titration is performed on cell culture (VERO) from a series of dilutions of reason 10 of the viral suspension to be titrated. The titer is determined according to the method of REED and MUENCH (Am J Trop Med Hyg, 127, 493, 1938) and expresses in cytopathic dose 50 (DCP50) per ml. 5) Measurement of Viral Antigen Expression The expression of the viral nucleocapsid (N) and matrix (M) proteins was measured by flow cytometry using specific monoclonal antibodies: 38-4 protein-specific antibody N from PPRV; IVB2-4 antibody specific for RPV N protein; 19-6 antibody specific for the PPRV M protein and the VPV M protein (LIBEAU et al., Journal of Veterinary Medicine and Veterinary Medicine of the Tropics, 50, 181-190, 1997), and antibody 11/295 Specifically, the porcine lymphocyte CD8 molecule (SAALMULLER et al., Vet Immunol Immunopathol, 43, 249, 1994) uses as isotype specificity control of labeling.

  The adherent cells are removed from the plastic by incubation with a solution containing trypsin and EDTA for 5 minutes at 37 ° C. The cells are washed in phosphate buffer PBS, 0.1% sodium azide, 5% horse serum and 0.0062% saponin (weight / volume). They are counted and distributed at the rate of 106 cells per well of a 96-well plate. All markings are made in plate. After sedimentation of the cells by centrifugation and removal of the supernatant, 100 μl of a dilution anti-N monoclonal antibody, anti-M or additions and mechanically re-suspended cells. The cells are incubated. They are then washed twice in phosphate and then incubated for 30 minutes at +4 ° C., appropriate dilution of an anti-mouse antibody conjugated to the fluorescene (BIORAD, France). The cells are washed twice and then fixed for 15 minutes at room temperature with 100 μl of a paraformaldehyde solution. The cells are resuspended in 400 μl of FACS FLOW PBS (Becton Dickinson, USA). Cell analysis is performed at FACsort (Becton Dickinson, USA). Cell debris was removed from FACS analysis by a window on FSCxSSC (FSC: Forward SCatter, SSC: Side SCatter), and fluorescence was measured on 20,000 cells. 6) Quantitative measurement of viral RNA synthesis The cells and the culture supernatant are harvested 96 hours after infection and frozen at -70 ° C.

  The total RNA is extracted from 100 l of this cell suspension mixed with the lysis solution of the RNeasy kit, according to the manufacturer's instructions (Qiagen, Courtaboeuf, France). The rest of the protocol corresponds to the manufacturer's instruction manual. The extracted RNA is stored at -70 ° C. The RNA is quantitated by QRT-PCR in one step (Brilliant SYBR Green QRT-PCR Master Mix, 1 step, Stratagene). The primers used are NP3bis = 5'-GTCTCGGAAATCGCCTCACAG-3 '(sense) (SEQ ID NO: 43) and NP4bis = 5'-CCTCCTCCTGGTCCTCCAGAA-3' (antisense) (SEQ ID NO: 44).

  These primers were derived from the previously described primers (COUACY-HYMANN et al., J. Virol Methods, 100, 17, 2002) by addition of a 5 'G of NP3bis and deletion of four bases at 3' and deletion of three. 3 'bases of NP4bis. These primers amplify a fragment of 351 bases. A main reaction solution consisting of 12.5 l SYBR Green Master mix (2x), 2.5 l of 1 M of each primer, 0.0625 l of Stratascript Reverse Transcriptase RT and 2.4375 l of water is deposited in a Microtube, then 5 μl of the appropriate isotype RNA is stirred for minutes at +4 C. the same buffer in 50 l of an extract is added. The QRT-PCR is carried out on a Mx3000P apparatus (Stratagene, Amsterdam, NL) according to the following protocol: reverse transcription at 50 ° C. for 30 minutes - denaturation at 95 ° C. for 10 minutes amplification with 35 cycles [95 ° C., 30 seconds] ; 55 C, 1 minute; 72 C, 30 seconds] dissociation with a cycle [95 C, 1 minute; 55 C, 30 seconds; 95 C, 30 seconds] The standard standard for quantitation is the PPRV pro-Lein N gene inserted into a pBluescript KS + plasmid (COUACY-HYMANN et al., 2002, supra). For quantitation, the plasmid is linearised, purified by alcohol precipitation, and serially diluted 10-fold. The amount of RNA detected in the samples submitted for analysis is reduced to the number of N gene copies incorporated in the assay. series of dilutions of the standard standard. EXAMPLE 1: SELECTION AND SYNTHESIS OF siRNAs The conserved regions of the N gene were localized by multiple alignment of the corresponding sequences from different morbilliviruses using a sequence analysis software (Vector NTI package, Informax Inc.). This multiple sequence alignment was performed on the N gene sequences of the PPRV morbilliviruses (Nigeria strain 75/1, GenBank X74443), RPV (GenBank Z30697), MV (GenBank Z66517), CDV (GenBank NC 001921), DMV ( GenBank AJ608288) and PDV (GenBank X75717). In areas with the highest degree of homology, 7 target sequences have been defined for PPRV. The siRNAs derived from the N gene of the PPRV virus, and containing sequences identical to the target sequences defined in this gene, are referred to as NPPR1, and NPPR5 to 10; a siRNA known as NPPR2, having at most only 58% identity with the sequence of the N gene of the PPRV virus (at positions 256-274, 751-769, and 850-869 of the cDNA sequence), and a siRNA denotes NPPR3, derived from the same target sequence as siRNA NPPR10, but having a difference of one base with said target sequence (C in the target sequence replaced by G at the 5 'end of siRNA sense strand NPPR3) have also been synthesized.

  For the RPV virus, 2 target sequences were defined at the locus corresponding to the sequencecible siRNA-NPPR1 sequence. The corresponding siRNAs are referred to as NRP1 and NRP2. All the siRNAs mentioned above were chemically synthesized by Ambion (Europe) (Cambridgeshire, UK). To test the specificity of the interference, a siRNA targeting the sequence of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used: the sequence and siRNA are available from Ambion (Europe) (SilencerTM GAPDH siRNA (Human) Control 4605). All identified siRNA sequences were genomically searched for in Genbank using Blast software. The maximum identity observed between the siRNA-GAPDH and the pro-Leine N coding sequence of the PPRV virus is 72%. The sequences of the sense and antisense strands of the various siRNAs mentioned above, as well as their position relative to the sequence of the N gene and with respect to the sequence of the reading frame of said gene, are indicated in Table I below. . The target sequence positions of siRNAs NPPR1, NPPR3, NPPR5 to NPPR10, and NRP1 and NRP2 are shown in Figure 2.

  EXAMPLE 2 Effect of siRNAs NPPR1, 2 and 3 and siRNAGAPDH on the CYTOPATHIC EFFECT OF SMALL RUMINANT FIRE FIGHTING VIRUS.

  VERO cells were transfected with different doses of NPPR1, 2, and 3 siRNAs or siRNA-GAPDH, and then infected with PPRV, according to the protocol described in the section "Materials and Methods"; The cytopathic effect was evaluated 4 days after infection, independently by two different persons. Figure 3 illustrates the results observed: Among the siRNAs tested, the siRNA NPPR1 is the one that inhibits the most ECP, whatever the dose used. At the dose of 100 nM, an inhibition was observed TABLE I Sequence siRNA name Position in the cDNA / in the siRNA-NPPR1 reading frame direction: 5'-GGAUCAACUGGUUUGAGAAtt -3 '(SEQ ID NO: 45) (position 480 -498 / 428-446) antisense: 5'-UUCUCAAACCAGUUGAUCCTT 3 '(SEQ ID NO: 46)

  siRNA-NPPR2 sense: 5'-GCUCACCUUCAUUCUUUUCTT -3 '(SEQ ID NO: 47) (only 63% homology) antisense: 5'-GAAAAGAAUGAAGGUGAGCtc -3' (SEQ ID NO: 48)

  siRNA-NPPR3 Sense: 5'-GGCCAGUUUCAUUCUUACACT -3 '(SEQ ID NO: 49) (position 850-868 / 798-816) antisense: 5'-AGUAAGAAUGAAACUGGCCtc -3' (SEQ ID NO: 50)

  siRNA-NPPR5 sense: 5'-GAGAACUCAAUUCAGAACAtt -3 '(SEQ ID NO: 51) (position 1001-1019 / 949-968) antisense: 5'-UGUUCUGAAUUGAGUUCUCTT -3' (SEQ ID NO: 52)

  siRNA-NPPR6 sense: 5'-GGCGGUUCAUGGUAUCUCUtt -3 '(SEQ ID NO: 53) (position 741-759 / 689-707) antisense: 5'-AGAGAUACCAUGAACCGCCTT -3' (SEQ ID NO: 54)

  siRNA-NPPR7 sense: 5'-GCAUUAGGCCUUCACGAGUtt -3 '(SEQ ID NO: 55) (position 869-917 / 847-865) antisense: 5'-ACUCGUGAAGGCCUAAUGCTT -3' (SEQ ID NO: 56)

  siRNA-NPPR8 sense: 5'-GUAUCAACAGCUAGGAGAGTT -3 '(SEQ ID NO: 57) (position 958-976 / 906-924) antisense: 5'-CUCUCCUAGCUGUUGAUACTT -3' (SEQ ID NO: 58)

  siRNA-NPPR9 sense: 5'-GAACUUUGGCAGGUCAUAUtt -3 '(SEQ ID No: 59) (position 1102-1120 / 1050-1068) antisense: 5'- AUAUGACCUGCCAAAGUUCTT -3' (SEQ ID NO: 60)

  siRNA-NPPRIO sense: 5'-CGCCAGUUUCAUUCUUACACT -3 '(SEQ ID NO: 61) (position 850-868 / 798-816) antisense: 3'-AGUAAGAAUGAAACUGGCGTT -3' (SEQ ID NO: 62)

  SiRNA-NRP 1 sense: 5'-GCAGUCUUACUGGUUUGAGTT -3 '(SEQ ID NO: 63) (position 478-496 / 426-444) antisense: 5'-CUCAAACCAGUAAGACUGCTT -3' (SEQ ID NO: 64)

  siRNA-NRP2 sense: 5'-CAGUCUUACUGGUUUGAGAtt -3 '(SEQ ID NO: 65) (position 479-497 / 427-445) antisense: 5'-UCUCAAACCAGUAAGACUGtc -3' (SEQ ID NO: 66)

  siRNA-GAPDH sense: 5'-AAGGUCAUCCAUGACAACUTT -3 '(SEQ ID NO: 67) antisense: 5'-AGUUGUCAUGGAUGACCUUtt -3' (SEQ ID NO: 68) complete with ECP. The siRNA NPPR3, whose sequence varies one nucleotide with respect to the target sequence defined on the virus (see Example 1) inhibits the CEP less marked than NPPR1. The siRNA NPPR10, which directs against the same target sequence as the siRNA NPPR3, but which, unlike the latter is perfectly complementary to said target sequence, also inhibits the ECP in a less marked way than NPPR1 (non-clocked results). As expected, siRNA-NPPR2 as well as siRNA-GAPDH produce no neutralization of the CEP. EXAMPLE 3 Effect of SiRNA on the Expression of the Protein N and the Protein M of PPRV and RPV VERO cells were transfected with different doses of NPPR1 siRNA, NPPR2 siRNA or siRNA GAPDH, and then infected with PPRV as described in the "Materials and Methods" section; 4 to 5 days after infection, the production of protein N in the cells is measured by flow cytometry, as described in the section "Materials and Methods". FIG. 4 (a) illustrates the results observed in cells transfected with 100 nM of NPPR1 siRNA or NPPR2 siRNA, as well as in non-infected and non-transfected cells, and in non-transfected and infected cells, used in vitro. title of control. A very strong inhibition of pro-Lein N expression was observed in infected cells transfected with NPPR1 siRNA, whereas in those transfected with NPPR2 siRNA, the expression profile of N protein was very similar to that observed with non-transfected infected cells, although some decrease in expression is observed. Figure 4 (b) illustrates the results observed in cells transfected with different doses of NPPR1 siRNA or GAPDH siRNA. A very significant decrease in the percentage of cells expressing the N protein was observed from 12.5 nM of siRNA NPPR1.

  In a second series of experiments, the expression of pro-Leine N and pro-Leine M was measured: Bans of VERO cells transfected with the optimal dose for each of the siRNAs tested, namely 100 nM for siRNAs NPPR1, 2, 3, or 5 to 9, or 25 nM for siRNA NPPR10, then infected with PPRV or with RPV; in VERO cells transfected with 100 nM of NRP NRP1 or NRP2 siRNAs, then infected with PPRV or with RPV. Measurement of the expression of pro-Leine M makes it possible to verify that the inhibition of the expression of pro-Lein N causes an inhibition of the other viral proteins, which reflects an effect on the replication of the viral genome. The results are summarized in Table II below. TABLE II Inhibition of Inhibition of Inhibition of Inhibition of the Expression of the Expression of the Expression of the Nucleoprotein Protein Protein Nucleoprotein PPRV (in PPRV Protein RPV (%)) (in%) %) RPV matrix (in%) siRNA-NPPR1 (100 nM) 90 89 0 0 siRNA-NPPR2 (100 nM) 25 11 nt nt siRNA-NPPR3 (100 nM) 40 30 nt nt siRNA-NPPR5 (100 nM) 16 nt siRNA-NPPR6 (100 nM) and siRN-NPPR7 (100 nM) 63 nt siRNA-NPPR8 (100 nM) 34 nt siRNA-NPPR9 (100 nM) 35 nt siRNA-NPPR10 ( 25 nM) 60 siRNA-NRP1 (100 nM) 0 nt 35 31 siRNA-NRP2 (100 nM) 0 nt 97 92.5 siRNA-GAPDH (100 nM) 0 5 13 3 nt: not tested Four of the siRNAs tested (siRNAs NPPR1, 6, 7 and 10) have a significant effect on PPRV, of which only NPPR1 siRNA achieves 90% inhibition of pro-Lein N expression ( the other siRNAs do not achieve 70% inhibition of expression) and a percentage of 89% inhibition of Expression of pro-Lein M. With regard to RPV, only siRNA NRP2 has a significant effect. The siRNA NRP1, which does not contain the totality of the siRNA (optimal concentration), has a very reduced effect. These results show that the locus recognized by the siRNAs NPPR1, and NRP2 constitutes a particularly interesting target for the extinction of the gene of the N protein of morbilliviruses. This target is very circumscribed, since a missing base considerably reduces the effects obtained. EXAMPLE 4: INHIBITOR EFFECT OF siRNA NPPR1 ON THE SYNTHESIS OF VIRAL RNA The effect of NPRP1 siRNA on viral replication was evaluated by quantifying the synthesis of viral RNA, as described in the "Materials and Methods" section, in non-transfected PPRV-infected VERO cells, or transfected with 100 nM of one of the siRNAs NPPR1, NPPR3, NPPR2 or GAPDH, as well as in non-infected VERO cells. The results are shown in Fig. 5. In non-transfected infected cells, viral replication results in a quantity of viral RNA 100 times greater than that provided by the initial inoculum. Transfection with NPPR2 and GAPDH siRNAs has no significant effect on viral replication. In cells transfected with NPPR3, the amount of viral RNA remained more than 10-fold higher than that provided by the initial inoculum. On the other hand, in the cells transfected with NPPR1, the amount of viral RNA corresponds to that brought by the initial inoculum, which confirms that this siRNA inhibits the viral replication. EXAMPLE 5: INHIBITOR EFFECT OF NPPR1 siRNA ON VIRAL TIR-MEASURED REPLICATION The effect of NPR1 siRNA on viral replication was evaluated by measuring the viral titre, as described in Materials and Methods, 4 days after 1 PPRV infection of VERO cells transfected with 6.25, 12.5, 25, 50 or 100 nM of one of the siRNAs NPPR1, NPPR2, NPPR3, NPPR10 or GAPDH. The results are illustrated in Figure 6.

  For the cells transfected with one of the siRNAs NPPR2, NPPR3 and NPPR10, there is a slight decrease in the viral titre compared with the control (cells transfected with the siRNA GAPDH), which does not vary significantly as a function of the concentration tested. For the cells transfected by NPPR1, the viral titer decreases very significantly from 12.5 nM of siRNA. At 100 nM siRNA, complete inhibition of viral replication is observed.

Claims (8)

  1) interfering RNA, characterized in that it is directed against a region of the mRNA of the N gene coding for the nucleoprotein of a morbillivirus, said region containing the motif defined by the following general sequence: RRWYNNRHUGGUUHGARA (SEQ ID NO: 1) in which: A = Adenine C = Cytosine G = Guanine U = Uracil R = A or GY = C or UW = A or UH = A or C or UN = A or C or G or U.   2) interfering RNA according to claim 1, characterized in that said unit is defined by a target sequence chosen from: - the sequence GGUUCGGAUGGUUCGAGA (SEQ ID NO: 2) - the sequence AGUCUUACUGGUUUGAGA (SEQ ID NO:   3) - the sequence GGAUCAACUGGUUUGAGA (SEQ ID NO:   4) - the sequence AAUUAGGCUGGUUAGAGA (SEQ ID NO:   5) - the sequence AAAUGGGCUGGUUAGAAA (SEQ ID NO:   6); the sequence GAACCCAUUGGUUUGAGA (SEQ ID NO:   7) 3) Interfering RNA according to claim 1, characterized in that it comprises a 19 to 29 bp portion of an antisense sequence of that of the N gene of a morbillivirus, said portion containing a pattern defined by the sequence General UYUCDAACCADYNNRWYY (SEQ ID NO:   8), wherein A, C, G, U, R, Y, W and N are as defined above, and D = G, A or U. 4) Interfering RNA according to any one of claims 1 to 3, characterized in that it is selected from siRNAs, shRNAs and pre-miRNAs. 5) An expression vector containing a transcriptionally transposable DNA sequence of an interfering RNA according to any one of claims 1 to 4, placed under the control of an appropriate promoter. 6) Use of an interfering RNA according to any one of claims 1 to 4 for obtaining a medicament for treating or preventing a morbillivirus infection. 7) The use according to claim 6, characterized in that said interfering RNA is used in combination with at least one other interfering RNA selected from: an interfering RNA directed against a region of the mRNA of the N gene containing a motif defined by the general sequence GSMGRUUYAUGGUVKCDYU (SEQ ID NO: 15), wherein A, C, G, U, R, Y, and D are as defined above and M = A or C, K = G or U, S = G or C, V = G, A or C; an interfering RNA directed against a region of the mRNA of the N gene containing a motif defined by the general sequence GCHYUDGGNYUDCAYGARU (SEQ ID NO: 16), in which A, C, G, U, R, Y, D, H, and N are as defined above. 10) Use according to any one of claims 6 or 7, characterized in that said morbillivirus is selected from the group consisting of measles virus (NV), rinderpest virus (RPV), peste des petits virus ruminants (PPRV), Carre's disease virus (CDV), seal plague virus (PDV), and dolphin morbillivirus (DMV). 11) A pharmaceutical composition comprising an interfering RNA according to any one of claims 1 to 4. 10) A pharmaceutical composition according to claim 9, characterized in that it further comprises at least one interfering RNA as defined in claim 7. 11) Pharmaceutical composition according to any one of claims 9 or 10, characterized in that it is a vaccine.