SiRNA SEQUENCES TARGETING EXPRESSION OF HUMAN GENES JAK1 OR JAK3 FOR THERAPEUTIC USE

Abstract

To provide nucleotide sequences of small interfering RNAs specific to the human genes Janus kinase 1 (JAK1) and Janus kinase 3 (JAK3), respectively.SOLUTION: A double-stranded (ds) ribonucleic acid (RNA) is provided comprising a sense strand and an antisense strand, where: the sense strand comprises the nucleotide sequence SEQ ID NO: 5 and the antisense strand comprises the nucleotide sequence SEQ ID NO: 6; or the sense stand comprises the nucleotide sequence SEQ ID NO: 7 and the antisense strand comprises the nucleotide sequence SEQ ID NO: 8; or the sense strand comprises the nucleotide sequence SEQ ID NO: 1 and the antisense strand comprises the nucleotide sequence SEQ ID NO: 2; or the sense strand comprises the nucleotide sequence SEQ ID NO: 3 and the antisense strand comprises the nucleotide sequence SEQ ID NO: 4. The dsRNA of the present invention for use as a drug is provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the nucleotide sequences of small interfering RNAs specific for the human genes Janus kinase 1 (JAK1) and Janus kinase 3 (JAK3), respectively. These sequences were selected to maximize their potential for functional inhibition of the expression of the JAK1 or JAK3 gene while minimizing possible undesirable or adverse effects, with the aim of enabling their therapeutic use in humans. [Background technology]

[0002] RNA interference was first described in 1998 by Andrew Fire and Craig Mello. The process occurs when a double-stranded RNA has a complementary nucleotide sequence that matches that of an mRNA. This double-stranded RNA is then complementary to the mRNA and subsequently activates its degradation, thereby reducing the expression of the corresponding gene. In detail, double-stranded RNA present in a cell is first taken up by a ribonuclease III known as Dicer, or "slicer." Dicer cleaves the double-stranded RNA molecule into fragments of 21-25 base pairs. Dicer then transfers the small interfering RNA (siRNA) to the RNA-induced silencing complex (RISC), a large multiprotein complex. One of the strands of the siRNA, called the "passenger," is removed, while the other (called the "guide") directs RISC to a messenger RNA (mRNA, or coding RNA that carries the message from the gene into the cytoplasm) that has a complementary sequence that matches that of the guide strand. If the complementarity (or match) between the siRNA and the target mRNA is perfect, RISC cleaves the target mRNA, which is subsequently degraded and therefore no longer translated into protein. A small number of non-complementary (mismatched) bases are sufficient to prevent cleavage.

[0003] Small interfering RNAs, or "siRNAs", are synthetic duplexes of 21 nucleotides, which exhibit "seed" sequences in the 5'→3' orientation in the guide strand, particularly in positions 2 to 8, that are particularly important for ensuring their specificity. These sequences obey certain rules of thermodynamics, such as the presence of guanine or cytosine in position 1 of the passenger strand, the presence of adenine or uracil in position 1 of the guide strand, the composition of the "seed" sequence of the guide strand with a minimum guanine / cytosine level, and a guanine / cytosine balance for nucleotides 8 to 16 of the guide strand.

[0004] In 2018, the siRNA patisiran, marketed by Alnylam, was approved for market launch. This siRNA is the first therapeutic siRNA approved for use in humans. The in vitro concentration routinely used for cell lines is 10-20 nM.

[0005] The Janus kinase (JAK) family is composed of four non-receptor tyrosine kinases, JAK1, JAK2, JAK3 and Tyk2, which play a pivotal role in cytokine- and growth factor-induced signal transduction. Phosphorylated Janus kinases (JAKs) bind and activate various signal transducers and activators of transcription (STAT) proteins. These STAT proteins dimerize and translocate to the nucleus where they act as both signal transducing molecules and transcription factors, ultimately binding to specific DNA sequences found in the promoters of cytokine-responsive genes. Various immunodeficiencies and autoimmune diseases, such as allergies, asthma, alopecia areata, allograft rejection (allografting), rheumatoid arthritis, amyotrophic lateral sclerosis and multiple sclerosis, as well as solid and hematological cancers, are due to disruption of signal transduction in the JAK / STAT pathway. Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to a double-stranded (ds) ribonucleic acid (RNA) comprising a sense strand and an antisense strand, the sense strand comprises the nucleotide sequence of SEQ ID NO:5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:6; or the sense strand comprises the nucleotide sequence of SEQ ID NO:7 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:8; or the sense strand comprises the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2; or the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:4; Regarding dsRNA.

[0007] The present invention also relates to a dsRNA according to the invention for its use as a medicament.

[0008] The invention particularly relates to a dsRNA according to the invention for its use in the prevention and / or treatment of diseases associated with Janus kinase (JAK) gain-of-function disruption of the JAK signaling pathway.

[0009] The present invention further relates to a pharmaceutical composition comprising at least one dsRNA according to the invention and a pharma- ceutically acceptable carrier.

[0010] The present invention also provides an in vitro JAK inhibition method for inhibiting expression of Janus kinase 1 (JAK1) or Janus kinase 3 (JAK3) in a cell, comprising: a. introducing the dsRNA according to the invention into said human cells; and b. maintaining the cells produced in step (a) for a period of time sufficient to effect degradation of the mRNA of the JAK1 gene or the JAK3 gene, thereby inhibiting expression of JAK1 or JAK3 in the cells; The present invention also relates to a method comprising the steps of: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present inventors have generated several siRNA sequences that follow the main principles of RNA interference. The siRNA according to the present invention thus: i. The guide strand of the siRNA shows 50% to 100% sequence homology to the target mRNA (human JAK1 or JAK3); ii. known nucleotide motifs for Toll-like receptor (TLR) activation are removed; iii. The 3' and 5' GC / AU composition is optimized to favor incorporation of the guide strand in RISC onto the passenger strand; iv. The expected sequence homology between the "seed" region of the siRNA guide strand and the genome-wide 3' untranslated region (3'-UTR) mRNA sequence is as small as possible in order to minimize the risk of obtaining microRNA-type gene regulation effects; v. Preferably, a phosphate is added to the 5' end of the siRNA guide strand to facilitate incorporation of the guide strand by RISC; It is designed in such a way.

[0012] A screening strategy was then developed that involved comparing a variety of different siRNA sequences in parallel based on their functional effectiveness in inhibiting gene expression, their specificity (not directly modulating other genes), and their reduction in undesirable or harmful effects (off-target effects, immunogenic effects, possible non-specific effects). The inventors therefore selected two siRNA sequences that are able to specifically reduce the expression of the human JAK1 or JAK3 genes, and thus the human JAK1 or JAK3 proteins, respectively, without showing undesirable effects (in particular, no effect on the expression of JAK2), with the common interest here being their therapeutic potential in treating diseases associated with Janus kinase (JAK) gain-of-function disruption of the JAK signaling pathway, in particular inflammatory diseases (including chronic inflammatory bowel disease) or certain cancers.

[0013] definition The term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two strands of substantially complementary nucleic acid in an antiparallel arrangement. Generally, each strand is composed primarily or entirely of ribonucleotides, although one or both strands may contain at least one non-ribonucleotide base, e.g., deoxyribonucleotides, and / or modified nucleotides, e.g., modified by chemical modification. The two strands forming the duplex structure may be different parts of a larger RNA molecule, or may be separate RNA molecules (siRNA). When the two strands are different parts of a larger RNA molecule, the 3' end of one strand may be linked to the 5' end of the other strand by a nucleotide linkage to form a hairpin loop structure (small hairpin RNA, or shRNA). Alternatively, the two strands may be linked by a linking means other than a nucleotide linkage ("linker").

[0014] "Antisense strand" refers to the strand of a dsRNA that includes a region of complementarity that is substantially complementary to a target sequence, according to the present invention, a target sequence on a JAK1 or JAK3 mRNA.

[0015] "Sense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to a region of the antisense strand.

[0016] Two sequences are fully complementary if their bases match each other over their entire length. A sequence can also be substantially complementary to a second sequence if the two sequences hybridize and are fully complementary, or are fully complementary over a portion of their length (e.g., in the case of a double-stranded complex with an overhang), or if they have a mismatch of up to 4, 3, 2, or 1 base.

[0017] Throughout this application, the term "comprising" should be interpreted to include all specifically mentioned characteristic features as well as, optionally, additional unspecified characteristic features. As used herein, use of the term "comprising" also describes embodiments in which no characteristic features are present (i.e., "consisting of") other than the specifically mentioned characteristic features.

[0018] Double-stranded ribonucleic acid As described in more detail below, double-stranded (ds) ribonucleic acid (RNA) molecules are provided for the purpose of inhibiting the JAK / STAT signaling pathway, particularly in humans having a disease associated with a Janus kinase (JAK) gain-of-function disruption of the JAK signaling pathway, the dsRNA comprising an antisense strand that is complementary to mRNA generated by transcription of the JAK1 gene or the JAK3 gene.

[0019] According to one embodiment, the two strands of dsRNA are separate RNA molecules (siRNA). Each strand of dsRNA comprises 21 nucleotides, preferably up to 24 nucleotides, 23 nucleotides or 22 nucleotides. The two strands of dsRNA are the same or different length, but preferably the same length.

[0020] Preferably, each strand of the dsRNA has a 3' overhang of two or more nucleotides, preferably two nucleotides.

[0021] According to one embodiment, the two strands of the dsRNA are part of a larger RNA molecule, for example constituting the stem of a hairpin-like structure. According to this embodiment, the dsRNA preferably comprises a total of up to 60 nucleotides, for example 55-60 nucleotides.

[0022] According to one embodiment, the dsRNA comprises or consists of a sense strand and an antisense strand, the sense strand consists of the nucleotide sequence of SEQ ID NO:1 and the antisense strand consists of the nucleotide sequence of SEQ ID NO:2; or the sense strand consists of the nucleotide sequence of SEQ ID NO:3 and the antisense strand consists of the nucleotide sequence of SEQ ID NO:4; or the sense strand consists of the nucleotide sequence of SEQ ID NO:5 and the antisense strand consists of the nucleotide sequence of SEQ ID NO:6; or the sense strand consists of the nucleotide sequence of SEQ ID NO: 7, the antisense strand consists of the nucleotide sequence of SEQ ID NO: 8, and The nucleotide at the 5' end of the antisense strand is phosphorylated.

[0023] According to one embodiment, the dsRNA is a dsRNA that reduces the expression of the human Janus kinase 3 (JAK3) gene, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2 (HJ3D41), or the sense strand comprises the nucleotide sequence of SEQ ID NO: 3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 4 (HMJ3D1). Preferably, in the dsRNA, the sense strand comprises the nucleotide sequence of SEQ ID NO: 1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 2 (HJ3D41). The dsRNA (HMJ3D1) that comprises the sense strand comprising the nucleotide sequence of SEQ ID NO: 3 and the antisense strand comprising the nucleotide sequence of SEQ ID NO: 4 is additionally specific to the mouse JAK3 gene and can reduce the expression of this gene.

[0024] According to one embodiment, the dsRNA is a dsRNA that reduces the expression of the human Janus kinase 1 (JAK1) gene, and the sense strand comprises the nucleotide sequence of SEQ ID NO: 5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 6 (HJ1D2), or the sense strand comprises the nucleotide sequence of SEQ ID NO: 7 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 8 (HJ1D8). Preferably, in the dsRNA, the sense strand comprises the nucleotide sequence of SEQ ID NO: 7 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 8 (HJ1D8).

[0025] dsRNA can be chemically modified, particularly to increase its stability or to increase the uptake of the guide strand by RISC. Examples of modified dsRNA molecules include dsRNA that contain unnatural backbone modifications or internucleotide bonds or that contain modified nucleotide bases.

[0026] In one preferred mode, the dsRNA is a dsRNA in which the nucleotide at the 5' end of the antisense strand is phosphorylated.

[0027] The dsRNA according to the invention can also be chemically modified for the purpose of forming conjugates, ie, covalently linked to one or more groups that enhance the activity, cellular distribution or cellular uptake of the dsRNA.

[0028] dsRNA can be prepared by any method known to those skilled in the art, for example by chemical synthesis or by genetic engineering.In the latter case, dsRNA is expressed from a transcription unit inserted into one or more DNA or RNA vectors.These transgenes can be introduced in the form of linear constructs, circular plasmids or viral vectors and then integrated into host cells, where they are integrated into the host cell genome, or they can be present in the form of extrachromosomal plasmids in other cases.Any viral vector that can accept a sequence encoding dsRNA can be used, such as vectors derived from adenoviruses (AV), adeno-associated viruses (AA V), retroviruses (e.g., lentiviruses, rhabdoviruses), herpes viruses, etc.The tropism of the virus can be modified, for example, by pseudotyping the vector by using envelope proteins or other surface antigens derived from other viruses.

[0029] The two strands of dsRNA can be transcribed under the control of promoters from two separate expression vectors that are co-transfected into the host cell, or actually under the control of multiple promoters, each on the same expression vector. dsRNA can also be expressed as two inverted repeats connected by a linker polynucleotide sequence to form a stem-loop structure (small hairpin RNA, or shRNA).

[0030] The promoter present in the DNA plasmid or viral vector controlling expression of the dsRNA can be a eukaryotic RNA polymerase I, II or III promoter (eg, U6) or a prokaryotic promoter (eg, T7 promoter).

[0031] The DNA plasmid for expressing dsRNA is typically introduced into target cells by transfection with the aid of a method and carrier well known to those skilled in the art.The effectiveness of transfection can be monitored, for example, by using a fluorescent label such as green fluorescent protein (GFP) as a reporter gene.

[0032] Medical Indication The dsRNA according to the present invention can be used as a drug, for therapy, particularly for human therapy, because of the above-mentioned iv properties that guided its design and the way in which the inventors selected it from 43 siRNAs originally generated to block expression of the human and / or mouse JAK1 or JAK3 genes.

[0033] This dsRNA is more particularly indicated for the treatment of diseases associated with Janus kinase (JAK) gain-of-function disruption of the JAK, particularly the JAK1 and / or JAK3, signaling pathways.

[0034] There is therefore provided a method for the treatment of a disease associated with Janus kinase (JAK) gain-of-function disruption of the JAK signaling pathway, in which a therapeutically effective amount of at least one dsRNA according to the invention is administered to a subject suffering from a disease associated with Janus kinase (JAK) gain-of-function disruption of the JAK, particularly the JAK1 and / or JAK3, signaling pathway.

[0035] The subject is a mammal, preferably a primate, and in one preferred embodiment is a human subject. The subject may also be a rodent, preferably a mouse.

[0036] Diseases associated with Janus kinase (JAK) gain-of-function disruption of JAK, particularly JAK1 and / or JAK3 signaling pathways, include in particular chronic inflammatory diseases of the intestine (Crohn's disease and ulcerative colitis), rheumatoid arthritis, alopecia areata, uveitis, atopic dermatitis, ankylosing spondylitis, psoriasis, lupus erythematosus, lupus nephritis, immune-mediated inflammatory diseases, including myelofibrosis, graft rejection and graft-versus-host disease, as well as cancer, especially leukemia (particularly acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML)) and solid tumor cancer. In one preferred mode, the dsRNA according to the invention is indicated for the treatment of immune-mediated inflammatory diseases, including chronic inflammatory diseases of the intestine, myelofibrosis, graft rejection, and cancer.

[0037] For its therapeutic use, the dsRNA is generally formulated in a pharmaceutical composition comprising at least one dsRNA according to the invention and a pharma- ceutically acceptable carrier.

[0038] According to one embodiment, the pharmaceutical composition comprises at least one dsRNA which is a JAK1 inhibitor according to the present invention.

[0039] According to another embodiment, the pharmaceutical composition comprises at least one dsRNA which is a JAK3 inhibitor according to the present invention.

[0040] According to yet another embodiment, the pharmaceutical composition comprises at least one JAK1 inhibitor dsRNA according to the invention and at least one JAK3 inhibitor dsRNA, e.g. - a dsRNA, the sense strand of which comprises the nucleotide sequence of SEQ ID NO: 1 and the antisense strand of which comprises the nucleotide sequence of SEQ ID NO: 2 (HJ3D41), and (i) a dsRNA, the sense strand of which comprises the nucleotide sequence of SEQ ID NO: 7 and the antisense strand of which comprises the nucleotide sequence of SEQ ID NO: 8 (HJ1D8) or (ii) a dsRNA, the sense strand of which comprises the nucleotide sequence of SEQ ID NO: 5 and the antisense strand of which comprises the nucleotide sequence of SEQ ID NO: 6 (HJ1D2), or - a dsRNA (HMJ3D1), the sense strand of which comprises the nucleotide sequence of SEQ ID NO: 3 and the antisense strand of which comprises the nucleotide sequence of SEQ ID NO: 4, and (i) a dsRNA (HJ1D8), the sense strand of which comprises the nucleotide sequence of SEQ ID NO: 7 and the antisense strand of which comprises the nucleotide sequence of SEQ ID NO: 8, or (ii) a dsRNA (HJ1D2), the sense strand of which comprises the nucleotide sequence of SEQ ID NO: 5 and the antisense strand of which comprises the nucleotide sequence of SEQ ID NO: 6, Includes.

[0041] A pharmaceutical composition according to the present invention may also comprise at least one dsRNA according to the present invention, at least one other active ingredient, such as an anti-cancer agent, and a pharma- ceutically acceptable carrier.

[0042] According to yet another embodiment, the present invention relates to the JAK1 inhibitor dsRNA according to the present invention for its use as a drug, in combination with the JAK3 inhibitor dsRNA according to the present invention or another active ingredient, for example an anticancer drug, or for its use in the prevention and / or treatment of diseases associated with Janus kinase (JAK) gain-of-function disruption of the JAK signaling pathway. According to this embodiment, the JAK1 inhibitor dsRNA and the JAK3 inhibitor dsRNA according to the present invention or another active ingredient can be formulated separately in the same given pharmaceutical composition, or in other cases actually in separate individual pharmaceutical compositions. They can be administered simultaneously or separately, for example in a manner that is spaced apart over time. According to this embodiment, the JAK1 inhibitor dsRNA is administered to a subject being treated with the JAK3 inhibitor dsRNA according to the present invention or another active ingredient.

[0043] According to yet another embodiment, the present invention relates to the JAK3 inhibitor dsRNA according to the present invention for its use as a drug in combination with the JAK1 inhibitor dsRNA according to the present invention or another active ingredient, for example an anticancer drug, or for its use in the prevention and / or treatment of diseases associated with Janus kinase (JAK) gain-of-function disruption of the JAK signaling pathway. According to this embodiment, the JAK3 inhibitor dsRNA and the JAK1 inhibitor dsRNA according to the present invention or another active ingredient can be formulated separately in the same given pharmaceutical composition, or in other cases actually in separate individual pharmaceutical compositions. They can be administered simultaneously or separately, for example in a manner that is spaced apart over time. According to this embodiment, the JAK3 inhibitor dsRNA is administered to a subject being treated with the JAK1 inhibitor dsRNA according to the present invention or another active ingredient.

[0044] Pharmaceutical compositions are formulated based on their route of administration, which may be, for example, local administration or systemic administration, such as parenteral or intravenous (iv) routes.

[0045] The pharmaceutical composition is administered at a dosage of dsRNA sufficient to induce inhibition of expression of JAK1 and / or JAK3.

[0046] Determining the appropriate route of administration and dosage for a given subject is well within the capabilities of one of ordinary skill in the art.

[0047] Methods for inhibiting JAK1 or JAK3 in a cell The present application also provides a JAK inhibition method for inhibiting expression of Janus kinase 1 (JAK1) or Janus kinase 3 (JAK3) in a cell, either in vivo or in vitro, comprising: a. introducing the dsRNA according to the invention into said human cells; and b. maintaining the cells produced in step (a) for a period of time sufficient to effect degradation of the mRNA of the JAK1 gene or the JAK3 gene, thereby inhibiting expression of JAK1 or JAK3 in the cells; A method is also described that includes:

[0048] The cells are preferably human or mouse cells.

[0049] According to one embodiment, in step a, the dsRNA is contacted with the cells at a concentration greater than or equal to 5 pM, preferably between 5 pM and 10 nM, or indeed even between 5 pM and 1 nM, or indeed even between 5 pM and 100 pM, or indeed even between 5 pM and 50 pM, even more preferably between 5 pM and 25 pM.

[0050] A transfection agent (eg, a cationic lipid such as lipofectamine) is typically used to facilitate transfection of cells with dsRNA.

[0051] If the method is carried out in vitro, in step b) the cells are maintained in a culture medium suitable for their survival and / or proliferation. The choice of such a medium is well within the capabilities of the skilled artisan.

[0052] In the following, the invention is explained in more detail by means of figures and examples. [Brief description of the drawings]

[0053] [Figure 1] 1 shows a typical structure of an siRNA according to the invention, comprising a dinucleotide 3' overhang and preferably a phosphate group at the 5' end of the guide strand. The passenger strand corresponds to the sense strand and the guide strand corresponds to the antisense strand. [Diagram 2] The hybridization regions on the coding portion of the JAK1 gene of the various JAK1-inhibitor siRNAs tested are depicted. [Diagram 3] The hybridization regions on the coding portion of the JAK3 gene of the various JAK3-inhibitor siRNAs tested are depicted. [Figure 4] The results of three independent experiments to measure the levels of JAK1, JAK2, JAK3 or TYK2 gene expression in Caco-2 cells transfected with 10 nM siRNA (targeting JAK1) are presented. [Diagram 5] The results of three independent experiments to measure the levels of JAK1, JAK2, JAK3 or TYK2 gene expression in Caco-2 cells transfected with 10 nM siRNA (targeting JAK3) are presented. [Figure 6] 4 presents the results of quantification of JAK1 expression in PC3 cells transfected with 12.5 pM siRNA. [Figure 7] Quantification of JAK3 expression in PC3 cells stably overexpressing JAK3 and transfected with 1 nM siRNA is presented. [Figure 8] 1 shows Western blot analysis of JAK1, JAK3 or GAPDH expression in PC3 cells stably overexpressing JAK3 and transfected with 0.5 pM to 12.5 pM siRNA. [Figure 9]1 shows the expression of potential target genes in T47D cells transfected with 10 nM of siRNA HJ1D2 targeting JAK1 or control siRNA (siAS). [Figure 10] 1 shows the expression of potential target genes in T47D cells transfected with 10 nM of siRNA HJ1D8 targeting JAK1 or control siRNA (siAS). [Figure 11] Figure 1 shows the expression of potential target genes in T47D or PC3 cells transfected with 10 nM of siRNA HJ3D41 targeting JAK3 or control siRNA (siAS). [Figure 12] Figure 1 shows the expression of potential target genes in T47D cells transfected with 10 nM of siRNA HMJ3D1 targeting JAK3 or control siRNA (siAS). [Figure 13] The effects of the siRNA according to the present invention or a control on cell proliferation (a), adenosine triphosphate (ATP) metabolism (b), and apoptosis (c), and (d) are shown. "opti" represents the medium OPTI-MEM used to prepare the mixture during transfection, and "lipo" represents the transfection agent used (Lipofectamine RNAimax). EXAMPLES

[0054] Example 1 siRNA identity Several siRNA sequences designed to inhibit the expression of human or human and mouse JAK1 or J1K3 genes were compared with each other, as well as with several commercially available siRNA sequences known to inhibit the expression of human JAK1 or JAK3 genes. The nomenclature of the siRNAs is as follows: H is human (Homo Sapiens) (species of interest), M is mouse (Mus musculus), J1 is the transcript targeted by JAK1 (JAK3 in the case of J3), followed by a unique number Dx that characterizes the sequence. Thus, siHJ1D8 is an siRNA sequence that targets the human JAK1 gene, and its unique number is D8. siHMJ3D1 is an siRNA sequence that targets the human and mouse JAK3 genes, and its unique number is D8. "A", "Q" and "H" represent siRNAs sold by Ambion, Qiagen and Dharmacon, respectively.

[0055] Several experimental approaches were combined to provide two siRNA sequences with the greatest possible efficacy and specificity while exhibiting the least possible unwanted or adverse effects, so as to enable their therapeutic use (see Tables 1 to 2).

[0056] [Table 1]

[0057] [Table 2]

[0058] The inventors compared dozens of unique or commercially available sequences with the aim of being able to derive therefrom two siRNAs capable of targeting JAK1 or JAK3 for therapeutic use.

[0059] It is interesting to note that among the siRNAs generated according to the principles of RNA interference, the siRNA sequence siHJ1D64 was indeed not effective in inhibiting the expression of JAK1.

[0060] The locations of the tested siRNAs on the JAK1 and JAK3 genes are shown in FIG. 2 and FIG.

[0061] [Table 3A]

[0062] [Table 3B]

[0063] [Table 4A]

[0064] [Table 4B]

[0065] [Table 4C]

[0066] Example 2 siRNA efficacy a. Assays to confirm specificity of gene inhibition: The specificity of the siRNA sequences was assessed using various molecular biology approaches by analyzing the gene expression of four members of the JAK protein family: JAK1, JAK2, JAK3 and TYK2 via RTqPCR. Experiments were performed at a concentration of 10 nM siRNA to promote potential non-specific effects that are known to be dose-dependent.

[0067] Caco-2 cells were transfected with 10 nM siRNA targeting JAK1 or JAK3 using Lipofectamine RNAimax for 48 hours. siRNA siHJ1D8 and siHJ1D2 target JAK1, and siRNA siHJ3D41 and HMJ3D1 target JAK3. RNA was extracted, reverse transcribed to cDNA, and tested by quantitative reverse transcription polymerase chain reaction (RTqPCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal gene, and untreated cells served as an external calibrator. siRNA "siAS" is a transfection control with no homology to the human genome. The results of three independent experiments to measure the levels of JAK1, JAK2, JAK3, or TYK2 gene expression in Caco-2 cells are shown in Figures 4 and 5.

[0068] These results were also confirmed at the protein level by Western blot studies. PC3 cells transfected with vector pCDNA3.1, which contains a sequence encoding a C-terminal GFP fusion of JAK3, were transfected with 10 nM siRNA targeting JAK1 or JAK3 for 48 h using Lipofectamine RNAimax to specifically allow monitoring of JAK3 protein. JAK-siRNA only affects the expression of their targets.

[0069] b. Assays to confirm susceptibility: Studies were conducted to determine the minimum effective dose of siRNA required to block endogenous expression of the JAK1 or JAK3 genes (si-JAK1: <12.5 pM in the human epithelial cell line PC3; si-JAK3: <1 nM in the human epithelial cell line PC3 overexpressing JAK3 by plasmid introduction).

[0070] PC3 cells were transfected with 12.5 pM siRNA using Lipofectamine RNAimax for 48 hours. Cells were lysed using standard conventional lysis buffer. Protein lysates were then analyzed for JAK1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression by Western blotting. Figure 6 shows the quantification of JAK1 expression normalized to GAPDH.

[0071] PC3 cells overexpressing JAK3-GFP were transfected with 1 nM siRNA using Lipofectamine RNAimax for 48 hours. Cells were lysed using standard conventional lysis buffer. Protein lysates were then analyzed for JAK3 and GAPDH expression by Western blotting. Figure 7 shows the quantification of JAK3 expression.

[0072] Finally, PC3 cells overexpressing JAK3-GFP were transfected with various concentrations of siRNA (0.5 pM-12.5 pM) using Lipofectamine RNAimax for 48 h. Cells were lysed using standard conventional lysis buffer. Protein lysates were subsequently analyzed for expression of JAK3 or JAK1 and GAPDH by Western blotting. Figure 8 presents the results of a Western blot experiment representative of three independent experiments.

[0073] Thus, the provided siRNA sequences maintain efficacy at doses well below those recommended for the already commercially available sequences (rarely below 10 nM), and the resulting sequences continue to demonstrate high JAK inhibitory efficacy for inhibiting expression of JAK1 or JAK3 at concentrations of 5 pM (see FIG. 8).

[0074] c. Consideration of Undesirable / Adverse Effects: i. Action on microRNA The present inventors searched the entire human genome for the presence of 3'UTR nucleotide sequences that could be recognized by siRNA "seed" sequences.

[0075] [Table 5]

[0076] [Table 6]

[0077] The results obtained are summarized in Table 5 for si-JAK1 and in Table 6 for si-JAK3. The selected si-JAKs presented a very low risk of activating the microRNA response.

[0078] ii. Possible direct effects on other genes For all human genes, si-JAK sequence homology was searched using the Basic Local Alignment Search Tool / Nucleotide by the US National Center for Biotechnology Information (NCBI), i.e., BLASTN 2.7.0. + The in silico analysis was carried out by using the program (freely available). This approach made it possible to investigate the sequence homology of the passenger and guide strands of the siRNAs to the entire human genome.

[0079] Potential targets with partial sequence homology to the siRNA were thus identified.

[0080] The set of all genes with partial sequence homology to siHJ1D2 is summarized in Table 7.

[0081] [Table 7]

[0082] Potential specific targets found by in silico analysis were experimentally validated by RTqPCR. To do this, T47D cells were transfected with 10 nM siRNA using Lipofectamine RNAimax for 48 h. RNA was extracted, reverse transcribed to cDNA, and tested by quantitative reverse transcription polymerase chain reaction (RTqPCR). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the internal gene, and untreated cells served as external calibrators. The siRNA "siAS" is a transfection control with no homology to the human genome. The results shown in Figure 9 represent four independent dot plot experiments, with the average values ​​overlaid.

[0083] Although partial sequence homology was detected in silico between the genes FECH, TAF4 and ABLIM1 and siHJ1D2, this latter siRNA does not affect the expression of these genes.

[0084] The set of all genes with partial sequence homology to siHJ1D8 is summarized in Table 8.

[0085] [Table 8]

[0086] Potential specific targets found by in silico analysis were experimentally validated by RTqPCR. To do this, T47D cells were transfected with 10 nM siRNA using Lipofectamine RNAimax for 48 h. RNA was extracted, reverse transcribed to cDNA, and tested by quantitative reverse transcription polymerase chain reaction (RTqPCR). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the internal gene, and untreated cells served as external calibrators. The siRNA "siAS" is a transfection control with no homology to the human genome. The results shown in Figure 10 represent four independent dot plot experiments, with the average values ​​overlaid.

[0087] Although partial sequence homology was detected in silico between genes ZNF782, ZFYVE1, ARL14EP, CADPS2, OAS1 and ZDHHC23 and siHJ1D8, this latter siRNA does not affect the expression of these genes.

[0088] The set of all genes with partial sequence homology to siHJ3D41 is summarized in Table 9.

[0089] [Table 9]

[0090] Potential specific targets found by in silico analysis were experimentally verified by RTqPCR. To do this, 10 nM siRNAs were transfected into T47D cells (genes ZSWIM4, RNPEP and PHF21A) or PC3 cells (gene LRCH2, which is not expressed by T47D cells and therefore could not be investigated in this line) for 48 h using Lipofectamine RNAimax. RNA was extracted, reverse transcribed to cDNA and tested by quantitative reverse transcription polymerase chain reaction (RTqPCR). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the internal gene and cells not subjected to any treatment served as external calibrator. siRNA "siAS" is a transfection control with no homology to the human genome. The results shown in Figure 11 represent four independent dot plot experiments, with the average values ​​overlaid.

[0091] siIHJ3D41 appears to reduce gene expression of ZSWIM4 and LRCH2, but not RNPEP and PHF21A.

[0092] The set of all genes with partial sequence homology to siHMJ3D1 is summarized in Table 10.

[0093] [Table 10]

[0094] Potential specific targets found by in silico analysis were experimentally validated by RTqPCR. To do this, T47D cells were transfected with 10 nM siRNA using Lipofectamine RNAimax for 48 h. RNA was extracted, reverse transcribed to cDNA, and tested by quantitative reverse transcription polymerase chain reaction (RTqPCR). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the internal gene, and untreated cells served as external calibrators. The siRNA "siAS" is a transfection control with no homology to the human genome. The results shown in Figure 12 represent four independent dot plot experiments, with the average values ​​overlaid.

[0095] Partial sequence homology was detected in silico between genes FAM213A, KANK3, TMEM120B and POLDIP3 and siHMJ3D1, but this latter siRNA does not affect the expression of these genes.

[0096] iii. Phenotypic effects The effect of the use of siRNA on key cellular functions (proliferation, apoptosis and ATP metabolism) was analyzed.

[0097] Caco-2 cells were transfected with 10 nM siRNA using Lipofectamine RNAimax.

[0098] The transfected cells were then incubated for a period of 5 hours in the presence of EdU (5 μM), a fluorescent substance that intercalates into the DNA of proliferating cells. The cells were then detached and subsequently permeabilized before analysis by flow cytometry. siRNA targeting the EG5 gene was used as an experimental internal control, since this gene is directly involved in cell proliferation. The results in Figure 13a represent four independent dot plot experiments, with the average values ​​overlaid. *" indicates significant p-value in a paired non-parametric statistical test. The siRNA of interest has no effect on proliferation.

[0099] Furthermore, after 48 hours of transfection, cellular ATP levels were measured using the Vialight kit (FIG. 13b).

[0100] During the transfection, cells were in the presence of CellEvent, a marker of activated caspase-3 and caspase-7, which allows apoptosis to be quantified. Cells were analyzed by flow cytometry after permeabilization. Figure 13c shows the percentage of cells in a state of apoptosis, while Figure 13d shows the number of fluorescent molecules on the surface of positive cells.

[0101] These siRNAs did not reduce cell proliferation, increase apoptosis, or alter ATP metabolism in any statistically significant manner.

Claims

1. A pharmaceutical composition for use as a medicament, comprising a double-stranded (ds) ribonucleic acid (RNA) comprising a sense strand and an antisense strand, the sense strand comprises the nucleotide sequence of SEQ ID NO:5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:6; or the sense strand comprises the nucleotide sequence of SEQ ID NO:7 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:8; or the sense strand comprises the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2; or the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:4; Pharmaceutical compositions.

2. A pharmaceutical composition for use in the prevention and / or treatment of a disease associated with a Janus kinase (JAK) gain-of-function disruption of the Janus kinase 1 (JAK1) or Janus kinase 3 (JAK3) signaling pathway, comprising a double-stranded (ds) ribonucleic acid (RNA) comprising a sense strand and an antisense strand, the sense strand comprises the nucleotide sequence of SEQ ID NO:5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:6; or the sense strand comprises the nucleotide sequence of SEQ ID NO:7 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:8; or the sense strand comprises the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2; or the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:4; Pharmaceutical compositions.

3. 3. The pharmaceutical composition of claim 2, wherein the disease is selected from the group consisting of immune-mediated inflammatory diseases and cancer.

4. The dsRNA reduces the expression of Janus kinase 1 (JAK1), the sense strand comprises the nucleotide sequence of SEQ ID NO:5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:6; or the sense strand comprises the nucleotide sequence of SEQ ID NO: 7 and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 8; A pharmaceutical composition according to any one of claims 1 to 3.

5. The dsRNA reduces the expression of Janus kinase 3 (JAK3), the sense strand comprises the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2; or the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:4; A pharmaceutical composition according to any one of claims 1 to 3.

6. 6. A pharmaceutical composition according to any one of claims 1 to 5 for use in a human subject.

7. A double-stranded (ds) ribonucleic acid (RNA) comprising a sense strand and an antisense strand, which can be used for human therapy, the sense strand comprises the nucleotide sequence of SEQ ID NO:5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:6; or the sense strand comprises the nucleotide sequence of SEQ ID NO:7 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:8; or the sense strand comprises the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2; or the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:4; dsRNA.

8. At least one double-stranded (ds) ribonucleic acid (RNA) comprising a sense strand and an antisense strand; A pharma- ceutically acceptable carrier; A pharmaceutical composition comprising: the sense strand comprises the nucleotide sequence of SEQ ID NO:5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:6; or the sense strand comprises the nucleotide sequence of SEQ ID NO:7 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:8; or the sense strand comprises the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2; or the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:4; Pharmaceutical compositions.

9. 1. An in vitro method for inhibiting expression of Janus kinase 1 (JAK1) or Janus kinase 3 (JAK3) in a cell, comprising: a. introducing into said cells a double-stranded (ds) ribonucleic acid (RNA) comprising a sense strand and an antisense strand, the sense strand comprises the nucleotide sequence of SEQ ID NO:5 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:6; or the sense strand comprises the nucleotide sequence of SEQ ID NO:7 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:8; or the sense strand comprises the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2; or The sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:4; Process, and b. maintaining the cells produced in step (a) for a period of time sufficient to effect degradation of the mRNA of the JAK1 gene or the JAK3 gene, thereby inhibiting expression of JAK1 or JAK3 in the cells; An in vitro method comprising:

10. The in vitro method according to claim 9, wherein in step a, the dsRNA is contacted with the cells at a concentration of 5 pM to 10 nM.

11. 11. The pharmaceutical composition of any one of claims 1 to 6 and 8, the dsRNA of claim 7, or the method of claim 9 or 10, wherein the nucleotide at the 5' end of the antisense strand of the dsRNA is phosphorylated.

12. 12. The pharmaceutical composition of any one of claims 1 to 6, 8 and 11, the dsRNA of claim 7 or 11, or the method of any one of claims 9 to 11, wherein each strand of the dsRNA comprises at most 24, 23 or 22 nucleotides.

13. 13. The pharmaceutical composition of any one of claims 1 to 6, 8, 11 and 12, the dsRNA of claim 7, 11 or 12, or the method of any one of claims 9 to 12, wherein the two strands of the dsRNA are the same length.

14. 14. The pharmaceutical composition of any one of claims 1 to 6, 8 and 11 to 13, the dsRNA of any one of claims 7 and 11 to 13, or the method of any one of claims 9 to 13, wherein the dsRNA comprises at most 60 nucleotides.

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