SIRNA SEQUENCES TARGETING THE EXPRESSION OF HUMAN GENES JAK1 OR JAK3 FOR A THERAPEUTIC USE

Abstract

A double-stranded (ds) ribonucleic acid (RNA) comprising a sense strand and an antisense strand, wherein: 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 stand comprises the nucleotide sequence SEQ ID NO: 3 and the antisense strand comprises the nucleotide sequence SEQ ID NO: 4; or 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 strand comprises the nucleotide sequence SEQ ID NO: 7 and the antisense strand comprises the nucleotide sequence SEQ ID NO: 8. The dsRNA therefrom may be used as a drug.

Claims

1. (canceled)

2. A method of preventing and/or treating a disease associated with a Janus kinase (JAK) gain of function disruption of the Janus kinase 1 (JAK1) or Janus kinase 3 (JAK3) signalling pathway, the method comprising: administering to a subject in need thereof a therapeutically effective amount of at least one the double-stranded (ds) ribonucleic acid (RNA) of claim 7.

3. The method of claim 2, wherein the disease comprises an immune-mediated inflammatory disease, cancer, or a combination of two or more of any of these.

4. The method of claim 2, wherein the dsRNA reduces the expression of Janus kinase 3 (JAK3) and wherein: 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 anti sense strand comprises the nucleotide sequence SEQ ID NO: 4.

5. The method of claim 2, wherein the dsRNA reduces the expression of Janus kinase 1 (JAK1) and wherein: 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 strand comprises the nucleotide sequence SEQ ID NO: 7 and the antisense strand comprises the nucleotide sequence SEQ ID NO: 8.

6. The method of claim 2, wherein the subject is a human subject.

7. A double-stranded (ds) ribonucleic acid (RNA) comprising a sense strand and an antisense strand, adapted for use in human therapy, wherein: 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; or 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 strand comprises the nucleotide sequence SEQ ID NO: 7 and the antisense strand comprises the nucleotide sequence SEQ ID NO: 8.

8. A pharmaceutical composition comprising: a pharmaceutically acceptable carrier; and a double-stranded (ds) ribonucleic acid (RNA) comprising a sense strand and an antisense strand wherein: 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; or 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 strand comprises the nucleotide sequence SEQ ID NO: 7 and the antisense strand comprises the nucleotide sequence SEQ ID NO: 8.

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

10. The method of claim 9, wherein, in the introducing (a), the dsRNA is brought into contact with the cell at a concentration in a range of from 5 pM to 10 nM.

11. The method of claim 2, wherein the nucleotide at the 5′ end of the antisense strand of dsRNA is phosphorylated.

12. The method of claim 2, wherein each strand of the dsRNA comprises at most 24 nucleotides.

13. The method of claim 2, wherein the two strands of the dsRNA are of identical length.

14. The method of claim 2, wherein the dsRNA comprises at most 60 nucleotides.

15. The dsRNA of claim 7, wherein the nucleotide at the 5′ end of the antisense strand of dsRNA is phosphorylated.

16. The dsRNA of claim 7, wherein each strand of the dsRNA comprises at most 24 nucleotides.

17. The dsRNA of claim 7, wherein the two strands of the dsRNA are of identical length.

18. The dsRNA of claim 7, wherein the dsRNA comprises at most 60 nucleotides.

19. The composition of claim 8, wherein the nucleotide at the 5′ end of the antisense strand of dsRNA is phosphorylated.

20. The composition of claim 8, wherein each strand of the dsRNA comprises at most 24 nucleotides.

21. The composition of claim 8, wherein the two strands of the dsRNA are of identical length.

Description

FIGURES

[0076] FIG. 1 represents the typical structure of an siRNA according to the invention, comprising a 3′ overhang, of 2 unpaired nucleotides, 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 to the antisense strand.

[0077] FIG. 2 represents the hybridisation regions, on the JAK1 gene encoding part, of the different JAK1-inhibitor siRNAs tested.

[0078] FIG. 3 represents the hybridisation regions, on the JAK3 gene encoding part, of the different JAK3-inhibitor siRNAs tested

[0079] FIG. 4 presents the results of 3 independent experiments for measuring the level of JAK1, JAK2, JAK3 or TYK2 gene expression in Caco-2 cells transfected with 10 nM of siRNA (targeting JAK1).

[0080] FIG. 5 presents the results of 3 independent experiments for measuring the level of JAK1, JAK2, JAK3 or TYK2 gene expression in Caco-2 cells transfected with 10 nM of siRNA (targeting JAK3).

[0081] FIG. 6 presents the results of quantification of JAK1 expression in PC3 cells transfected with 12.5 pM of siRNA.

[0082] FIG. 7 presents the results of quantification of JAK3 expression in PC3 cells that stably overexpress JAK3, and were transfected with 1 nM of siRNA.

[0083] FIG. 8 shows the analysis by Western blot of JAK1, JAK3, or GAPDH expression in PC3 cells that stably overexpress JAK3, and were transfected with 0.5 pM to 12.5 pM of siRNA.

[0084] FIG. 9 shows the expression of potential target genes in T47D cells transfected with 10 nM of siRNA HJ1D2 targeting JAK1 or a control siRNA (siAS).

[0085] FIG. 10 shows the expression of potential target genes in T47D cells transfected with 10 nM of siRNA HJ1D8 targeting JAK1 or a control siRNA (siAS).

[0086] FIG. 11 shows the expression of potential target genes in T47D or PC3 cells transfected with 10 nM of siRNA HJ3D41 targeting JAK3 or a control siRNA (siAS).

[0087] FIG. 12 shows the expression of potential target genes in T47D cells transfected with 10 nM of siRNA HMJ3D1 targeting JAK3 or a control siRNA (siAS).

[0088] FIG. 13 shows the effect of the siRNAs according to the invention or control on cell proliferation (a), adenosine triphosphate (ATP) metabolism (b), and apoptosis (c), and (d). “opti” denotes the medium OPTI-MEM used to produce the mixture during transfection, and “lipo” denotes the transfection agent used (LipofectamineRNAimax).

EXAMPLES

Example 1: Identity of siRNAs

[0089] Several siRNA sequences designed to inhibit expression of the human, or human and murine JAK1 or J1K3 gene, were compared with each other as well as with several commercially available siRNA sequences known to inhibit expression of the human JAK1 or JAK3 genes. The nomenclature of siRNAs is as follows: H for Homo Sapiens (the species concerned), M for Mus musculus, J1 for JAK1: targeted transcript (and J3 for JAK3), then a unique number Dx to characterise the sequence. Thus siHJ1D8 is the siRNA sequence which targets the human JAK1 gene, the unique number whereof is D8. siHMJ3D1 is the siRNA sequence that targets the human and murine JAK3 genes, the unique number whereof is D8. “A”, “Q” and “H” denote the siRNAs marketed respectively by the companies Ambion, Qiagen, and Dharmacon.

[0090] Several experimental approaches have been combined with a view to providing two sequences of siRNA that have the greatest efficacy and specificity possible while presenting the least possible undesirable or adverse effects such as to make possible the therapeutic use thereof (see Tables 1-2).

TABLE-US-00001 TABLE 1 siRNA Sequences Targeting Human JAK3 Antisense Sense Sequence Sequence Name (Passenger) (Guide) HJ3D41 CGACUUUCCA UACGAUUUCU GAAAUCGUAG GGAAAGUCGC A A (SEQ ID (SEQ ID NO: 1) NO: 2) HMJ3D1 GCGUGGAGCU UAGCGGCACA GUGCCGCUAU GCUCCACGCU G G (SEQ ID (SEQ ID NO:3) NO: 4)

TABLE-US-00002 TABLE 2 siRNA Sequences Targeting Human JAK1 Antisense Sense Sequence Sequence Name (Passenger) (Guide) HJ1D2 GGAUUACAAG UUCGUCAUCC GAUGACGAAG UUGUAAUCCA G U (SEQ ID (SEQ ID NO: 5) NO: 6) HJ1D8 GGACAUCAGC UCGCUUGUAG UACAAGCGAU CUGAUGUCCU A U (SEQ ID (SEQ ID NO: 7) NO: 8)

[0091] The inventors compared dozens of original or already marketed sequences, in order to be able to derive therefrom two siRNAs capable of targeting JAK1 or JAK3 for therapeutic use.

[0092] It is interesting to note that among the siRNAs generated according to the principles of RNA interference, the siRNA sequence siHJ1D64 was in fact not effective in inhibiting the expression of JAK1.

[0093] The position of the tested siRNAs on the JAK1 and JAK3 genes is shown in FIGS. 2 and 3.

TABLE-US-00003 TABLE 3 Synthesis of the experiments carried out to test the siRNAs directed against human JAK1. Average RT-QPCR Weighted Western Blot Average Efficacy Caco2 Caco2 Caco2 Caco2 Caco2 Average Caco2 Caco2 Caco2 Inhibition Score with with Caco2 with with with Inhibition with with with of Jak1 WB 10 nM 10 nM with 0.2 nM 0.2 nM 0.2 nM of Jak1 at 0.2 nM 0.2 nM 0.2 nM mRNA at 0.2 nM@3, siRNA siRNA 1 nM siRNA siRNA siRNA 0.2 nM siRNA siRNA siRNA 0.2 nM QPCR Exp1 Exp2 siRNA Exp1 Exp2 Exp3 Exp1-2-3 Exp1 Exp2 Exp3 n = 3 0.2 nM@2 siRNA % Average % Average % Inhibition inhib. % inhib. % inhib. inhib. Weighted % inhib. % inhib. % inhib. % inhib. % inhib. inhib. of Jak1 at Jak1 Jak1 Jak1 Jak1 Efficacy Jak1 Jak1 Jak1 Jak1 Jak1 Jak1 0.2 nM mRNA mRNA mRNA mRNA Score AS  0.0  0.0  0.0  0.0  0.0  0.0  0.0  0.0  0.0  0.0  0.0  0.0 HJ1A46 96.7 NA 92.3 73.8 NA NA NA 58.8 NA NA NA NA HJ1A47 97.5 NA 93.2 82.0 NA NA NA NA NA NA NA NA HJ1A48 NA 97.0 91.3 33.5 NA NA NA NA NA NA NA NA HJ1Q1 94.0 89.4 91.6 72.1 NA NA NA NA NA NA NA NA HJ1Q3 95.1 96.1 90.2 69.1 NA NA NA NA NA NA NA NA HJ1Q6 94.9 93.7 93.7 78.2 NA NA NA 70.6 NA NA 70.6 NA HJ1Q12 95.0 88.8 83.4 72.3 NA NA NA  0.0 NA NA  0.0 NA HJ1H9 96.7 98.9 85.0 78.1 82.1 88.2 82.8 70.1 45.9 89.0 68.3 77.0 HJ1H10 80.2 85.6 NA NA NA NA NA NA NA NA NA NA HJ1H11 92.3 92.9 81.8 62.7 NA NA 62.7 NA NA NA NA NA HJ1H12 94.6 90.3 83.6 75.1 NA NA 75.1 NA NA NA NA NA HJ1D2 NA NA 91.2 76.5 94.2 92.7 87.8 77.8 91.4 90.6 86.6 87.3 HJ1D4 NA NA 93.1 85.5 97.0 91.9 91.5 73.1 80.7 88.3 80.7 87.1 HJ1D8 NA NA 79.3 88.3 93.5 94.9 92.2 71.0 85.2 93.3 83.2 88.6 HJ1D13 NA NA 88.0 76.6 61.7 95.9 78.1 43.5 55.8 83.9 61.0 71.3 HJ1D15 NA NA 88.8 80.3 79.3 98.0 85.9 43.0 79.9 86.7 69.9 79.5 HJ1D17 NA NA 87.7 74.3 96.7 99.9 90.3 48.4 84.0 83.5 71.9 83.0 HJ1D21 NA NA 84.0 81.2 92.7 99.5 91.1 50.2 83.3 90.3 74.6 84.5 HJ1D26 NA NA 76.5 65.3 40.7 97.5 67.8 37.9 74.5 63.1 58.5 64.1 HJ1D64 NA NA  −7.9   NA NA NA NA −97.4   NA NA NA NA HJ1D73 NA NA NA 60.8 87.7 98.9 82.5 36.2 90.0 77.9 68.0 76.7 HMJ1D1 NA NA NA 83.2 95.4 95.6 91.4 74.1 81.2 76.8 77.4 85.8 HMJ1D2 NA NA NA NA NA 82.0 82.0 NA NA NA NA NA MJ1D11 NA NA NA NA 86.9 82.0 84.4 NA 79.2 79.2 79.2 82.3

TABLE-US-00004 TABLE 4 Synthesis of the experiments carried out to test the siRNAs directed against human JAK3. Fluorescence Quantification (Cellinsight Microscope) Weighted Average Western Blot PC3* Zscore PC3* PC3* PC3* Average PC3* with Exp w/ PC3* PC3* with with with Inhibition with 1 nM 10 nM = 1 with with 0.2 nM 0.2 nM 0.2 nM of JAK3 10 nM siRNA and Exp 1 nM 0.2 nM siRNA siRNA siRNA at 0.2 nM siRNA Z- w/1 nM = 2 siRNA siRNA Exp1 Exp2 Exp3 Exp1-2-3 siRNA Avg. Weighted % % % % % Inhib. Average inhib. inhib. inhib. inhib. inhib. Jak3 at Zscore Zscore Zscore Jak3 Jak3 Jak3 Jak3 Jak3 0.2 nM AS 0.0 0.0 0.0 0.0 0.0  0.0  0.0  0.0  0.0 HJ3A52 −0.5 −1.2 −1.0 61.0 72.0 NA NA NA NA HJ3A53 −1.0 −1.2 −1.1 78.9 85.8 85.4 74.3 85.8 81.8 HJ3A54 −0.9 −1.3 −1.2 58.5 74.2 NA NA NA NA HJ3Q1 −1.0 −2.2 −1.8 78.7 83.3 83.0 82.3 77.7 81.0 HJ3Q2 1.2 1.0 1.0 −30.2 −0.4 NA NA NA NA HJ3Q6 −0.4 −0.8 −0.7 59.8 52.1 38.3  3.0 NA 20.6 HJ3Q11 1.0 1.2 1.1 20.1 −4.3 NA NA NA NA HJ3H9 −0.5 −1.2 −1.0 83.0 72.4 81.6 83.3 89.7 84.9 HJ3H10 −0.1 −1.1 −0.8 80.7 68.2 40.8 76.7 74.8 64.1 HJ3H11 −0.3 −0.7 −0.5 74.0 73.3 NA NA NA NA HJ3H12 −0.4 −1.1 −0.8 83.3 82.6 79.8 23.2 93.3 65.4 HJ3D1 −0.5 −1.8 −1.3 67.9 70.6 77.7 75.1 85.0 79.3 HJ3D5 −0.5 −3.3 −2.4 53.9 64.1 81.3 78.3 86.7 82.1 HJ3D8 −0.3 0.6 0.3 23.2 57.8 65.0 58.1 71.1 64.7 HJ3D11 −0.5 −1.7 −1.3 31.2 62.3 74.2 72.2 82.1 76.2 HJ3D12 −1.2 −2.2 −1.9 57.7 63.3 76.5 81.1 73.2 76.9 HJ3D15 −0.3 −1.2 −0.9 54.7 75.0 74.1 75.8 79.6 76.5 HJ3D18 −0.3 −1.2 −0.9 20.8 63.8 40.3 34.8 59.0 44.7 HJ3D23 −0.4 −2.2 −1.6 44.3 65.2 80.1 81.7 80.9 80.9 HJ3D38 0.0 −1.8 −1.2 56.7 69.4 81.6 64.7 78.4 74.9 HJ3D41 −0.4 −1.8 −1.3 71.3 83.1 84.2 88.3 87.3 86.6 HMJ3D1 −0.1 −1.8 −1.2 76.1 68.6 85.5 88.3 77.7 83.8 Weighted Efficacy Score with HCS@1 FACS@1, FACS RT-QPCR WB_E3-1 PC3* PC3* PC3* PC3* PC3* PC3* Average 0.2 nM@2, with with with Average with with with Inhibition WB_E8 0.2 nM 0.2 nM 0.2 nM inhibition 0.2 nM 0.2 nM 0.2 nM of JAK3 0.2 nM@3, siRNA siRNA siRNA at 0.2 nM siRNA siRNA siRNA at 0.2 nM QPCR Exp1 Exp2 Exp3 Exp1-2-3 Exp1 Exp2 Exp3 Exp1-2-3 0.2@3 siRNA Avg. Avg. % % % Inhib. inhib. inhib. inhib. Inhib. Weighted inhib. inhib. inhib Jak3 at Jak3 Jak3 Jak3 Jak3 at Efficacy Fluo. Fluo. Fluo. 0.2 nM mRNA mRNA mRNA 0.2 nM Score AS  0.0  0.0  0.0  0.0  0.0  0.0  0.0  0.0  0.0 HJ3A52 NA NA NA NA NA NA NA NA NA HJ3A53 54.1 51.3 55.0 53.5 94.1 89.6 93.1 92.3 74.8 HJ3A54 NA NA NA NA NA NA NA NA NA HJ3Q1 54.1 57.1 64.4 58.5 89.2 60.3 93.6 81.0 71.3 HJ3Q2 NA NA NA NA NA NA NA NA NA HJ3Q6 NA NA NA NA NA NA NA NA NA HJ3Q11 NA NA NA NA NA NA NA NA NA HJ3H9 NA NA NA NA NA NA NA NA NA HJ3H10 NA NA NA NA NA NA NA NA NA HJ3H11 NA NA NA NA NA NA NA NA NA HJ3H12 51.4 52.4 56.4 53.4 86.7 95.3 95.2 92.4 69.3 HJ3D1 49.2 50.8 54.0 51.3 96.8 81.7 93.5 90.7 70.4 HJ3D5 42.1 45.0 51.5 46.2 92.4 67.2 95.2 84.9 67.8 HJ3D8 NA NA NA NA NA NA NA NA NA HJ3D11 NA NA NA NA 92.0 93.6 93.5 93.0 NA HJ3D12 44.8 43.9 52.0 46.9 77.6 86.6 87.9 84.0 65.8 HJ3D15 NA NA NA NA 78.3 79.4 87.0 81.6 NA HJ3D18 NA NA NA NA NA NA NA NA NA HJ3D23 41.0 37.0 45.0 41.0 90.4 97.1 94.4 94.0 69.8 HJ3D38 39.9 41.3 48.5 43.2 86.0 40.8 91.9 72.9 62.7 HJ3D41 47.5 51.3 53.5 50.8 89.7 69.2 89.1 82.7 72.6 HMJ3D1 48.6 55.6 58.4 54.2 92.1 91.1 91.1 91.4 71.8 *PC3 = PC3-HsJak3-GFP cells E8 clone

Example 2: Efficacy of the siRNAs

[0094] a. Analysis to Ascertain the Specificity of Gene Inhibition:

[0095] The specificity of the siRNA sequences was evaluated using various molecular biology approaches, by analysing via RTqPCR the gene expression of the 4 members of the JAK protein family: JAK1, JAK2, JAK3 and TYK2. The experiments were performed with a concentration of 10 nM siRNA in order to promote potential non-specific effects which are known to be dose-dependent.

[0096] The Caco-2 cells were transfected with 10 nM of siRNA targeting JAK1 or JAK3 using Lipofectamine RNAimax for 48 hours. The siRNAs siHJ1D8 and siHJ1D2 target JAK1 and the siRNAs siHJ3D41 and HMJ3D1 target JAK3. The RNAs were extracted, reverse transcribed into cDNA and tested by Quantitative Reverse Transcription Polymerase Chain Reaction (RTqPCR) technique. The gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal gene, and the cells not subjected to any treatment served as an external calibrator. The siRNA “siAS” is a transfection control that has no homology to the human genome. The results of 3 independent experiments for measuring the level of JAK1, JAK2, JAK3 or TYK2 gene expression in Caco-2 cells are shown in FIGS. 4 and 5.

[0097] These results were also confirmed at the protein level by Western blot testing. The PC3 cells transfected with a vector pCDNA3.1 in which the sequence encoding JAK3 fused at its C-terminus with the GFP, were transfected with 10 nM of siRNA targeting JAK1 or JAK3 using Lipofectamine RNAimax for 48 hours in order to enable monitoring of the JAK3 protein in particular. The JAK-siRNAs only impact the expression of their target.

[0098] b. Analysis to Ascertain the Sensitivity:

[0099] Investigation was performed to determine the minimum effective dose of siRNA necessary to block the endogenous expression of the JAK1 or JAK3 genes (si-JAK1: <12.5 pM in the human epithelial line PC3; si-JAK3: <1 nM in the human epithelial line PC3 overexpressing JAK3 by means of the introduction of a plasmid).

[0100] The PC3 cells were transfected with 12.5 pM of siRNA using Lipofectamine RNAimax for 48 hours. The cells were lysed using a standard conventional lysis buffer. By means of Western blotting, the protein lysates were then analysed for the expression of JAK1 and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). FIG. 6 represents the quantification of the expression of JAK1, normalised to GAPDH.

[0101] The PC3 cells overexpressing JAK3-GFP were transfected with 1 nM of siRNA using Lipofectamine RNAimax for 48 hours. The cells were lysed using a standard conventional lysis buffer. By means of Western blotting, the protein lysates were then analysed for the expression of JAK3 and GAPDH. FIG. 7 shows the quantification of the expression of JAK3.

[0102] The PC3 cells overexpressing JAK3-GFP were finally transfected with different concentrations of siRNA (from 0.5 pM to 12.5 pM) using Lipofectamine RNAimax for 48 hours. The cells were lysed using a standard conventional lysis buffer. By means of Western blotting, the protein lysates were then analysed for the expression of JAK3 or JAK1 and GAPDH. FIG. 8 presents the results of a Western blot experiment representative of 3 independent experiments.

[0103] The siRNA sequences provided therefore remain effective at doses that are well below those recommended for the sequences already being commercially marketed (rarely less than 10 nM). At a concentration of 5 pM, the sequences generated indeed continue to exhibit high JAK inhibition efficacy for inhibiting the expression of JAK1 or JAK3 (see FIG. 8).

[0104] c. Investigation of Undesirable/Adverse Effects: [0105] i. microRNA Effects

[0106] The inventors investigated the presence of 3′ UTR nucleotide sequences from the entire human genome that can be recognised by the “seed” sequences of siRNAs.

TABLE-US-00005 TABLE 5 The number of times where the “seed” sequence of si-JAK1 could exhibit sequence complementarity with a 3′UTR nucleotide sequence was investigated in silico over the entire human genome. Predicted Sequence Complementarity siRNA 1 hit 2 hits >=3 hits HJ1D2 339 7 0 HJ1D8 287 13 0

TABLE-US-00006 TABLE 6 The number of times where the “seed” sequence of si-JAK3 could exhibit sequence complementarity with a 3′UTR nucleotide sequence was investigated in silico over the entire human genome. Predicted Sequence Complementarity siRNA 1 hit 2 hits >=3 hits HJ3D41  527 8 1 HMJ3D1 1161 48 3

[0107] The results obtained have been synthesised in Table 5 for the si-JAK1s and in Table 6 for the si-JAK3s. The selected si-JAKs present a very moderate risk of activating a microRNA response.

[0108] ii. Potential Direct Effects on Other Genes

[0109] The si-JAK sequence homology for all of the human genes was analysed in silico by making use of the Basic Local Alignment Search Tool/Nucleotide, ie BLASTN 2.7.0+ program (freely available) from the US National Center for Biotechnology Information (NCBI). This approach made it possible to study the sequence homologies of the passenger and guide strands of the siRNAs with the entire human genome.

[0110] Potential targets having partial sequence homology with the siRNAs were thus identified.

[0111] The set of all genes having partial sequence homology to siHJ1D2 has been compiled in Table 7.

TABLE-US-00007 TABLE 7 The genes exhibiting partial sequence homology with the passenger and guide strands of siHJ1D2 have been compiled in this table. The “seed” sequence of siHJ1D2 is underlined, the sequence homology between the gene and siHJ1D2 is in bold italics. Position of Sequence Homology (“Seed” Sequence of siHJ1D2 Gene % Homologous (SEQ ID NO: 6) Name Homology Nucleotides Underlined) NPBWR2 80% 17/17 UU FECH 71% 15/15 UUCG TAF4 71% 15/15 U UCCAU ADAMTSL2 66% 14/14 AAUCCAU ABLIM 1 66% 14/14 UUCGUC U

[0112] Certain potential targets found by the in silico analysis have been experimentally validated by RTqPCR. In order to do this, the T47D cells were transfected with 10 nM of siRNA using Lipofectamine RNAimax for 48 hours. The RNAs were extracted, reverse transcribed into cDNA and tested by Quantitative Reverse Transcription Polymerase Chain Reaction (RTqPCR) technique. The gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal gene, and the cells not subjected to any treatment served as an external calibrator. The siRNA “siAS” is a transfection control that has no homology to the human genome. The results shown in FIG. 9 represent 4 independent dot plot experiments with the mean overprinted.

[0113] Although partial sequence homology between the genes FECH, TAF4 and ABLIM1 and the siHJ1D2 has been detected in silico, this latter siRNA has no impact on the expression of these genes.

[0114] The set of all genes having partial sequence homology to siHJ1D8 has been compiled in Table 8.

TABLE-US-00008 TABLE 8 The genes exhibiting partial sequence homology with the passenger and guide strands of siHJ1D8 have been compiled in this table. The “seed” sequence of siHJ1D8 is underlined, the sequence homology between the gene and siHJ1D8 is in bold italics. Position of Sequence Homol- Homology (“Seed” % ogous Sequence of siHJ1D8 Gene Homol- Nucleo- (SEQ ID NO: 8) Name ogy tides Underlined) ZFYVE1 71% 15/15 UCGC UU ZNF782 66% 14/14 UCGC CUU TPMT 66% 14/14 UCGCUUG OAS1 66% 14/14 UCGCUUG FLO11- 61% 13/13 UCGCU CUU like MFSD14B 61% 13/13 UCGCU CUU BACH2 61% 13/13 UCG UCCUU CADPS2 61% 13/13 UCGCU CUU ARL14EP 61% 13/13 AUGUCCUU NCSTN 61% 13/13 UCGCUUGU RNH1 61% 13/13 UCGCUUGU MEGF10 61% 13/13 UCGCUU U FAM160A1 76% 13/13 UCGCUU UU ZDHHC23 61% 13/13 UCGC CCUU

[0115] Certain potential targets found by the in silico analysis have been experimentally validated by RTqPCR. In order to do this, the T47D cells were transfected with 10 nM of siRNA using Lipofectamine RNAimax for 48 hours. The RNAs were extracted, reverse transcribed into cDNA and tested by Quantitative Reverse Transcription Polymerase Chain Reaction (RTqPCR) technique. The gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal gene, and the cells not subjected to any treatment served as an external calibrator. The siRNA “siAS” is a transfection control that has no homology to the human genome. The results shown in FIG. 10 represent 4 independent dot plot experiments with the mean overprinted.

[0116] Although partial sequence homology between the genes ZNF782, ZFYVE1, ARL14EP, CADPS2, OAS1 and ZDHHC23, and the siHJ1D8 has been detected in silico, this latter siRNA has no impact on the expression of these genes.

[0117] The set of all genes having partial sequence homology to siHJ3D41 has been compiled in Table 9.

TABLE-US-00009 TABLE 9 The genes exhibiting partial sequence homology with the passenger and guide strands of siHJ3D41 have been compiled in this table. The “seed” sequence of siHJ3D41 is underlined, the sequence homology between the gene and siHJ3D41 is in bold italics. Position of Sequence Homol- Homology (“Seed” % ogous Sequence of siHJ3D41 Gene Homol- Nucleo- (SEQ ID NO: 2) Name ogy tides Underlined) ZSWIM4 76% 16/16 UA GCA LRCH2 71% 15/15 U UCGCA RNPEP 66% 14/14 UAC CGCA PHF21A 66% 14/14 UAC CGCA

[0118] Certain potential targets found by the in silico analysis have been experimentally validated by RTqPCR. In order to do this, the T47D cells (genes ZSWIM4, RNPEP and PHF21A) or PC3 cells (the gene LRCH2, because it is not expressed by the T47D cells and therefore could not be studied in this line) were transfected with 10 nM of siRNA using Lipofectamine RNAimax for 48 hours. The RNAs were extracted, reverse transcribed into cDNA and tested by Quantitative Reverse Transcription Polymerase Chain Reaction (RTqPCR) technique. The gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal gene, and the cells not subjected to any treatment served as an external calibrator. The siRNA “siAS” is a transfection control that has no homology to the human genome. The results shown in FIG. 11 represent 4 independent dot plot experiments with the mean overprinted.

[0119] The silHJ3D41 appears to decrease the gene expression of ZSWIM4 and LRCH2, but not that of RNPEP and PHF21A.

[0120] The set of all genes having partial sequence homology to siHMJ3D1 has been compiled in Table 10.

TABLE-US-00010 TABLE 10 The genes exhibiting partial sequence homology with the passenger and guide strands of siHMJ3D41 have been compiled in this table. The “seed” sequence of siHMJ3D41 is underlined, the sequence homology between the gene and siHMJ3D41 is in bold italics. Position of Sequence Homol- Homology (“Seed” % ogous Sequence of siHMJ3D1 Gene Homol- Nucleo- (SEQ ID NO: 4) Name ogy tides Underlined) KANK3 71% 15/15 UAG CUG DNAH9 66% 14/14 UAG GCUG FARP1 66% 14/14 UAGC CUG TMEM120B 85% 17/18 UAGC FAM213A 66% 14/14 UAG GCUG CHD7 61% 13/13 UAGC GCUG TMEM267 61% 13/13 UA ACGCUG MAST4 61% 13/13 UAG CGCUG DGKQ 61% 13/13 UAG CGCUG GSDMD 61% 13/13 UAGCGGCA POLDIP3 61% 13/13 U CACGCUG GRIP2 61% 13/13 UAGCGGCA TEKT4 61% 13/13 UAG CGCUG HAUS8 61% 13/13 UAGC CUG YJEFN3 61% 13/13 UAGCG CUG FCH01 61% 13/13 U CACGCUG ELAC2 61% 13/13 UAG CGCUG SGSM3 61% 13/13 UAGCGGCA

[0121] Certain potential targets found by the in silico analysis have been experimentally validated by RTqPCR. In order to do this, the T47D cells were transfected with 10 nM of siRNA using Lipofectamine RNAimax for 48 hours. The RNAs were extracted, reverse transcribed into cDNA and tested by Quantitative Reverse Transcription Polymerase Chain Reaction (RTqPCR) technique. The gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal gene, and the cells not subjected to any treatment served as an external calibrator. The siRNA “siAS” is a transfection control that has no homology to the human genome. The results shown in FIG. 12 represent 4 independent dot plot experiments with the mean overprinted.

[0122] Although partial sequence homology between the genes FAM213A, KANK3, TMEM120B and POLDIP3, and the siHMJ3D1 has been detected in silico, this latter siRNA has no impact on the expression of these genes.

[0123] iii. Phenotypic Effects

[0124] The impact of the use of siRNAs on major cellular functions (proliferation, apoptosis, and ATP metabolism) was analysed.

[0125] The Caco-2 cells were transfected with 10 nM of siRNA using Lipofectamine RNAimax.

[0126] The transfected cells were then incubated for a period of 5 hours in the presence of EdU (5 μM), which is a fluorescent agent that brings about intercalation in the DNA of cells that proliferate. The cells were thereafter detached and then permeabilised before being analysed by flow cytometry. The siRNA targeting the EG5 gene is an experimental internal control since this gene is directly involved in cell proliferation. The results in FIG. 13a represent 4 independent dot plot experiments with the mean overprinted; “*” signifies a significant p-value in a paired non-parametric statistical test. The siRNAs of interest have no impact on proliferation.

[0127] Furthermore, after 48 hours of transfection, the level of cellular ATP was measured using a Vialight kit (FIG. 13b.)

[0128] During the transfection, the cells were placed in the presence of CellEvent, a marker of activated capase-3 and capase-7. This marker makes it possible to quantify apoptosis. The cells were analysed after permeabilisation by flow cytometry. FIG. 13c represents the percentage of cells in apoptosis while FIG. 13d represents the number of fluorescent molecules on the surface of positive cells.

[0129] The siRNAs did not reduce cell proliferation, or increase apoptosis, or alter ATP metabolism in any statistically significant manner.