HYBRID htiRNA / NANOPARTICLE COMPLEX AND USE THEREOF FOR TREATING A DISEASE OF THE DIGESTIVE SYSTEM

Abstract

A hybrid DNA/RNA molecule, or a complex thereof with at least one nanoparticle, may be used for the prevention or treatment of a disease, in particular a disease of the digestive system.

Claims

1. A hybrid DNA/RNA molecule, called htiRNA hybrid, comprising: two antisense RNA strands of same sequences and able to modulate endogenous mechanisms of RNA interference, each comprising a number of nucleotides nRNA in a range of from 18 to 30, and having a phosphorylated 5′ end; a first DNA strand, having a nucleotide sequence comprising a nucleotide sequence S1 linked via its 3′ end to the 5′ end of a nucleotide spacer arm L1 linked by its 3′ end to the 5′ end of a nucleotide sequence SC1; and a second DNA strand, having a nucleotide sequence comprising a nucleotide sequence S2 linked via its 3′ end to the 5′ end of a nucleotide spacer arm L2 inked via its 3′ end to the 5′ end of a nucleotide sequence SC2, wherein the nucleotide sequence SC1 comprises a number n.sub.SC1 of nucleotides with nSC1 from (n.sub.RNA−3) to (n.sub.RNA+3), and has at least 40% sequence identity, determined by a global alignment method, with the complementary sequence of the antisense RNA, so that the nucleotide sequence SC1 is hybridized via complementarity to a first of the two antisense RNA strands, wherein the nucleotide sequence SC2 comprises a number n.sub.SC2 of nucleotides with nSC2 from (n.sub.RNA−3) to (n.sub.RNA+3), and has at least 40% sequence identity, determined by a global alignment method, with the complementary sequence of the antisense RNA, so that the nucleotide sequence SC2 is hybridized via complementarity to the second of the two antisense RNA strands, wherein the nucleotide sequence S1 of the first DNA strand is complementary to the nucleotide sequence S2 of the second DNA strand, so that the nucleotide sequence S1 of the first DNA strand is hybridized via complementarity to the nucleotide sequence S2 of the second DNA strand, wherein the two nucleotide sequences S1 and S2 have the same number n.sub.S1-S2 of nucleotides, n.sub.S1-S2 being a number in a range of from 16 to 30, wherein the nucleotide spacer arm L1 of the first DNA strand comprises a number n.sub.L1 of nucleotides and the nucleotide spacer arm L2 of the second DNA strand comprises a number n.sub.L2 of nucleotides, n.sub.L1 and n.sub.L2 independently being a number in a range of from 1 to 15, and wherein the two antisense RNA strands, the first DNA strand, and/or the second DNA strand optionally carry one or more lipid groups.

2. The htiRNA hybrid of claim 1, wherein n.sub.S1-S2 is an integer in a range of from 18 to 22; and/or wherein n.sub.L1 and n.sub.L2 are independently an integer in a range of from 1 to 15, in particular of 2 to 10; and/or wherein the sum n.sub.SC1+n.sub.L1+n.sub.S1+nRNA and/or the sum n.sub.SC2+n.sub.L2+n.sub.S2+n.sub.RNA are higher than or equal to 60.

3. A complex, comprising: a nanoparticle; and the htiRNA hybrid of claim 1.

4. The complex of claim 3, wherein the nanoparticle is cationic.

5. The complex of claim 3, wherein the nanoparticle is an inorganic, organic, or hybrid organic/inorganic nanoparticle.

6. The complex of claim 3, wherein the nanoparticle is a cationic organic nanoparticle comprising a lipid.

7. The complex of claim 3, in the form of a nanoemulsion comprising a continuous aqueous phase and a dispersed phase, the dispersed phase comprising: (a) an amphiphilic lipid in at least 5 mole %; (b) a cationic surfactant in a range of from 15 to 70 mole %, the cationic surfactant comprising: (i) a lipophilic group comprising: (bi-1) a group R, which is a linear hydrocarbon chain comprising 11 to 23 carbon atoms; (bi-2) a lipophilic group comprising (bi-1) R—(C═O)—, R being a linear hydrocarbon chain comprising 11 to 23 carbon atoms; (bi-3) a fatty acid ester comprising 12 to 24 carbon atoms and phosphatidylethanolamine; (bi-4) a fatty acid amide comprising 12 to 24 carbon atoms and phosphatidylethanolamine, and/or (bi-5) a poly(propylene oxide); and (ii) a hydrophilic group comprising comprising: (bii-1) a linear alkyl group comprising 1 to 12 carbon atoms and interrupted and/or substituted by at least one cationic group; (bii-2) a branched alkyl group comprising 1 to 12 carbon atoms and interrupted and/or substituted by at least one cationic group; and/or (bii-3) a polymeric hydrophilic group comprising at least one cationic group; and (c) a co-surfactant in a range of from 10 to 55 mole %, the co-surfactant comprising a poly(ethylene oxide) chain comprising at least 25 ethylene oxide units; (d) a solubilizing lipid comprising a fatty acid glyceride; (e) optionally, a helper lipid; (f) the htiRNA hybrid, wherein the molar percentages of the amphiphilic lipid, the cationic surfactant, and the co-surfactant are relative to a molar sum of the amphiphilic lipid, cationic surfactant, co-surfactant, and helper lipid.

8. A method of stabilizing a complex of an antisense RNA and a nanoparticle, the method comprising: complexing the htiRNA hybrid of claim 1 with the nanoparticle.

9. A method for inserting in a eukaryote cell an antisense RNA able to modulate endogenous mechanisms of RNA interference, the method comprising: placing the eukaryote cell in contact with the complex of claim 3.

10. A method of preventing or treating a disease, the method comprising: administering to a mammal in need thereof an effective amount of the complex of claim 3.

11. The htiRNA hybrid of claim 1, wherein n.sub.S1-S2 is an integer in a range of from 18 to 22.

12. The htiRNA hybrid of claim 1, wherein n.sub.L1 and n.sub.L2 are independently an integer in a range of from 1 to 15.

13. The htiRNA hybrid of claim 1, wherein n.sub.L1 and n.sub.L2 are independently an integer in a range of from 2 to 10.

14. The htiRNA hybrid of claim 1, wherein the sum n.sub.SC1+n.sub.L1+n.sub.S1+nRNA and/or the sum n.sub.SC2+n.sub.L2+n.sub.S2+n.sub.RNA are higher than or equal to 60.

15. The complex of claim 3, wherein the nanoparticle is a cationic organic nanoparticle comprising a liposome.

16. The complex of claim 3, wherein the nanoparticle is a droplet of a nanoemulsion comprising a continuous aqueous phase and a dispersed lipid phase.

17. The method of claim 3, wherein the disease comprises a digestive system disease.

18. The method of claim 3, wherein the disease comprises an intestinal chronic inflammatory disorder.

19. The method of claim 3, wherein the disease comprises Crohn's disease or haemorrhagic rectocolitis.

Description

[0290] The invention is illustrated in connection with the following Figures and examples. Figures

[0291] FIG. 1: Structure of the htiRNA hybrid.

[0292] FIG. 2: Structure of the two monomer units of the htiRNA hybrid, seen as a dimer.

[0293] FIG. 3: Structure of an example of htiRNA hybrid («htiGFP») of the invention

[0294] FIG. 4: Structure of a monomer unit of the htiGFP hybrid (comparative).

[0295] FIG. 5: Structure of a dimer of a first DNA strand/second DNA strand of a htiGFP hybrid, the two strands being hybridized by S1 and S2 (comparative).

[0296] FIG. 6: Structure of control htiRNA («htiCTRL»), in which the antisense RNA of sequence SEQ ID NO:7 is unable to modulate the endogenous mechanisms of RNA interference (control).

[0297] [FIG. 7]: Structure of a monomer unit of the htiCTRL hybrid.

EXAMPLES

Example 1: htiGFP Hybrid

[0298] 1.1. Preparation of the htiGFP Hybrid

[0299] Hybridization of the two monomer units to a dimer ([FIG. 2] FIG. 2) was conducted by heating, following the protocol given in Table 2, to form the htiGFP hybrid having the structure detailed in [FIG. 3] FIG. 3. An interfering RNA targeting the messenger RNA of the gene coding for GFP was used as antisense RNA.

TABLE-US-00002 TABLE 2 Protocol for preparing a hybrid DNA-RNA structure « htiGFP » able to modulate endogenous mechanisms of RNA interference. Molar Hybridization Oligonucleotide Sequence Supplier equivalent buffer Protocol Antisense RNA SEQ ID IDT 2 100 mM KCl Heating: NO: 3 DNA 1 mM MgCl.sub.2 1) 5 minutes at 90° C. 1.sup.st DNA strand SEQ ID IDT 1 30 mM HEPES 2) 5 minutes at 80° C. NO: 1 DNA pH 7.3 3) 20 minutes at 70° C. 2.sup.nd DNA strand SEQ ID IDT 1 Leave to cool slowly NO: 2 DNA down to 25° C.

[0300] Effective hybridization of the two monomer units to a htiGFP hybrid dimer was controlled by electrophoresis on agarose (gel E-gel EX 4% Agarose Sybr Gold, Invitrogen). Migration time was15 minutes. A molecular weight marker (GeneRuler 50 bp DNA Ladder, Thermo Scientific) was used to demonstrate that the size of the structure obtained lies between the 100 base pair markers (100 bp) and 50 base pair markers 50 (50 bp). The structure of the htiGFP hybrid is 69 base pairs (n.sub.SC1+n.sub.L1+n.sub.S1+n.sub.RNA=n.sub.SC2+n.sub.L2+n.sub.S2+n.sub.RNA=69).

1.2. Preparation of Comparative Structures

[0301] To obtain comparative examples, the following were prepared: [0302] one monomer unit of the htiGFP hybrid ([FIG. 4] FIG. 4), [0303] a dimer composed of the first DNA strand and second DNA strand, the two strands being hybridized by S1 and S2 (dimer free of antisense RNA) ([FIG. 5] FIG. 5), [0304] a control hybrid htiCTRL, in which the antisense RNA is not able to modulate the endogenous mechanisms of RNA interference ([FIG. 6] FIG. 6). This non-targeted antisense RNA of sequence SEQ ID NO:7 (Kim et al., 2005) has no specific target on the human genome and is used as control. The first DNA strand of the htiCTRL hybrid has the sequence SEQ ID NO:8, and the second strand has the sequence SEQ ID NO:9. The antisense RNAs and SC1 and SC2 differ between htiGFP and htiCTRL. On the other hand, the spacer arms L1 and L2 and nucleotide sequences S1 and S2 of the htiCTRL hybrid are the same as those of the htiGFP hybrid.
1.3. Functional Activity of the htiGFP Hybrid

[0305] A line of prostate cancer tumour cells over-expressing the gene coding for Green Fluorescent Protein (PC3-GFP) was used to validate the functional activity of RNA interference of the different structures described in 1.1 et 1.2.

[0306] Each interfering RNA was transfected into the PC3-GFP cell line to a final concentration of 5 to 20 nM using Lipofectamine RNAimax reagent following the manufacturer's instructions.

[0307] Fluorescence at 510 nm (GFP emission wavelength) of each cell was quantified under confocal microscopy (>300 cells per condition). Each condition was reproduced at N=3 independent experiments, +/−standard deviation. The results are given in Table 3.

TABLE-US-00003 TABLE 3 GFP fluorescence intensity per cell as a function of the structure used. GFP fluorescence intensity per cell (Arbitrary unit, ±standard deviation, N = 3 independent experiments) Concentration 5 nM 10 nM 20 nM Non-treated 441.23 ± 41.46  474.68 ± 103.75 462.71 ± 57.27 Non-targeted 479.02 ± 26.48 529.92 ± 86.86 561.02 ± 31.85 antisense RNA siAllStar (Qiagen and its complementary strand) (comp.) Antisense RNA GFP 147.38 ± 13.94 141.95 ± 21.24 117.33 ± 27.66 siGFP (SEQ ID NO: 3 and its complementary strand (comp.) Monomer unit of the 456.09 ± 24.14 458.12 ± 48.42  540.6 ± 84.28 htiCTRL hybrid ([FIG. 7] FIG. 7) (comp.) Monomer unit of the 256.43 ± 20.37 245.89 ± 16.42 149.55 ± 29.65 htiGFP hybrid ([FIG. 4] FIG. 4) (comp.) Dimer of 1.sup.st DNA 523.38 ± 69.67 567.72 ± 57.99 519.98 ± 67.82 strand/2.sup.nd DNA strand of the htiGFP hybrid ([FIG. 5] FIG. 5) (comp.) htiCTRL ([FIG. 6] FIG. 6) 502.53 ± 38.22  519.1 ± 67.32 528.58 ± 46.05 (comp.) htiGFP ([FIG. 3] FIG. 3) 311.79 ± 11.7  309.41 ± 75.99 237.15 ± 15.5  (invention)

[0308] The activity of the htiGFP hybrid maintains activity similar to RNA interference compared with a siRNA having an identical antisense sequence SEQ ID NO:3. According to Table 3, the siRNA targeting the gene coding for GFP (siGFP) strongly decreases the expression of its target gene as seen by a reduction in GFP intensity per cell, when PC3-GFP cells are transfected with this siRNA. After integration of the same sequence of antisense RNA in a hybrid DNA/RNA structure by inverted tandem (htiGFP), and similar to its monomeric sub-unit (monomer unit of the htiGFP hybrid), activity similar to RNA interference is still observed. (Table 3). However, this activity is slightly less than with a siRNA composed solely of RNA (siGFP).

Example 2: Preparation of a Lipid Nanoparticle/htiRNA Hybrid Complex

2.1. Preparation of Lipid Nanoparticles

[0309] Lipid nanoparticles (LNPs) were prepared by mixing the organic and aqueous phases with a sonication method allowing the generation of nano-droplets. After homogenization at 55° C., the two phases were mixed and sonication cycles performed at 55° C. for 5 minutes (alternating 10 seconds of sonication and 30 seconds rest time). An ultrasonic processor with conical probe of 3 mm was used (AV505 Ultrasonic processor, Sonics), adjusted to 45% power.

[0310] The non-encapsulated components were separated from the LNPs by dialysis in a volume of PBS buffer (Phosphate-Buffered Saline) equivalent to 200 times the volume of the LNPs. The PBS buffer was changed twice throughout dialysis which lasted a total time of 24 h. After characterization, the LNPs were filtered on a cellulose membrane of 0.22 μm porosity.

[0311] The hydrodynamic diameter, polydispersity index (P01) and zeta potential of the lipid nanoparticles were measured on a Zeta Sizer Nano instrument (NanoZS, Malvern). Hydrodynamic diameter and polydispersity index were measured at a LNP concentration of 0.6 mg/mL in PBS buffer at 25° C. Zeta potential was measured at a concentration of 0.4 mg/mL int mM NaCl buffer, pH 7.4 at 25° C.

TABLE-US-00004 TABLE 4 Composition of emulsions comprising nanoparticles CL40 and CL80 Organic phase Aqueous phase SUPER MYRJ ™ LIPOID REFINED ® SOYBEAN SUPPOCIRE S40-PW- S PC-3 DOTAP DOPE USP EP-LQ-(MH) STANDARD (MV) PBS Glycerol CL40 % (Solid phase) 3.43 30.4 3.61 18.24 6.11 38.24 — — mg 3.7 32.8 3.9 19.7 6.6 41.3 1480 800 CL80 % (Solid phase) 1.47 10.01 — 44.88 14.95 28.77 — — mg 2.2 15 — 67.1 22.4 43.1 1480 800

TABLE-US-00005 TABLE 5 Lists of products used Trade name Supplier CAS No: Description SUPPOCIRE STANDARD Gattefossé 85665-33-4 Glycerides, C10-18; Triglycerides C10-C18 MYRJ ™ S40-PW-(MV) CRODA 9004-99-3 Polyoxyethylene fatty acid ester SUPER REFINED ® CRODA 232-274-4 Soybean oil SOYBEAN USP EP-LQ-(MH) LIPOID S PC-3 Lipoid 97281-48-6 Phosphatidylcholine hydrogenated (phospholipids) DOTAP MERCK 132172-61-3 1,2-dioleoyl-3-trimethylammonium-propane DOPE AvantiPolar 4004-05-1 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine Phosphate Buffered Saline Sigma — Buffer, neutral pH Glycerol Sigma 56-81-5 —

2.2. Preparation of a Lipid Nanoparticle/htiRNA Hybrid Complex

[0312] The htiRNA hybrid in Example 1 was complexed with the nanoparticles prepared in Example 3.1.

[0313] The complexes were formed between the different nucleic acids and lipid nanoparticles for 20 minutes in a reaction buffer (154 mM NaCl, 10 mM HEPES, pH 7.2). The quantities of lipid nanoparticles were adjusted to maintain a constant N/P ratio with N/P=36 (N: DOTAP/DOPE amine group; P: phosphate group of the corresponding nucleic acid). A quantity of artificial SHIME medium (Prodigest) was added to a final concentration ranging from 0 to 90% SHIME medium (% V/V). The results are given in Table 6.

TABLE-US-00006 TABLE 6 Stability according to proportion of SHIME medium. Buffer 154 mM % Stability NaCl, (Ratio of Volume Volume 10 mM SHIME Volume siAllStar htiGFP Hepes, SHIME Total % control + LNP 20 μM 20 μM pH 7.2 medium volume Ratio SHIME interfering (μL) (μL) (μL) (μL) (μL) (μL) N/P medium RNA) 1 0 1 39.0 0 40.00 36 0.0% — 2 0 1 19.0 20 40.00 36 50.0% — 3 0.7 1 38.3 0 40.00 36 0.0% 92.85 4 0.7 1 18.3 20 40.00 36 50.0% 46.11 5 0.7 1 14.3 24 40.00 36 60.0% 20.48 6 0.7 1 10.3 28 40.00 36 70.0% 2.093 7 0.7 1 6.3 32 40.00 36 80.0% 5.114 8 0.7 1 2.3 36 40.00 36 90.0% 3.368 9 0 1 39.0 0 40.00 36 0.0% — 10 0 1 19.0 20 40.00 36 50.0% — 11 2 1 37.0 0 40.00 36 0.0% 96.78 12 2 1 17.0 20 40.00 36 50.0% 95.62 13 2 1 13.0 24 40.00 36 60.0% 95.82 14 2 1 9.0 28 40.00 36 70.0% 94.51 15 2 1 5.0 32 40.00 36 80.0% 93.13 16 2 1 1.0 36 40.00 36 90.0% 92.89
2.3. Increase in the Stability of the Nanoparticle/htiGFP Complex Compared with a Nanoparticle/siGFP Complex, in a Complex Biological Fluid.

[0314] The stability: [0315] of the nanoparticle/control siRNA complex (siAllstar, Qiagen, 21 base pairs) or [0316] of the nanoparticle/htiGFP complex was monitored by electrophoresis on agarose gel (E-gel 2% Agarose Ethidium Bromide, Invitrogen, migration time 10 minutes) after exposure of the complexes to increasing concentrations of artificial SHIME medium (Prodigest) to reproduce physiological conditions at the distal colon (100% SHIME).

[0317] When the concentration of SHIME medium is increased, de-complexing of the nanoparticle/siAllStar complexes can be observed on and after a concentration of 50% SHIME medium, since migration of siAllStar is observed on agarose gel. On the other hand, by means of greater electrostatic interaction, the nanoparticle/htiGFP complex remains stable in this biological fluid as indicated by the absence of migration of htiGFP on agarose gel.