CAPPED AND UNCAPPED RNA MOLECULES AND BLOCK COPOLYMERS FOR INTRACELLULAR DELIVERY OF RNA

Abstract

The present invention relates to the use of at least one tetrafunctional non-ionic amphiphilic block copolymer as a vehicle for capped or uncapped mRNA for intracellular delivery for gene therapy.

Claims

1-17. (canceled)

18. A method for intracellular delivery, comprising one or more than one administration to a subject of a composition comprising a tetrafunctional non-ionic amphiphilic block copolymer as a vehicle for capped or uncapped mRNA; wherein said tetrafunctional non-ionic amphiphilic block copolymer comprises hydrophilic blocks comprising polyethylene oxide units and hydrophobic blocks comprising polypropylene oxide units, and is selected from the group consisting of: ##STR00042## ##STR00043## and mixtures thereof.

19. The method according to claim 18, wherein said mRNA is a modified mRNA.

20. The method according to claim 18, wherein said mRNA is a capped and modified mRNA.

21. The method according to claim 18, wherein said mRNA is a messenger .sub.5′pppRNA, .sub.5′ppRNA, .sub.5′pRNA or .sub.5′OHRNA.

22. The method according to claim 18, wherein said tetrafunctional non-ionic amphiphilic block copolymer is selected from: ##STR00044## and mixtures thereof.

23. The method according to claim 18, wherein said tetrafunctional non-ionic amphiphilic block copolymer is: ##STR00045##

24. The method according to claim 18, wherein said tetrafunctional non-ionic amphiphilic block copolymer is: ##STR00046##

25. The method according to claim 18, wherein said tetrafunctional non-ionic amphiphilic block copolymer is: ##STR00047##

26. The method according to claim 18, wherein at least one terminal block of the tetrafunctional non-ionic amphiphilic block copolymer is glycosylated and/or functionalized.

27. The method according to claim 18, wherein said tetrafunctional non-ionic amphiphilic block copolymer comprises at least one terminal hydrophilic or hydrophobic block conjugated with at least one glycosyl moiety.

28. The method according to claim 18, wherein said method comprises more than one administration to the subject of the composition comprising a tetrafunctional non-ionic amphiphilic block copolymer.

29. The method according to claim 19, wherein said method comprises more than one administration to the subject of the composition comprising a tetrafunctional non-ionic amphiphilic block copolymer.

30. A pharmaceutical composition comprising a tetrafunctional non-ionic amphiphilic block copolymer, in combination with at least one capped modified mRNA or uncapped modified mRNA; wherein said tetrafunctional non-ionic amphiphilic block copolymer comprises hydrophilic blocks comprising polyethylene oxide units and hydrophobic blocks comprising polypropylene oxide units, and is selected from the group consisting of: ##STR00048## ##STR00049## and mixtures thereof.

31. The pharmaceutical composition according to claim 30, wherein the tetrafunctional non-ionic amphiphilic block copolymer comprises at least one terminal block which is glycosylated and/or functionalized.

32. The pharmaceutical composition according to claim 30, wherein said tetrafunctional non-ionic amphiphilic block copolymer and mRNA are formulated in a Tyrode's medium or an equivalent medium.

33. A method for increasing, improving, and/or maintaining the expression of a protein in an eukaryotic host, comprising a step of transfecting into said host at least one tetrafunctional non-ionic amphiphilic block copolymer, as a vehicle for at least one capped or uncapped mRNA; wherein said tetrafunctional non-ionic amphiphilic block copolymer comprises hydrophilic blocks comprising polyethylene oxide units and hydrophobic blocks comprising polypropylene oxide units, and is selected from the group consisting of: ##STR00050## ##STR00051## and mixtures thereof.

34. A tetrafunctional non-ionic amphiphilic block copolymer of formula: ##STR00052## or one of its pharmaceutically acceptable salts.

35. The tetrafunctional non-ionic amphiphilic block copolymer according to claim 34, comprising at least one terminal block which is glycosylated and/or functionalized.

36. The tetrafunctional non-ionic amphiphilic block copolymer according to claim 35, comprising at least one terminal block which is glycosylated and/or functionalized.

Description

FIGURES

[0309] FIGS. 1A-1D. Influence of the mRNA capping and/or nucleotide modification on β-galactosidase expression in C57Bl6 mouse skeletal muscle and immune reaction against β-galactosidase. FIG. 1A β-galactosidase activity one day after intramuscular injection of 20 μg uncapped fully modified or unmodified mRNA either naked or formulated with 20.Math.10.sup.−4% 704. FIG. 1B β-galactosidase activity one day after intramuscular injection of 20 μg capped fully modified or unmodified mRNA either naked or formulated with 20.Math.10.sup.−4% 704. FIG. 1C Humoral response at 42 days after a vaccination scheme consisting in one intramuscular injections at day 0 and 21 of 20 μg capped or uncapped modified and/or unmodified mRNA either naked or formulate with 20.Math.10.sup.−4% 704. Each column represents the mean antibody titer determined by ELISA of at least eight individual mice. FIG. 1D Specific CD8+IFNγ+β-galactosidase cells percentage. Splenocytes were prepared at day 42 stimulated overnight with murine dendritic cell line (Jaws) transfected with ICAFectin®441 and plasmid DNA encoding β-galactosidase or as control with plasmid DNA encoding the AlphaFetoproteine. After washing, cells were stained with an anti-CD8 antibody and anti-IFNγ. Each column represents the percentage of CD8+IFNγ cells in total splenic CD8++/−SEM of at least six individual mice.

[0310] FIG. 2. Influence of the mRNA capping and/or nucleotide modification on β-galactosidase expression in cells in culture. Hela (upper part of FIG. 2), C2C12 (middle of FIG. 2) and JAWII cells (bottom of FIG. 2) were transfected with aminoglycoside lipidic derivative DOST at a charge ratio of 5 (+/−) with 0.5 μg of formulated mRNA or formulated DNA encoding beta galactosidase. RNA molecules were either capped or uncapped with fully modified U and C by Pseudo-U or 5-Methyl-Cytosine or unmodified U and C. After 24h, cells were collected and beta galactosidase expression was measured. Results are expressed by pg of beta galactosidase per mg of cellular protein (pg/mg prot). Data are shown as the average±SEM of the (beta gal (pg/mg prot) of the transfected cells. As control, cells transfected with RNA alone or DNA alone gave no significant expression of the beta galactosidase.

[0311] FIGS. 3A-3C. Mouse hematocrit as a function of time after injection of block copolymer formulations. FIG. 3A Mouse hematocrit as a function of time after intramuscular injection of 704/RNA formulations containing 20 μg mRNA encoding murine EPO and 10×10.sup.−4% (open circles), 20×10.sup.−4% (open squares) or 100×10.sup.−4% (open triangles) 704. As control, mice were also injected with 20 μg of naked RNA encoding murine EPO (solid circles). As control a group of mice was also uninjected (solid squares). FIG. 3B mouse hematocrit as a function of time after intramuscular injected of 704/DNA formulation containing 10 μg of plasmid DNA encoding murine EPO and 0.15% 704 (open circles). As control, mice were also injected with 10 μg of naked DNA encoding murine EPO (close circles). As control a group of mice was also uninjected (solid squares). FIG. 3C mouse hematocrit as a function of time after intramuscular injection of 904/RNA formulations containing 20 μg mRNA encoding murine EPO and 10×10.sup.−4% 4 (open circles), 20×10.sup.−4% (open squares) or 100×10.sup.−4% (open triangles) 904. As control, mice were also injected with 20 μg of naked RNA encoding murine EPO (solid circles). As control a group of mice was also uninjected (solid squares).

[0312] FIGS. 4A-4D. Modulation of the hematocrit in mice intramuscularly injected with block copolymer/RNA formulations and 704/DNA formulations (B). FIG. 4A Six mice were treated 2 times at day 0 and 42 with 2 successive injections with one week interval. Treatments consisted of 20 μg mRNA encoding murine EPO either naked (open triangles) or formulated with and 100×10.sup.−4% (solid squares) 904 and 20×10.sup.−4% (solid circles) 704. At day 100 after the beginning of the treatment mice received a single injection consisting of 20 μg mRNA encoding EPO either naked (open triangles) or formulated with 100×10.sup.−4% (solid squares) 904 and 20×10.sup.−4% (w/v) (solid circles) 704. At day 134 after the beginning of the treatment, mice received a single injection of 20 μg mRNA encoding the murine EPO with 100×10.sup.−4% 904 (solid squares). As control, mice were also uninjected (open squares). Dotted lines represent the fluctuation over the of the hematocrit of healthy non injected mice. (FIG. 4B Six mice were treated at day 0, 56 and 100 with 10 μg naked DNA (open diamond) or formulated with 0,15% 704 (solid diamond). As control, mice were also uninjected (open squares). Dotted lines represent the fluctuation over the of the hematocrit of healthy non injected mice. FIG. 4C Murine EPO expression measured at day 135 in serum of mice 24 hours after injection of 20 μg RNA encoding murine EPO either naked of formulated with 100×10.sup.−4% 904 and 10 μg plasmid DNA encoding EPO either naked or formulated with 0.15% 704. Each bar represents the mean+/−SEM of 6 individual values. FIG. 4D Humoral response at 170 days after the beginning of the treatment with either DNA or mRNA encoding the murine EPO formulated with tetra functional block copolymers. Each column represents the mean antibody amount against murine EPO measured in the serum of mice injected with the various compositions, determined by ELISA using a standard curve made of known amount of commercially available antibodies against murine EPO.

[0313] FIG. 5: Luciferase expression in mouse skeletal muscle after intramuscular injection of mRNA formulated with 704 at 20.Math.10.sup.−4%. Luciferase activity 24 hours after intramuscular injection of 10 μg mRNA formulated with 704 in various medium buffered either with Hepes, NaHCO.sub.3 or sodium Lactate corresponding respectively to pH of 7.4, 7.4 and 6.7. The effect of the concentration of CaCl.sub.2 ranging from 1 to 5 mM on luciferase expression was also measured on two different medium buffered either by Hepes or NaHCO.sub.3. As a control a group of mice was injected with a medium consisting of 150 mM NaCl. Each column represents the mean+/−SEM of at least six individual values.

[0314] FIG. 6: Luciferase expression in mouse skeletal muscle after intramuscular injection of mRNA formulated with 704 at various concentrations. Luciferase activity 24 hours after intramuscular injection of 10 μg mRNA formulated with 704 at various concentrations ranging from 10.Math.10.sup.−4 to 1000.Math.10.sup.−4% (w/v). Each column represents the mean+/−SEM of at least six individual values.

[0315] FIG. 7: Luciferase expression in mouse skeletal muscle after intramuscular injection of capped modified mRNA either naked or formulated with tetrafunctional PEO-PPO amphilic block copolymer of 14 463 g/mol. Luciferase activity 24 hours after intramuscular injection of 5 μg mRNA formulated with the block copolymer at various concentrations ranging from 5 to 1000.Math.10.sup.−4% (w/v). Each column represents the mean+/−SEM of at least six individual values. The column “naked” relates to the expression of luciferase after administration of the same mRNA but without the block copolymer. The column “ref” relates to the expression of luciferase after administration of the same mRNA in combination with the tetrafunctional block copolymer 704. when administered at a concentration of about 20.Math.10.sup.−4% (w/v).

[0316] FIG. 8: Luciferase expression in mouse skeletal muscle after intramuscular injection of capped modified mRNA either naked or formulated with tetrafunctional PPO-POE amphilic block copolymer of 7423 g/mol. Luciferase activity 24 hours after intramuscular injection of 5 μg mRNA formulated with the block copolymer at various concentrations ranging from 5 to 1000.Math.10.sup.−4% (w/v). Each column represents the mean+/−SEM of at least six individual values. The column “naked” relates to the expression of luciferase after administration of the same mRNA but without the block copolymer. The column “ref” relates to the expression of luciferase after administration of the same mRNA in combination with the tetrafunctional block copolymer 704. when administered at a concentration of about 20.Math.10.sup.−4% (w/v).

[0317] FIG. 9: Luciferase expression in mouse skeletal muscle after intramuscular injection of capped modified mRNA either naked or formulated with tetrafunctional PLA-POE amphilic block copolymer of 8996 g/mol. Luciferase activity 24 hours after intramuscular injection of 5 μg mRNA formulated with the block copolymer at 10 and 100.Math.10.sup.−4% (w/v). Each column represents the mean+/−SEM of at least six individual values. The column “naked” relates to the expression of luciferase after administration of the same mRNA but without the block copolymer.

[0318] FIG. 10: Luciferase expression in mouse skeletal muscle after intramuscular injection of capped modified mRNA either naked or formulated with tetrafunctional POE-PPO amphilic block copolymer of 7332 g/mol. Luciferase activity 24 hours after intramuscular injection of 5 μg mRNA formulated with the block copolymer at 10 and 100.Math.10.sup.−4% (w/v). Each column represents the_mean+/−SEM of at least six individual values. The column “naked” relates to the expression of luciferase after administration of the same mRNA but without the block copolymer.

[0319] FIG. 11A-11C: Mouse hematocrit and EPO levels as a function of time after injection of block copolymer formulations. FIG. 11A Mouse hematocrit as a function of time after intramuscular injection of 704/RNA formulations containing 20 μg mRNA encoding murine EPO and 10×10.sup.−4% (open circles), 20×10.sup.−4% (open squares) or 100×10.sup.−4% (open triangles) 704. As control, mice were also injected with 20 μg of naked RNA encoding murine EPO (solid circles). As control a group of mice was also uninjected (solid squares). FIG. 11B mouse hematocrit as a function of time after intramuscular injection of 904/RNA formulations containing 20 μg mRNA encoding murine EPO and 10×10.sup.−4% 4 (open circles), 20×10.sup.−4% (open squares) or 100×10.sup.−4% (open triangles) 904. As control, mice were also injected with 20 μg of naked RNA encoding murine EPO (solid circles). As control a group of mice was also uninjected (solid squares). FIG. 11C mouse EPO as a function of time after intramuscular injection of 704/RNA formulations containing 20×10.sup.−4% 704 and various amounts of mRNA encoding murine including 1 (open circles), 5 (open diamonds), 10 (open triangles) and 50 μg (open octagon). As control, mice were also injected with 1 (solid circles), 5 (solid diamonds), 10 (solid triangles) and 50 μg (solid octagon) of naked RNA encoding murine EPO. As control a group of mice was also uninjected (solid squares, dotted line). FIG. 11D Hematocrit level as a function of time of the same described in FIG. 11C. After 10 hours, mouse EPO levels were measured in serum FIG. 11E and in the muscles FIG. 11F of mice injected intramuscularly either with plasmid DNA encoding murine EPO formulated with 704 (solid symbols) or with mRNA formulated with 704 (empty symbols). Each symbol represents the mean+/−SEM of at least 6 individual mice.

[0320] FIG. 12: β-galactosidase expression after intramuscular injection of uncapped modified mRNA in combination with block copolymers of the invention. The y-axis represents the β-galactosidase expression in cps. The x-axis represents from left to right, the datasets corresponding to block copolymers 704; 10257; 3648; 1614 and 7426.

[0321] FIG. 13: Humoral response in mice at day 35 after 2 intramuscular injections of capped unmodified mRNA at day 0 and 21, in the presence of block copolymers 904 and 10257. The y-axis represents the mean antibody titer determined ELISA of at least six individual mice. The x-axis represents from left to right the datasets corresponding to naked mRNAs, block copolymers 904 an 10257. For each dataset, the column on the left represents DNA, and the column on the right RNA.

EXAMPLES

Material and Methods

Nucleic Acids Molecules

[0322] mRNA either capped or not and fully substituted or not for every U or C by Pseudo-U and 5-methyl-C respectively, encoding β-galactosidase, luciferase, Erythropoietin (EPO) were purchased at Trilink (San Diego, USA). The plasmid containing the murine EPO cDNA under the control of the cytomegalovirus (CMV) IE1 promoter/enhancer was constructed by recovering mEPO cDNA by PCR from plasmid pTetO-mEPO (Richard et al., Human Gene Therapy 2005) and introduced into the pcDNA-3 vector (Invitrogen, Cergy Pontoise, France). The pCMV-bGal plasmid (Clontech, St Germain en Laye, France) coding for b-galactosidase controlled by the human cytomegalovirus immediate-early gene promoter was used as antigen. Plasmids were purified from transformed recombinant Escherichia coli by means of EndoFree plasmid purification columns (Qiagen, Chatsworth, Calif., EISA).

Animals Experiments and Nucleic Acids Formulations

[0323] All animal experiments were performed in accordance with the guidelines of the French Institut National de la Santé et de la Recherche Médicale. Eight-week old female Swiss and C57bl/6 mice were obtained from Janvier (Le Genest Saint Isle, France). At least six to eight mice were injected in each experimental group. For intramuscular injections, mice were anaesthetized. Fifty microliters of synthetic formulations were injected into shaved tibial anterior muscles at a single site, using a microfine syringe (U100, Becton Dickinson, Rungis, France). Stock solutions of block copolymers were prepared at 2% (w/v) in water and stored at 4° C. Formulations of DNA and mRNA with block copolymer were prepared by equivolumetric mixing of block copolymer in water at the desired concentration with plasmid DNA solution at the desired concentration in buffer.

Cell Culture

[0324] Hela, C2C12, JAW II were grown at 37° C., 5% CO2 in Dublecco's modified Eagles medium supplementaed with penicillin, streptomycine, L glutamine and 10% fetal calf serum. One day before transfection, cells were plated in 1 mL complete growth medium so that cells reach 70-80% confluence at the time of transfection (0.5-2×10.sup.5 cells per well). One day after transfection, cells were harvested and Reporter Lysis Buffer (Promega) supplemented with a protease inhibitor cocktail (Roche Diagnostics) was added to each wells. After centrifugation at 10,000 rpm for 4 min, luciferase activity was measured from an aliquot of supernatant with Victor.sup.2 (PerkinElmer), using a Luciferase Assay System (Promega). Luciferase activity was determined by measuring the light emission after addition of 100 μl of luciferase assay substrate to 10 μl of supernatant.

EPO Expression Analysis

[0325] Hematocrit values were measured by microcapillary centrifugation. At different time points after intramuscular injection, mouse blood was collected from the retro-orbital cavity and serum obtained by centrifugation (3 minutes at 1000 g). For plasma samples, blood was collected from the retro-orbital sinus in heparinized tubes and centrifuged 3 minutes at 1000 g. Mouse serum EPO levels were measured by Enzyme Linked-ImmunoAssay (ELISA) following the instructions provided by the manufacturer (R&D Systems).

Anti-Murine EPO Specific Immune Response

[0326] Humoral immune responses were measured by ELISA. Briefly, 96-well plates (Nunc Maxisorp) were coated overnight at 4° C. with recombinant murine EPO in 50 mM NaHCO.sub.3 pH 9.5, then blocked for 1 hour at room temperature with PBS 0.05% Tween-20 1% bovine serum albumin (BSA) before distributing diluted sera in triplicate. Plates were incubated at 37° C. for 90 minutes, then EPO specific IgG was detected using peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch, Newmarket, UK) diluted 1/5000 in PBS 0.05% Tween-20 1% BSA. Plates were washed three times in PBS 0.05% Tween-20 between steps, and peroxidase activity was revealed with 1 mg/mL OPD in pH5 citrate buffer. Reactions were stopped by addition of 1M H.sub.2SO.sub.4, then absorption was measured at 492 nm. Sera were tested at 1/100, 1/1000 and 1/10000, and anti-murine EPO antibody amount was calculated with respect to a standard curve consisting of fixed known amounts of increasing anti-murine EPO commercially available antibodies present in each ELISA plate.

Luciferase Expression

[0327] Luciferase protein expression was evaluated by live animal imaging using a PhotonIMAGER Optima system (http://www.biospacelab.com). Briefly, 2 mg of in-vivo luciferase substrate (beetle luciferin substrate, Promega) was injected intraperitoneally in mice and after 10 minutes, mice were anesthetized and luminescent signal will be measured until the baseline was stable. After stabilization of the luminescent signal, measurement of the luminescent was performed for 30s.

β-Gal Expression

[0328] β-Gal expression was quantified in muscle extracts using the BetaGlo Assay System (Promega, Charbonnieres, France) according to the manufacturer's protocol.

Anti-β-Gal Specific Immune Response

[0329] Humoral immune responses were measured by ELISA. Briefly, 96-well plates (Nunc Maxisorp) were coated overnight at 4° C. with recombinant bGal in 50 mM NaHCO.sub.3 pH 9.5, then blocked for 1 hour at room temperature with PBS 0.05% Tween-20 1% bovine serum albumin (BSA) before distributing diluted sera in triplicate. Plates were incubated at 37° C. for 90 minutes, then bGal specific IgG was detected using peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch, Newmarket, UK) diluted 1/5000 in PBS 0.05% Tween-20 1% BSA. Plates were washed three times in PBS 0.05% Tween-20 between steps, and peroxidase activity was revealed with 1 mg/mL OPD in pH5 citrate buffer. Reactions were stopped by addition of 1M H.sub.2SO.sub.4, then absorption was measured at 492 nm. Sera were tested at 1/100, 1/1000 and 1/10000, and titres were calculated with respect to doubling dilutions of a control serum present in each ELISA plate.

[0330] To measure the percentage of CD8 cell expressing IFNg in the total of splenic CD8 cells, splenocytes were cultured at 5×10.sup.6 cells/mL in complete medium. A murine dendritic cell line (JAWS) was transfected with ICAFectin®441 with plasmid DNA encoding either b-galactosidase or murine AlphaFetoprotein, and cells were incubated at 37° C. and 5% C02. Cells were harvested at 24 hours, then stained with an anti-CD8 antibody and anti-IFNγ and quantified by FACS.

Protocols for the Functionalization of Block Copolymers 704 at their Terminal Blocks

I— Preparation of 704-Me

[0331] 704 (1.07 g, 0.19 mmol, 1 eq.) was dried for 30 min under vacuum, and then dissolved in dry THF (25 mL). At 0° C., NaH (95%, 56 mg, 2.33 mmol, 12 eq.) was added and the mixture was stirred for 30 min at rt. Iodomethane (0.14 mL, 2.33 mmol, 12 eq.) was then added and the mixture stirred at rt overnight. After concentration, the residue was purified by flash chromatography (DCM/MeOH) to give 704-Me (0.93 g, 88%).

II— Preparation of 704-NH.SUB.2

[0332] To a solution of 704 (4.7 g, 0.85 mmol, 1 eq.) in DCM (120 mL) was added p-toluenesulfonyl chloride (1.95 g, 10.25 mmol, 12 eq.). Powdered KOH (0.77 g, 13.67 mmol, 16 eq.) was then added portionwise over 30 min and the mixture was stirred at rt for 2 days. DCM (100 mL) was added and the mixture washed with H.sub.2O, brine, dried over MgSO.sub.4 and concentrated. The residue was purified by flash chromatography (DCM/MeOH) to give 704-Tos (4.37 g, 84%).

[0333] For reference, 704-Tos is of formula:

##STR00035##

wherein TsO refers to a tosyl group.

[0334] To a solution of 704-Tos (4.37 g, 0.71 mmol, 1 eq.) in EtOH (100 mL) was added sodium azide (1.16 g, 17.85 mmol, 25 eq.). The mixture was refluxed for 20 h. After cooling to rt, volatiles were evaporated. The residue was taken up with DCM (100 mL), washed with NaHCO.sub.3 sat, H.sub.2O, brine, dried over MgSO.sub.4 and concentrated. The residue was purified by flash chromatography (DCM/MeOH) to give 704-N.sub.3 (3.30 g, 82%).

[0335] For reference, 704-N.sub.3 is of formula:

##STR00036##

[0336] To a solution of 704-N.sub.3 (3.30 g, 0.58 mmol, 1 eq.) in EtOH (60 mL) was added Pd/C (10%, 0.75 g, 0.11 mmol, 0.2 eq.). 3 cycles of vacuum/N.sub.2 were applied, followed by 3 cycles of vacuum/H.sub.2. The mixture was stirred at rt for 2 days, then filtered over a pad of celite, washed with MeOH and concentrated. The residue was purified by flash chromatography (DCM/MeOH) to give 704-NH.sub.2 (2.81 g, 88%).

III— Preparation of 704-NOx

[0337] To a solution of 704-NH.sub.2 (0.2 g, 0.036 mmol, 1 eq.) in DCM (20 mL) were successively added Et.sub.3N (0.06 mL, 0.36 mmol, 10 eq.) and succinic anhydride (0.036 g, 0.36 mmol, 10 eq.). The mixture was stirred at rt overnight, then washed with HCl 1M, H.sub.2O, dried over MgSO.sub.4 and concentrated. The residue was purified by flash chromatography (DCM/MeOH) to give 704-NOx (0.185 g, 87%).

IV— Preparation of 704-Paromo

[0338] To a solution of 704-NOx (0.185 g, 0.031 mmol, 1 eq.) in DMF (15 ml) were successively added Paromo(Teoc)-NH.sub.2 (0.224 g, 0.188 mmol, 6 eq.), HBTU (0.083 g, 0.220 mmol, 7 eq.) and DMAP (0.053 g, 0.440 mmol, 14 eq.). The mixture was stirred at 50° C. for 48h, then concentrated and purified by flash chromatography (DCM/MeOH) to give 704-Paromo(Teoc) (0.149 g, 48%).

[0339] For reference, 704-Paromo(Teoc) is of formula:

##STR00037##

[0340] To a solution of 704-Paromo(Teoc) (0.149 g, 0.014 mmol) in DCM (3 mL) was added trifluoroacetic acid (4 mL) at 0° C. After 30 min at 0° C., the mixture was stirred for 1h at rt, then concentrated. The residue was purified by flash chromatography (DCM/MeOH) to give 704-Paromo (0.042 g, 39%).

V— Preparation of 704-OOx

[0341] To a solution of 704 (2 g, 0.36 mmol, 1 eq.) in pyridine (15 mL) was added succinic anhydride (0.36 g, 3.63 mmol, 10 eq.). The mixture was stirred at 55° C. overnight, and then concentrated. The residue was taken up with EtOAc (100 mL), washed with HCl 1M, H.sub.2O, brine, dried over MgSO.sub.4 and concentrated. The residue was purified by flash chromatography (DCM/MeOH) to give 704-OOx (1.78 g, 84%).

Example 1: In Vivo Transfection of Skeletal Muscles and Immunogenicity, Using Different mRNA Structures and Sequences

[0342] Purpose: this experiment provides a comparative study of the influence of mRNA capping and nucleotide modification on protein expression on a C57BI6 skeletal muscle, and also to assess the importance of immune reaction after injection.

[0343] As shown from figures A1-1D, transfection of a mRNA encoding a β-galactosidase using the tetrafunctional block copolymer 704 as a vehicle allows both (i) efficient protein expression and (ii) minimal immune reaction.

Example 2: In Vitro Transfection of Cultured Cells, Using Different mRNA Structures and Sequences

[0344] Purpose: this experiment (see FIG. 2) provides evidence that aminoglycoside lipid derivatives are not so satisfactory for mRNA transfection on three different cell lines

Example 3: Secretion of Murine Erythropoietin

[0345] Purpose: this experiment provides a follow-up, over 20 days, of the injection of an mRNA coding for murine EPO using block copolymer 704 as a vehicle (see FIGS. 3A-3C).

Example 4: Repeated mRNA and DNA Injection and Mouse Murine EPO Expression

[0346] Purpose: this experiment provides a follow-up, over 180 days, of the injection of an mRNA coding for murine EPO using block copolymers 704 or 904 as vehicles, and with repeated mRNA injections. A comparative study is further provided which shows the efficiency of block copolymers 704 and 904 as vehicles for RNA transfection (see FIGS. 4A-4D).

Example 5: Influence of the Medium of Complexation on Luciferase Expression

[0347] Purpose: using Luciferase as a reporter gene, this comparative study provides good evidence that Tyrode's medium and equivalents are endowed with excellent properties regarding RNA transfection using block copolymers of the invention (see FIG. 5).

Example 6: Influence of the Concentration of 704 on Transfection Efficiency

[0348] Purpose: This comparative study provides evidence that block copolymers of the invention are very efficient for transfection of RNA molecules even at low concentrations of block copolymers (see FIG. 6).

Example 7: Influence of the Concentration of a Tetrafunctional PEO-PPO Non-Ionic Amphiphilic Block Copolymer of 14463 g/Mol on Transfection Efficiency

[0349] Purpose: This comparative study provides evidence of the efficiency of a block copolymer of general formula:

##STR00038##

[0350] Indeed, it is observed that block copolymers of the invention are very efficient for transfection of RNA molecules even at low concentrations of block copolymers (see FIG. 7) after intramuscular administration in mouse skeletal muscle. What is more, it has been found that the expression of luciferase after administration of this particular block copolymer is also significantly higher than the expression after administration of the tetrafunctional block copolymer 704.

Example 8: Influence of the Concentration of a Tetrafunctional PEO-PPO Non-Ionic Amphiphilic Block Copolymer of 7423 g/Mol on Transfection Efficiency

[0351] Purpose: This comparative study provides evidence of the efficiency of a block copolymer of general formula:

##STR00039##

[0352] Indeed, it is observed that block copolymers of the invention are very efficient for transfection of RNA molecules even at low concentrations of block copolymers (see FIG. 8) after intramuscular administration in mouse skeletal muscle. What is more, it has been found that the expression of luciferase after administration of this particular block copolymer is also significantly higher than the expression after administration of the tetrafunctional block copolymer 704.

Example 9: Influence of the Concentration of a Tetrafunctional PLA-POE Non-Ionic Amphiphilic Block Copolymer of 8996 g/Mol on Transfection Efficiency

[0353] Purpose: This comparative study provides evidence of the efficiency of a block copolymer of general formula:

##STR00040##

[0354] Indeed, it is observed that block copolymers of the invention are very efficient for transfection of RNA molecules even at low concentrations of block copolymers (see FIG. 9) after intramuscular administration in mouse skeletal muscle.

Example 10: Influence of the Concentration of a Tetrafunctional POE-PPO Non-Ionic Amphiphilic Block Copolymer of 7332 g/Mol on Transfection Efficiency

[0355] Purpose: This comparative study provides evidence of the efficiency of a block copolymer of general formula:

##STR00041##

[0356] Indeed, it is observed that block copolymers of the invention are very efficient for transfection of RNA molecules even at low concentrations of block copolymers (see FIG. 10) after intramuscular administration in mouse skeletal muscle.

Example 11: In Vivo Effect of an Intramuscular Administration of Block Copolymers Formulations with an mRNA Encoding EPO, on the Level of Hematocrit in Mice

[0357] Purpose: This study provides evidence of the variation of EPO and hematocrit in mice over time, after intramuscular administration in mice (see FIGS. 11A-11F).

Example 12: Influence of Block Copolymers of the Invention as Vehicles for Uncapped Modified mRNAs

[0358] Purpose: This study provides evidence that block copolymers of the invention are particularly efficient for promoting the expression of uncapped modified mRNAs in an eukaryotic host (see FIG. 12).

[0359] β-galactosidase activity one day after intramuscular injection of 15 μg uncapped modified mRNA encoding the β-galactosidase formulated with 704 at 20×10.sup.−4% as reference and 10257 at 100×10.sup.−4%, 3648 at 10×10.sup.−4%, 1614 at 20×10.sup.−4% and 7426 at 100×10.sup.−4%.

[0360] The modified RNAs which were used were modified on all Uracile and Cytosine bases, respectively using pseudouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate nucleotides. Twenty four hours after injection, muscles were harvested and frozen in liquid nitrogen. Beta gal expression was assessed with the help of beta-Glo assay system following manufacturer's instructions (Promega #E4720) in pure muscle extract. The results show that block copolymers 10257, 3648, 1614 and 7426 are particularly efficient as vehicles, even in comparison to the 704 block copolymer.

Example 13

[0361] Purpose: This study provides evidence of the lack of immune response after administration of RNA molecules in combination with block copolymers of the invention (see FIG. 13). Mice were injected with 20 μg of plasmid DNA or capped unmodified mRNA encoding beta-galactosidase either naked or formulated with tetrafunctional block copolymer 904 or 10257. Each column represents the mean antibody titer determined by ELISA of at least six individual mice. It is observed that said block copolymers are particularly efficient for intracellular delivery of RNA molecules and for gene therapy.

TABLE-US-00001 SEQUENCE LISTING SEQ ID No 1: nucleic acid coding for β-galactosidase E. Coli ATGTCGTTTACTTTGACCAACAAGAACGTGATTTTCGTTGCCGGTCTGGGA GGCATTGGTCTGGACACCAGCAAGGAGCTGCTCAAGCGCGATCCCGTCGTT TTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTT GCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCC TGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTT CCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTAC GATGCGCCCATCTACACCAACGTAACCTATCCCATTACGGTCAATCCGCCG TTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTT GATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTT AACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAG GACAGTCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGA GAAAACCGCCTCGCGGTGATGGTGCTGCGTTGGAGTGACGGCAGTTATCTG GAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTG CTGCATAAACCGACTACACAAATCAGCGATTTCCATGTTGCCACTCGCTTT AATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGC GAGTTGCGTGACTACCTACGGGTAACAGTTTCTTTATGGCAGGGTGAAACG CAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGT GGTGGTTATGCCGATCGCGTCACACTACGTCTGAACGTCGAAAACCCGAAA CTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCGGTGGTTGAACTGCAC ACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGATGTCGGTTTCCGC GAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTG ATTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATG GATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTT AACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTG TGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCAC GGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCGGCG ATGAGCGAACGCGTAACGCGAATGGTGCAGCGCGATCGTAATCACCCGAGT GTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGAC GCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGCAGTAT GAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTAC GCGCGCGTGGATGAAGACCAGCCCTTCCCGGCTGTGCCGAAATGGTCCATC AAAAAATGGCTTTCGCTACCTGGAGAGACGCGCCCGCTGATCCTTTGCGAA TACGCCCACGCGATGGGTAACAGTCTTGGCGGTTTCGCTAAATACTGGCAG GCGTTTCGTCAGTATCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTG GATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTAC GGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGT CTGGTCTTTGCCGACCGCACGCCGCATCCAGCGCTGACGGAAGCAAAACAC CAGCAGCAGTTTTTCCAGTTCCGTTTATCCGGGCAAACCATCGAAGTGACC AGCGAATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACTGGATGGTG GCGCTGGATGGTAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTCGCT CCACAAGGTAAACAGTTGATTGAACTGCCTGAACTACCGCAGCCGGAGAGC GCCGGGCAACTCTGGCTCACAGTACGCGTAGTGCAACCGAACGCGACCGCA TGGTCAGAAGCCGGGCACATCAGCGCCTGGCAGCAGTGGCGTCTGGCGGAA AACCTCAGTGTGACGCTCCCCGCCGCGTCCCACGCCATCCCGCATCTGACC ACCAGCGAAATGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTT AACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATAAAAAACAA CTGCTGACGCCGCTGCGCGATCAGTTCACCCGTGCACCGCTGGATAACGAC ATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGC TGGAAGGCGGCGGGCCATTACCAGGCCGAAGCAGCGTTGTTGCAGTGCACG GCAGATACACTTGCTGATGCGGTGCTGATTACGACCGCTCACGCGTGGCAG CATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGT AGTGGTCAAATGGCGATTACCGTTGATGTTGAAGTGGCGAGCGATACACCG CATCCGGCGCGGATTGGCCTGAACTGCCAGCTGGCGCAGGTAGCAGAGCGG GTAAACTGGCTCGGATTAGGGCCGCAAGAAAACTATCCCGACCGCCTTACT GCCGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCG TACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAAT TATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTAC AGTCAACAGCAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAA GAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGAC GACTCCTGGAGCCCGTCAGTATCGGCGGAATTACAGCTGAGCGCCGGTCGC TACCATTACCAGTTGGTCTGGTGTCAAAAATAA SEQ ID No 2: β-galactosidase protein sequence E. Coli MSFTLTNKNVIFVAGLGGIGLDTSKELLKRDPVVLQRRDWENPGVTQLNRL AAHPPFASWRNSEEARTDRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDL PEADTVVVPSNWQMHGYDAPIYTNVTYPITVNPPFVPTENPTGCYSLTFNV DESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYGQDSRLPSEFDLSAFLRAG ENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHKPTTQISDFHVATRF NDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIIDER GGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFR EVRIENGLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNF NAVRCSHYPNHPLWYTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPA MSERVTRMVQRDRNHPSVIIWSLGNESGHGANHDALYRWIKSVDPSRPVQY EGGGADTTATDIICPMYARVDEDQPFPAVPKWSIKKWLSLPGETRPLILCE YAHAMGNSLGGFAKYWQAFRQYPRLQGGFVWDWVDQSLIKYDENGNPWSAY GGDFGDTPNDRQFCMNGLVFADRTPHPALTEAKHQQQFFQFRLSGQTIEVT SEYLFRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIELPELPQPES AGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPHLT TSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQFTRAPLDND IGVSEATRIDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLITTAHAWQ HQGKTLFISRKTYRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVAER VNWLGLGPQENYPDRLTAACFDRWDLPLSDMYTPYVFPSENGLRCGTRELN YGPHQWRGDFQFNISRYSQQQLMETSHRHLLHAEEGTWLNIDGFHMGIGGD DSWSPSVSAELQLSAGRYHYQLVWCQK SEQ ID No 3: RIG-I helicase Homo Sapiens MTTEQRRSLQAFQDYIRKTLDPTYILSYMAPWFREEEVQYIQAEKNNKGPM EAATLFLKFLLELQEEGWFRGFLDALDHAGYSGLYEAIESWDFKKIEKLEE YRLLLKRLQPEFKTRIIPTDIISDLSECLINQECEEILQICSTKGMMAGAE KLVECLLRSDKENWPKTLKLALEKERNKFSELWIVEKGIKDVETEDLEDKM ETSDIQIFYQEDPECQNLSENSCPPSEVSDTNLYSPFKPRNYQLELALPAM KGKNTIICAPTGCGKTFVSLLICEHHLKKFPQGQKGKVVFFANQIPVYEQQ KSVFSKYFERHGYRVTGISGATAENVPVEQIVENNDIIILTPQILVNNLKK GTIPSLSIFTLMIFDECHNTSKQHPYNMIMFNYLDQKLGGSSGPLPQVIGL TASVGVGDAKNTDEALDYICKLCASLDASVIATVKHNLEELEQVVYKPQKF FRKVESRISDKFKYIIAQLMRDTESLAKRICKDLENLSQIQNREFGTQKYE QWIVTVQKACMVFQMPDKDEESRICKALFLYTSHLRKYNDALIISEHARMK DALDYLKDFFSNVRAAGFEEIEQDLTQRFEEKLQELESVSRDPSNENPKLE DLCFILQEEYHLNPETITILFVKTRALVDALKNWIEGNPKLSFLKPGILTG RGKTNQNTGMTLPAQKCILDAFKASGDHNILIATSVADEGIDIAQCNLVIL YEYVGNVIKMIQTRGRGRARGSKCFLLTSNAGVIEKEQINMYKEKMMNDSI LRLQTWDEAVFREKILHIQTHEKFIRDSQEKPKPVPDKENKKLLCRKCKAL ACYTADVRVIEECHYTVLGDAFKECFVSRPHPKPKQFSSFEKRAKIFCARQ NCSHDWGIHVKYKTFEIPVIKIESFVVEDIATGVQTLYSKWKDFHFEKIPF DPAEMSK SEQ ID No 4: EPO mus musculus ATGGGGGTGCCCGAACGTCCCACCCTGCTGCTTTTACTCTCCTTGCTACTG ATTCCTCTGGGCCTCCCAGTCCTCTGTGCTCCCCCACGCCTCATCTGCGAC AGTCGAGTTCTGGAGAGGTACATCTTAGAGGCCAAGGAGGCAGAAAATGTC ACGATGGGTTGTGCAGAAGGTCCCAGACTGAGTGAAAATATTACAGTCCCA GATACCAAAGTCAACTTCTATGCTTGGAAAAGAATGGAGGTGGAAGAACAG GCCATAGAAGTTTGGCAAGGCCTGTCCCTGCTCTCAGAAGCCATCCTGCAG GCCCAGGCCCTGCTAGCCAATTCCTCCCAGCCACCAGAGACCCTTCAGCTT CATATAGACAAAGCCATCAGTGGTCTACGTAGCCTCACTTCACTGCTTCGG GTACTGGGAGCTCAGAAGGAATTGATGTCGCCTCCAGATACCACCCCACCT GCTCCACTCCGAACACTCACAGTGGATACTTTCTGCAAGCTCTTCCGGGTC TACGCCAACTTCCTCCGGGGGAAACTGAAGCTGTACACGGGAGAGGTCTGC AGGAGAGGGGACAGGTGA SEQ ID No 5: Firefly luciferase ATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATGTCCG TTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAA TCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCG CGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAAC GTAAGCACCCTCGCCATCAGACCAAAGGGAATGACGTATTTAATTTTTAAG GTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTT CCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAA TCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGT CGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACG ATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGA ATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAA CTGCCTGCGTCAGATTCTCGCATGCCAGGTATGTCGTATAACAAGAGATTA AGTAATGTTGCTACACACATTGTAGAGATCCTATTTTTGGCAATCAAATCA TTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAA TGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGT ATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAAAATTC AAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTC TGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCG CACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGGTGAGTTAAGC GCATTGCTAGTATTTCAAGGCTCTAAAACGGCGCGTAGCTTCCATCTTCCA GGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATT ACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTT TTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAAT CAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATGTA AACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACAT TCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGAC CGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGTAATGAAGATTTTT ACATGCACACACGCTACAATACCTGTAGGTGGCCCCCGCTGAATTGGAATC GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCC CGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGG AAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTAAATGAAT TCGTTTTACGTTACTCGTACTACAATTCTTTTCATAGGTCAAGTAACAACC GCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGT CTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCC AAGAAGGGCGGAAAGTCCAAATTGTAAAATGTAACTGTATTCAGCGATGAC GAAATTCTTAGCTATTGTAATATTATATGCAAATTGATGAATGGTAATTTT GTAATTGTGGGTCACTGTACTATTTTAACGAATAATAAAATCAGGTATAGG TAACTAAAAA