RNA for treatment of autoimmune diseases

Abstract

The present invention relates to non-immunogenic RNA. This RNA forms the basis for the development of therapeutic agents for inducing tolerance towards an autoantigen and thus, for the treatment of autoimmune diseases.

Claims

1. A method of treating an autoimmune disease in a subject, comprising administering to the subject a composition comprising a non-immunogenic RNA encoding a peptide or polypeptide comprising an autoantigen or a fragment thereof, or a variant of the autoantigen or fragment, wherein the non-immunogenic RNA comprises 1-methyl-pseudouridine, wherein the non-immunogenic RNA is formulated in a F12 liposome, and wherein the composition does not comprise dsRNA.

2. A method of inducing tolerance to autoreactive T cells in a subject, comprising administering to the subject a composition comprising a non-immunogenic RNA encoding a peptide or polypeptide comprising an autoantigen or a fragment thereof, or a variant of the autoantigen or fragment, wherein the non-immunogenic RNA comprises 1-methyl-pseudouridine, wherein the non-immunogenic RNA is formulated in a F12 liposome, and wherein the composition does not comprise dsRNA.

3. The method of claim 2, wherein the subject has an autoimmune disease.

4. The method of claim 2, wherein the non-immunogenic RNA when administered does not result in activation of dendritic cells, activation of T cells and/or secretion of IFN-alpha.

5. The method of claim 2, wherein the non-immunogenic RNA is mRNA or in vitro transcribed RNA.

6. The method of claim 2, wherein the autoantigen is a T cell-antigen, CNS-derived, a myelin antigen, or Myelin Oligodendrocyte Glycoprotein (MOG).

7. The method of claim 2, wherein the peptide or polypeptide comprising an autoantigen or a fragment thereof, or a variant of the autoantigen or fragment comprises amino acids 35 to 55 of Myelin Oligodendrocyte Glycoprotein (MOG).

8. The method of claim 2, wherein the non-immunogenic RNA is formulated in a delivery vehicle.

Description

FIGURES

(1) FIG. 1: Activation of splenic immune cells, cytokine release and expression of protein after administration of luciferase-RNA complexed with F12 liposomes. BALB/c mice (n=7) were injected intravenously into the retro-orbital plexus with 10 μg non-immunogenic LUC mRNA-LPX and investigated 24 hours later (A) for maturation status of dendritic cells (n=3) (revealed by upregulation of CD86), T cells, B cells and NK cells (revealed by upregulation of CD69). 6 hours after mRNA-LPX injection into BALB/c mice (B) serum concentration of IFNα was assessed. (C) Luciferase activity determination in vivo 6, 24, 48 and 72 h after mRNA-LPX-injection into BALB/c mice (n=4) of luciferase-RNA by in vivo bioluminescence imaging. Data are depicted as mean±SD from n=3 mice/group. LLOD=lower limit of detection.

(2) FIG. 2: Dotblot analysis of non-immunogenic MOG35-55 mRNA purified by either Cellulose treatment or HPLC purification. Exposure time 20 sec.

(3) FIG. 3: MOG35-55 antigen presentation by DCs induces tolerance to EAE. EAE was actively induced by immunization with MOG35-55-CFA (day 0) and Pertussis Toxin (days 0 and 2) in C57BL/6 mice. Non-immunogenic MOG35-55 coding mRNA, purified either by Cellulose or HPLC method, was injected intravenously into the retro-orbital plexus at day 7 and day 10 after EAE induction. Control mice received 150 mM NaCl. Data are depicted as mean. n=6 mice/group.

(4) FIG. 4: MOG35-55 antigen presentation by APCs induces antigen-specific T cell proliferation. Naïve, cell-trace-violet (CTV) labeled, Thy1.1.sup.+ 2D2.sup.+ CD4.sup.+ T cells were adoptively transferred to Thy1.2.sup.+ C57BL/6 control mice. At the next day, mice were immunized with 10, 20 or 40 μg of non-immunogenic MOG35-55 coding mRNA. Control mice were treated with 20 μg non-immunogenic irrelevant control mRNA. A second control group received furthermore 150 mM NaCl. 4 days later, mice were sacrificed and spleen was analyzed for proliferating Thy1.1.sup.+ cells. Data are depicted as mean±SD from n=3 mice/group.

(5) FIG. 5: In vitro suppression of MOG35-55 T cell proliferation by induced antigen-specific Treg cells. (A, B) 2D2 Foxp3-eGFP mice were immunized 4 times (d0, d3, d6, d9) with non-immunogenic MOG35-55 coding mRNA. Control mice were treated with immunogenic MOG35-55 coding mRNA or non-immunogenic irrelevant control mRNA. A third control group received furthermore 150 mM NaCl. 4 days after the last mRNA treatment mice were sacrificed and MOG35-55 specific CD4.sup.+ cells were analyzed for Foxp3-eGFP expression (C). Cells of each treatment group (suppressors) were co-cultured with CTV labeled 2D2 CD4.sup.+ T cells of untreated naïve mice (responder cells) and restimulated in vitro with MOG35-55 peptide-loaded BMDCs for 72 h. Proliferation of responder cells was determined by CTV dilution by FACS (gated on CTV.sup.+ cells). The inhibitory capacity of suppressor cells was measured at 4 different ratios to responder cells. Data are depicted as mean±SD from n=4 mice/group. Statistical analysis of frequency of CD4.sup.+ Foxp3.sup.+ cells was assessed via one-way ANOVA and Tukey's multiple comparison test, ***p≤0.001.

(6) FIG. 6: MOG35-55 antigen presentation by DCs induces tolerance to EAE. EAE was actively induced by immunization with MOG35-55-CFA (day 0) and Pertussis Toxin (days 0 and 2) in C57BL/6 mice. Non-immunogenic MOG35-55 coding mRNA, as well as non-immunogenic irrelevant mRNA were injected intravenously into the retro-orbital plexus at day 7 and day 10 after EAE induction. Control mice received 150 mM NaCl. Data are depicted as mean. n=6-8 mice/group. Statistical analysis of maximal EAE score of each mouse per group was determined via one-way ANOVA and Tukey's multiple comparison test. Area under curve (AUC) was used to determine statistical significance via one-way ANOVA and Tukey's multiple comparison test of the different EAE disease development curves.

(7) FIG. 7: MOG35-55 antigen presentation by DCs induces tolerance to EAE in a real therapeutic setting. EAE was actively induced by immunization with MOG35-55-CFA (day 0) and Pertussis Toxin (days 0 and 2) in C57BL/6 mice. Non-immunogenic MOG35-55 coding mRNA, as well as non-immunogenic irrelevant mRNA were injected intravenously into the retro-orbital plexus after mice reached an EAE score of 1-2. Control mice received 150 mM NaCl. Data are depicted as mean. n=6-9 mice/group. Statistical analysis of maximal EAE score of each mouse per group was assessed via one-way ANOVA and Tukey's multiple comparison test Area under curve (AUC) was used to determine statistical significance via one-way ANOVA and Tukey's multiple comparison test of the different EAE disease development curves.

(8) FIG. 8: Antigen presentation by DCs induces tolerance to EAE. EAE was actively induced by immunization with MOG35-55-CFA in C57BL/6 mice and mice were treated at day 7 and day 10 after EAE induction with 20 μg of non-immunogenic MOG35-55-coding mRNA or 150 mM NaCl. At day 16 after disease induction organs were taken from EAE afflicted and mRNA treated mice and were cultured in the presence of MOG35-55 peptide for 6 h in the presence of Brefeldin A. The expression of CD40L was examined by intracellular cytokine staining of CD4+CD44.sup.+ T cells. Data are depicted as mean±SD from n=4 mice/group. Statistical analysis of CD4.sup.+ Teff cells was assessed via one-way ANOVA and Tukey's multiple comparison test, *p≤0.05.

(9) FIG. 9: MOG35-55 peptide specific Th1 and Th17 cells are significantly reduced in therapeutically treated mice (day 7/day 10). EAE was actively induced by immunization with MOG35-55-CFA in C57BL/6 mice and mice were treated at day 7 and day 10 after EAE induction with 20 μg of non-immunogenic MOG35-55-coding mRNA or 150 mM NaCl. Brain and spinal cord were harvested at day 16 after EAE induction and cells restimulated in vitro in the presence of MOG35-55 peptide for 6 h in the presence of Brefeldin A. CD4.sup.+ T cells were then analyzed for their expression of IFNγ and IL-17A by intracellular cytokine staining and percentages of IFNγ.sup.+ and IL-17A.sup.+ CD4.sup.+ T cells were calculated. Data are depicted as mean SD from n=4 mice/group. Statistical analysis was evaluated by one-way ANOVA and Tukey's multiple comparison test, *p≤0.05.

(10) FIG. 10: Activation of splenic immune cells and cytokine release after administration of different MOG35-55-mRNA mixes of non-immunogenic (m1Y) and immunogenic (U) mRNA complexed with F12 liposomes. C57BL/6 mice were injected intravenously into the retro-orbital plexus with 20 μg total MOG35-55 mRNA-LPX and investigated 24 hours later for maturation status of dendritic cells (revealed by upregulation of CD86), T cells and NK cells (revealed by upregulation of CD69). 6 hours after mRNA-LPX injection into C57BL/6 mice serum concentration of IFNα was assessed. Data are depicted as mean±SD from n=3 mice/group.

(11) FIG. 11: Dotblot analysis of different MOG35-55-mRNA mixtures of non-immunogenic and immunogenic mRNA. Exposure time 20 sec. Dashed line is set as 100% of Dot-intensity for 320 μg of dsRNA.

(12) FIG. 12: Non-immunogenic MOG35-55 mRNA induces full tolerance to EAE. EAE was actively induced by immunization with MOG35-55-CFA (day 0) and Pertussis Toxin (days 0 and 2) in C57BL/6 mice. Different mixtures of non-immunogenic (m1Y) and immunogenic (U) MOG35-55 coding mRNAs were injected intravenously into the retro-orbital plexus at day 7 and day 10 after EAE induction. Control mice received 150 mM NaCl. Data are depicted as mean. n=6-8 mice/group. Statistical analysis of maximal EAE score of each mouse per group was assessed via one-way ANOVA and Tukey's multiple comparison test, **p≤0.01.

(13) FIG. 13: Nucleoside-modification alone does not induce a loss of recognition. Activation of splenic immune cells and cytokine release after administration of different nucleoside-modified mRNAs complexed with F12 liposomes. C57BL/6 mice were injected intravenously into the retro-orbital plexus with 10 μg total mRNA-LPX and investigated 24 hours later for maturation status of dendritic cells (revealed by upregulation of CD86), T cells and NK cells (revealed by upregulation of CD69). 6 hours after mRNA-LPX injection into C57BL/6 mice serum concentration of IFNα was assessed. Data are depicted as mean±SD from n=3 mice/group.

(14) FIG. 14: Non-immunogenic MOG35-55 coding mRNA-LPX treatment does not lead to expansion of antigen-specific CD4.sup.+ effector cells. Naïve C57BL/6 mice were treated at day 0, 3, 7 and 10 with 20 μg non-immunogenic MOG35-55 coding mRNA. Control mice were treated with 20 μg non-immunogenic irrelevant control mRNA. A second control group received 150 mM NaCl. At day 13 after the first mRNA treatment, mice were sacrificed and splenocytes were analyzed for secretion of pro- and anti-inflammatory cytokines by IFNγ ELISpot (A) and detection of cytokines by Multiplex ELISA in the supernatant of MOG35-55-peptide restimulated cells (B and C). Data are depicted as mean±SD from n=4-5 mice/group.

(15) FIG. 15: Co-inhibitory molecules are upregulated upon treatment with non-immunogenic MOG35-55 coding mRNA-LPX. Naïve C57BL/6 mice were treated at day 0, 3, 7 and 10 with 20 μg non-immunogenic MOG35-55 coding mRNA. Control mice were treated with 20 μg non-immunogenic irrelevant control mRNA. Furthermore, a second control group received 150 mM NaCl. At day 13 after the first mRNA treatment, mice were sacrificed and MOG35-55 CD4.sup.+ T cells from the spleen were analyzed by flow cytometry for upregulation of co-inhibitory molecules. Data are depicted as mean±SD from n=4-5 mice/group. Statistical analysis was assessed via one-way ANOVA and Tukey's multiple comparison test.

(16) FIG. 16: Therapeutic vaccination with non-immunogenic MOG35-55 coding mRNA-LPX reduces infiltration of lymphocytes into the CNS. Naïve Thy1.1.sup.+ 2D2 CD4.sup.+ T cells were adoptively transferred into Thy1.2.sup.+ C57BL/6 mice at d-1. EAE was actively induced the day after by immunization with MOG35-55-CFA (day 0) and Pertussis Toxin (day 0 and 2) and mice were treated at day 7 and day 10 after EAE induction with 20 μg non-immunogenic MOG35-55-coding mRNA, 20 μg non-immunogenic irrelevant mRNA or 150 mM NaCl. At day 16 after disease induction organs were taken from differently treated mice. The infiltration of Thy1.1.sup.+ CD4.sup.+ MOG35-55 specific cells was examined and the total number of infiltrating cells was determined via flow cytometry using Trucount™ tubes. Data are depicted as mean±SD from n=4 mice/group. Statistical analysis of infiltrated CD4.sup.+ Teff cells was assessed via one-way ANOVA and Tukey's multiple comparison test.

(17) FIG. 17: MOG35-55 specific Th1 and Th17 cells are significantly reduced in therapeutically treated mice (day 7/day 10). EAE was actively induced by immunization with MOG35-55-CFA (day 0) and Pertussis Toxin (day 0 and 2) in C57BL/6 mice and mice were treated at day 7 and day 10 after EAE induction with 20 μg non-immunogenic MOG35-55-coding mRNA, 20 μg non-immunogenic irrelevant mRNA or 150 mM NaCl. Brain and spinal cord were harvested at day 16 after EAE induction and cells were restimulated in vitro in the presence of MOG35-55 peptide for 6 h in the presence of Brefeldin A. CD4.sup.+ T cells were then analyzed by flow cytometry for their expression of IFNγ and IL-17A by intracellular cytokine staining and percentages of IFNγ.sup.+ and IL-17A.sup.+ of CD4.sup.+ T cells were calculated. Data are depicted as mean±SD from n=4 mice/group. Statistical analysis was evaluated by one-way ANOVA and Tukey's multiple comparison test.

(18) FIG. 18: Co-inhibitory molecules are upregulated on MOG35-55-specific CD4.sup.+ T cells upon successful non-immunogenic MOG35-55-specific mRNA-LPX treatment EAE was actively induced by immunization with MOG35-55-CFA (day 0) and Pertussis Toxin (day 0 and 2) in C57BL/6 mice and mice were treated at day 7 and day 10 after EAE induction with 20 μg non-immunogenic MOG35-55-coding mRNA, 20 μg non-immunogenic irrelevant mRNA or 150 mM NaCl. At day 16 after disease induction spleens were dissected from differently treated groups. MOG35-55 specific CD4.sup.+ T cells were isolated by tetramer-specific MACS and phenotypically analyzed via flow cytometry by tetramer staining. Data are depicted as mean±SD from n=4 mice/group. Statistical analysis was evaluated by one-way ANOVA and Tukey's multiple comparison test.

(19) FIG. 19: PD-1 and CTLA-4 are required for maintenance of non-immunogenic mRNA induced antigen-specific tolerance. EAE was actively induced by immunization with MOG35-55-CFA (day 0) and Pertussis Toxin (day 0 and 2) in C57BL/6 mice and mice were treated at day 7 and day 10 after EAE induction with 20 μg non-immunogenic MOG35-55-coding mRNA or 20 μg non-immunogenic irrelevant mRNA (n=8 mice/group). mRNA treated mice were treated concomitantly with anti-PD-1 or anti-CTLA-4 blocking antibodies or corresponding IgG controls. EAE development was assessed by daily health monitoring. EAE area under curve (AUC) was used to determine statistical significance via one-way ANOVA and Tukey's multiple comparison test of the different disease development curves.

(20) FIG. 20: Antigen-specific tolerance induction by treatment with non-immunogenic antigen-specific mRNA is independent of the EAE disease model. (A) Therapeutic vaccination with non-immunogenic PLP139-151 coding mRNA-LPX induces tolerance to relapsing-remitting EAE. EAE was actively induced by immunization with PLP139-151-CFA (day 0) and Pertussis Toxin (day 0 and 2) in SJL mice. 20 μg non-immunogenic PLP139-151 coding mRNA as well as non-immunogenic irrelevant mRNA were injected i.v. into the retro-orbital plexus 2× per week starting from day 7 and day 10 after EAE induction. (B) Therapeutic vaccination with non-immunogenic multi-epitope coding mRNA-LPX induces tolerance in a complex EAE. EAE was actively induced by immunization with MOG35-55, PLP139-151, PLP178-191, MBP84-104 and MOBP15-36-CFA (day 0) and Pertussis Toxin (day 0 and 2) in F1 hybride mice from C57BL/6 and SJL/JRj. 40 μg non-immunogenic multi-epitope coding mRNA, 20 μg non-immunogenic MOG35-55 mRNA and 20 μg non-immunogenic irrelevant mRNA were injected i.v. into the retro-orbital plexus 2× per week starting from day 7 and day 10 after EAE induction. EAE development was assessed by daily health monitoring. Data are depicted as mean. n=6-8 mice/group. EAE area under curve (AUC) was used to determine statistical significance via one-way ANOVA and Tukey's multiple comparison test of the different disease development curves.

(21) FIG. 21: Cytokine co-delivery by non-immunogenic mRNA improves the tolerogenic potency of non-immunogenic antigen-specific mRNA-LPX treatment in EAE model. EAE was actively induced by immunization with MOG35-55-CFA (day 0) and Pertussis Toxin (day 0 and 2) in C57BL/6 mice and mice were treated at day 7 and day 10 after EAE induction with 5 μg non-immunogenic MOG35-55 mRNA together with 15 μg non-immunogenic mIL-10 (A) or mIL-27 (B) mRNA, respectively. Control mice received 5 μg non-immunogenic MOG35-55 mRNA together with 15 μg non-immunogenic irrelevant mRNA, 15 μg non-immunogenic cytokine-coding mRNA or 20 μg non-immunogenic irrelevant mRNA. EAE development was assessed by daily health monitoring. Data are depicted as mean. n=6-8 mice/group. EAE area under curve (AUC) was used to determine statistical significance via one-way ANOVA and Tukey's multiple comparison test of the different disease development curves.

EXAMPLES

Example 1: Material and Methods

(22) Animals

(23) C57BL/6, BALB/c, SJL/JRj wild-type mice and F1 hibrid mice from C57BL/6 and SJL/JRj were purchased from ENVIGO RMS gmbH, Netherlands and Janvier Laboratories, France respectively. Thy1.1.sup.+ 2D2 TCR MOG transgenic C57BL/6 mice, expressing a T cell receptor recognizing MOG35-55 in the context of MHC class II (I-A.sup.b), as well as Thy1.1.sup.+ 2D2 Foxp3-eGFP TCR MOG transgenic C57BL/6 mice that additionally include the knock-in Foxp3-eGFP, were kindly provided by the laboratory of Prof. Dr. Ari Waisman. Age and sex matched animals were used throughout the experiments and mice were maintained under specific pathogen-free (SPF) conditions at the animal facility of BioNTech AG Mainz.

(24) RNA Constructs and In Vitro Transcription

(25) In vitro transcription of LUC-mRNA were based on the pST1-hAg-CDS-FI-A30LA70 plasmid-backbone, which contains a 5′ human alpha globin UTR (hAg), a 3′ FI element and a poly(A) tail of 100 nucleotides, with a linker after 30 nucleotides. The LUC-construct contains the firefly luciferase gene. The MOG35-55, PLP139-151, PLP178-191 and MBP84-104 constructs were designed based on the same plasmid-backbone (pST1-hAg-CDS-FI-A30LA70) and additionally contained a mmsec(opt) and mmMITD(opt) sequence before and after the MOG35-55, PLP139-151, PLP178-191 and MBP84-104 coding sequences (pST1-hAg-mmsec(opt)-CDS-mmMITD(opt)-A30LA70), respectively. The addition of a leader peptide and a MHC trafficking signal (MITD) strongly improves antigen presentation by APCs (Kreiter, S. et al., 2007, J. Immunol. 180, 309-318). All antigen-sequences contain additionally seven flanking amino acids before and after the disease relevant epitope to facilitate antigen presentation. The antigen-sequence Myelin Oligodendrocyte Glycoprotein (MOG) has the peptide-sequence: MOG27-63: SPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEAQP (SEQ ID NO: 1). For the mouse myelin peptide Proteolipid Protein PLP139-151 also seven flanking amino acids were added for better peptide presentation (PLP131-159: AHSLERVCHCLGKWLGHPDKFVGITYALT; SEQ ID NO: 3). The Proteolipid Protein PLP178-191 includes the amino acids PLP170-199: AVPVYIYFNTWITCQSIAFPSKTSASIGSL (SEQ ID NO: 4) and the Myelin basic protein (MBP) 84-104 includes the amino acids MBP76-112: RTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFS (SEQ ID NO: 5). For control immunization with mRNA, empty vector was used, coding for no specific antigen-sequence (pST2-hAg-mmsec(opt)-emtpy-mmMITD(opt)-2hBg-A30LA70). Cytokine coding mRNAs for murine Interleukine 10 (mIL-10) and murine Interleukine 27 (mIL-27) were also synthesized based on the plasmid-backbone pST1-hAg-CDS-FI-A30LA70. RNA was generated by in vitro transcription as described by Kreiter et al., 2007 (Cancer Immunol. Immunother. 56, 1577-87). Purified mRNA was eluted in H.sub.2O and stored at −80° C. until further use. In vitro transcription of all described mRNA constructs was carried out by BioNTech RNA Pharmaceuticals GmbH.

(26) Generation of Non-Immunogenic mRNA

(27) To generate the non-immunogenic mRNA used, 1-methyl-pseudouridin was used during in vitro transcription instead of the normal nucleoside Uridin, and mRNA was further purified by HPLC or Cellulose treatment. For HPLC purification the protocol of Weissman et al., 2013 (Methods Mol Bio. 969, 43-43), was adapted and elution of the mRNA was performed with a gradient of 38%-70% of Buffer B. From all generated mRNAs quality controls were performed (Bioanalyser and Dot-Blot analysis) to ensure the purity and integrity of the mRNA.

(28) To generate the immunogenic mRNA used, the normal nucleoside Uridin was used for in vitro transcription and mRNA was not further purified by HPLC or Cellulose treatment.

(29) For investigating the influence of HPLC-purification or Cellulose-treatment on the activation of splenocytes, pseudouridine-modified mRNA (pU-mRNA) was used, that either received an additional HPLC purification (pU-HPLC mRNA) or not. The corresponding mRNA was generated as already described.

(30) Dotblot-Analysis for Quality Control of IVT mRNA

(31) In vitro transcribed, modified and HPLC-purified mRNA was analyzed for double-stranded mRNA (dsRNA) by Dotblot. 1 μg of different mRNA constructs were loaded on a NYTRAN SPC membrane (GE Healthcare), blocked and then incubated with J2 antibody (SCICONS English and Scientific Consulting) for the detection of dsRNA. As secondary antibody anti-mouse HRP antibody (Jackson ImmunoResearch) was used and membranes were analyzed with a BioRad ChemiDoc.

(32) Preparation and Injection of RNA-Lipoplexes

(33) RNA-lipoplexes (RNA-LPX) were prepared under sterile and RNase-free conditions. HEPES (10 mM)/EDTA (0.1 mM) was used to dilute the RNA to a concentration of 1 mg/ml. 1.5 M NaCl (5 M NaCl Stock; Ambion) were added to a specific volume of RNA to obtain a final concentration of 150 mM NaCl. After briefly vortexing, a specified volume of F12 liposomes was added and the RNA-liposome mixture (RNA-LPX) was again briefly vortexed and finally incubated for 10 min at RT to enable the formation of RNA-LPX (Kranz et al. 2016, Nature. 16, 396-401). 200 μl RNA-LPX solution was injected per mouse intravenously into the retro-orbital plexus of the mice.

(34) Flow Cytometric Analysis

(35) Fluorescence-activated cell sorting (FACS) surface and intracellular antibodies were purchased from eBioscience or BD Pharmingen and used in accordance with the manufacturer's protocol. Antibodies used were the following: CD11b, CD11c, CD19, CD25, CD4, CD44, CD40L, CD49b, CD69, CD8, CD86, CD90.1, Foxp3. ICOS, IFNγ, IL-17A, Lag-3, PD-1, TIGIT and Tim-3.

(36) Single cell suspensions from different organs were stained for 30 min at 4° C. for extracellular markers. For intracellular cytokine staining of IFNγ, IL-17A and CD40L, cells were isolated as described and additionally stimulated in culture medium containing MOG35-55 peptide (conc. 15 μg/ml), Monensin (GolgiStop, BD-Bioscience) and Brefeldin A at 37° C., 5% CO.sub.2 for 6 h. After performing live-dead staining (viability dye eFluor® 506, eBioscience) and staining of cell-surface markers, cells were fixed and permeabilized using Cytofix/Cytoperm and Perm/Wash buffer from BD Biosciences in accordance to the manufacturer's protocol. Cells were incubated for 30 min at 4° C. with intracellular antibodies and washed twice in Perm/Wash before FACS analysis. Samples were acquired on a BD LSR Fortessa or BD FACS Canto II and analyzed using FlowJo 7.6.5 or FlowJo 10.4 (Tree Star) software.

(37) For intracellular Foxp3 staining the Foxp3 transcription factor staining buffer set (Thermo Fisher Scientific) was used for fixation and permeabilization. All steps were performed according to the manufacturer's recommendation after extracellular staining.

(38) MOG35-55 specific CD4.sup.+ T cells were detected by tetramer staining. MOG35-55 specific APC-conjugated pMHC class II tetramer was obtained from the National Institutes of Health Tetramer Core Facility. Single cell suspensions of mouse splenocytes were incubated with MOG35-55 APC-conjugated tetramer for 1 h at room temperature in complete DC medium (for media composition see “Generation of bone marrow-derived DCs (BMDCs)”). Cells were washed and anti-APC staining and subsequent MACS were performed. Cells were incubated with extracellular or intracellular antibodies as described above.

(39) For determination of absolute CD4.sup.+ MOG35-55 specific cell counts (Thy1.1.sup.+ 2D2 CD4.sup.+ T cells) in the spleen, brain and spinal cord, Trucount™ Tubes (BD Biosciences) were used. Single cell suspensions were stained as described above, subsequently transferred into Trucount™ tubes and absolute cell counts were determined by flow cytometry.

(40) In Vivo Bioluminescence Imaging (BLI)

(41) Translation efficiency of non-immunogenic mRNA in spleen cells was investigated using Xenogen IVIS Spectrum in vivo imaging system (Caliper Life Sciences). 6 h, 24 h, 48 h and 72 h after RNA-LPX-immunization of non-immunogenic LUC-mRNA, mice were injected i.p. with an aqueous solution of D-luciferin (250 μl, 1.6 mg in PBS) (BD Biosciences). After 5 minutes, signal intensities from the spleen were defined and measured by in vivo bioluminescence in regions of interest (ROI) and quantified as total flux (photons/sec) using IVIS Living Image 4.0 Software. The acquisition time was 1 min at binning 4. During bioluminescent imaging of the live mice, mice were anesthetized with a dose of 2.5% isoflurane/oxygen mixture. The LUC signal intensity of emitted photons of live animals was depicted as a grayscale image, where black is the least intense and white the most intense bioluminescence signal. Images of mice were analyzed using the IVIS Living Image Software.

(42) Magnetic Activated Cell Sorting (MACS)

(43) Thy1.1.sup.+ 2D2 CD4.sup.+ T cells of Thy1.1.sup.+ 2D2 TCR MOG transgenic C57BL/6 mice or Thy1.1.sup.+ 2D2 Foxp3-eGFP TCR MOG transgenic C57BL/6 mice, were isolated from spleen and lymph node by positive selection using MACS (Miltenyi Biotec) according to the manufacturer's instruction. CD4.sup.+ T cells were purified using CD4 (L3T4) Micro Beads.

(44) CD4.sup.+ MOG35-55 specific T cells were MACS-enriched after MOG35-55 specific APC-conjugated pMHC class II tetramer staining using anti-APC Micro Beads. Enrichment was performed according to the manufacturer's protocol.

(45) Generation of Bone Marrow-Derived DCs (BMDCs)

(46) Bone marrow (BM) cells were extracted from femur and tibia of C57BL/6 mice and cultured in tissue culture flasks at 37° C. in RPMI 1640+GlutaMAX-I medium (Gibco) supplemented with 10% FBS (Biochrom), 1% Sodium Pyruvate 100× (Gibco), 1% MEM NEAA 100× (Gibco), 0.5% penicillin-streptomycin (Gibco), 50 μM 2-Mercaptoethanol (Life Technologies) and 1000 U/ml dendritic cell (DC) differentiation factor granulocyte-macrophage colony-stimulation factor (GM-CSF) (Peprotech). At day 6, 2×106 DCs/ml were pre-incubated for 3 h at 37° C. with MOG35-55 peptide (15 μg/ml) and then co-cultured with T cells in a total volume of 200 μl medium in a 96-well plate.

(47) In Vitro Treg Suppression Assay

(48) For in vitro Treg suppression studies, CTV-labeled (CellTrace Violet cell proliferation kit, Thermo Fisher Scientific) naïve CD4.sup.+ 2D2 transgenic (Thy1.1.sup.+) T cells (3×10.sup.4 cells per well) were co-culture with isolated and CFSE labeled (5 μM) CD4.sup.+ 2D2-Foxp3-eGFP transgenic cells of repetitively mRNA immunized mice in different ratios of suppressor and responder 2D2 T cells (1:1, 2:1, 4:1, 8:1). The 2D2-Foxp3-eGFP mice were injected intravenously into the retro-orbital plexus at day 0, day 3, day 6 and day 9 with non-immunogenic and immunogenic MOG35-55 mRNA as well as non-immunogenic irrelevant mRNA and saline. The proliferation of CTV-labeled responder 2D2 T cells was analyzed by flow cytometry 72 h after restimulation with MOG35-55 peptide-loaded BMDCs (final concentration 15 μg/ml MOG35-55 peptide).

(49) Adoptive T Cell Transfer and In Vivo Proliferation Assay

(50) For proliferation studies, enriched CD4.sup.+ 2D2 transgenic (Thy1.1V) T cells were CTV-labeled (CellTrace Violet cell proliferation kit, Thermo Fisher Scientific) according to the manufacturer's instruction, counted and 7×10.sup.6 cells injected i.v. into the retro-orbital plexus in 200 μl PBS into naïve C57BL/6 (Thy1.2.sup.+) recipient mice under anesthesia. At the next day C57BL/6 mice were immunized with 10, 20 or 40 μg of non-immunogenic mRNA coding for the MOG35-55 epitope. Control mice received either 20 μg of non-immunogenic irrelevant mRNA or saline. 4 days after T cell transfer, mice were sacrificed and cells were analyzed for proliferation by flow cytometry.

(51) For determination of absolute cell counts of CD4.sup.+ MOG35-55 specific T cells in the spleen, brain and spinal cord as well as phenotypic analysis of these cells after successful mRNA treatment at day 7 and day 10 of EAE mice, CD4.sup.+ 2D2 transgenic (Thy1.1+) T cells were isolated from Thy1.1.sup.+2D2 TCR MOG transgenic C57BL/6 mice, enriched for CD4 cells by MACS and 7-10×10.sup.6 cells in 200 μl PBS were injected i.v. into the retro-orbital plexus into naïve C57BL/6 (Thy1.2.sup.+) recipient mice under anesthesia using an oxygen-isoflurane vaporizer (2.5% isoflurane/oxygen). On the day after, EAE was actively induced in C57BL/6 mice.

(52) ELISA

(53) Detection of mouse IFN-α was performed by ELISA (PBL) 6 hours after RNA-immunization in mouse sera using standard ELISA assay according to manufacturer's instructions.

(54) Th1/Th2/Th9/Th17/Th22 and Treg cytokines present in cell culture supernatants of restimulated splenocytes of repetitively mRNA treated mice were quantified using a 17-plex mouse ELISA assay kit (Thermo Fisher Scientific). Single cell suspensions of splenocytes (5×10.sup.5 cells) isolated from mice were stimulated for 72 h with 0, 5, 10, 20 or 100 μg/ml of MOG35-55 peptide, and supernatants were analyzed for cytokine content.

(55) IFNγ Enzyme-Linked Immune Spot Assay (ELISpot)

(56) Frequencies of MOG35-55 responding T cells were examined by Interferon γ (IFNγ) ELISpot. Mice were treated at day 0, 3, 7 and 10 with non-immunogenic MOG35-55 mRNA, non-immunogenic irrelevant mRNA or saline. At day 13 after first treatment, spleens of different treatment groups were isolated and single cell suspensions were prepared. CD4.sup.+ T cells were purified by MACS using CD4 (L3T4) Micro Beads. Quantification of IFNγ responding antigen-specific T cells was performed upon restimulation with MOG35-55 peptide using the ELISpot kit. 96-well ELISpot plates (MultiScreenHTS IP Filter Plate, Merck Millipore) were coated over night with IFNγ-specific capture antibody (anti-mouse IFNγ antibody, Mabtech). After blocking with complete medium, plates were washed and 5×105 CD4.sup.+ T cells were plated together with 1×10.sup.5 MOG35-55 peptide-loaded BMDCs (peptide-concentration 15 μg/ml). As a control, CD4.sup.+ T cells were incubated with irrelevant peptide-loaded BMDCs. All cells were cultured for 16 h in a 37° C., 5% CO.sub.2 humidified incubator. Cells were washed and incubated for 2 h at 37° C. and 5% CO2 with the biotinylated anti-IFNγ detection antibody (anti-mouse IFNγ biotinylated antibody, Mabtech). This secondary antibody was visualized by adding ExtrAvidin-Alkaline phosphatase (Sigma) and after incubation for 1 h, BCIP/NBT liquid substrate (Sigma) was added and the reaction was stopped after 1-3 min by rinsing the plate under running tab water. Quantification of spots was performed on an ELISpot Reader (S6 Macro Analyzer: CTL).

(57) Induction of EAE and Treatment of Mice with mRNA

(58) EAE was actively induced in eight- to ten-week old female C57BL/6 mice with 50 μg of MOG35-55 peptide (amino acid sequence: MEVGWYRSPFSRVVHLYRNGK; SEQ ID NO: 2) emulsified in CFA (Difco Laboratories) supplemented with 10 mg/ml of heat-inactivated Mycobacterium tuberculosis H37RA (Difco Laboratories) subcutaneously (s.c.) at the base of the tail. The mice received 150 ng of Pertussis toxin (List Biological Laboratories, INC., Campbell, Calif.) intraperitoneally (i.p.) on the day of immunization and 2 days later. Mice were weighted and scored daily starting on day 10 after immunization according to the following criteria: 0, no disease; 1, decreased tail tone; 2, impaired righting reflex; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, hind limb paralysis with partial fore limb paralysis; and 6, moribund or dead.

(59) EAE was furthermore actively induced in female SJL/JRj mice (8-10 weeks) with 200 μg of PLP139-151 peptide (amino acid sequence: HSLGKWLGHPDKF; SEQ ID NO: 6) emulsified in CFA supplemented with 10 mg/ml of heat-inactivated Mycobacterium tuberculosis H37RA s.c. at the base of the tail. The mice received 200 ng PTX i.p. on the day of immunization and 2 days later. Mice were weighed and scored daily starting on day 8 after immunization according to the already described scoring criteria.

(60) A complex EAE was actively induced in female F1 hybrid mice from C57BL/6 and SJL/JRj (8-10 weeks) with 250 μg of peptide mixtures (MOG35-55, PLP139-151, PLP178-191 (amino acid sequence: NTWTTCQSIAFPSK; SEQ ID NO: 7), MBP84-104 (amino acid sequence: VHFFKNIVTPRTPPPSQGKGR; SEQ ID NO: 8), MOBP15-36 (amino acid sequence: QKFSEHFSIHCCPPFTFLNSKR; SEQ ID NO: 9)). Of each peptide 50 μg were used. The peptide mixture was emulsified in CFA supplemented with 10 mg/ml of heat-inactivated Mycobacterium tuberculosis H37RA and injected s.c. at the base of the tail. The mice received 200 ng PTX i.p. on the day of immunization and 2 days later. Mice were weighed and scored daily starting on day 8 after immunization according to the already described scoring criteria.

(61) To determine protective immunity in C57BL/6 mice, mice were treated on day 7 and day 10 or at an EAE score of 1-2 after disease induction with 20 μg non-immunogenic MOG35-55 coding, 20 μg non-immunogenic irrelevant control mRNA or saline.

(62) Therapeutic treatment of relapsing-remitting EAE in SJL mice was performed with 20 μg non-immunogenic PLP139-151 mRNA 2× per week starting from day 7 and day 10 after EAE induction. Control mice received 20 μg non-immunogenic irrelevant control mRNA.

(63) In experiments comparing non-immunogenic single-epitope mRNA treatment with non-immunogenic multi-epitope treatment, complex EAE mice were treated with 40 μg non-immunogenic multi-epitope coding mRNA (10 μg of each non-immunogenic epitope-coding mRNA (MOG35-55, PLP139-151, PLP178-191, MBP84-104) was used), 20 μg non-immunogenic MOG35-55 mRNA or 20 μg non-immunogenic irrelevant control mRNA. mRNA was injected 2× per week starting from day 7 and day 10 after EAE induction.

(64) For testing combinatorial effects of cytokine coding mRNA and antigen-specific mRNA treatment, 5 μg non-immunogenic MOG35-55 coding mRNA were injected together with 15 μg non-immunogenic mIL-10 or mIL-27 coding mRNA on day 7 and day 10 after EAE induction. Control mice received 5 μg non-immunogenic MOG35-55 mRNA together with 15 μg non-immunogenic irrelevant mRNA, 15 μg non-immunogenic cytokine-coding mRNA or 20 μg non-immunogenic irrelevant mRNA.

(65) For all experiments mRNA was injected i.v. into the retro-orbital plexus under anesthesia using an oxygen-isoflurane vaporizer (2.5% isoflurane/oxygen).

(66) Antibody Treatment

(67) C57BL/6 mice were treated with PD-1 (500 μg first treatment, 250 μg following treatments, clone RMP1-14, BioXcell), or CTLA-4 (500 μg first treatment, 250 μg following treatments, clone 9H10, BioXcell) blocking antibody or isotype-matched control antibodies (Rat IgG2a and syrian hamster IgG, BioXCell) on day 7, 10, 14 and 17 after EAE induction. Antibodies were diluted in PBS and applied i.p.

(68) Isolation of Splenic, LN and CNS Infiltrates

(69) All cell-based analyses were performed on single cell suspension of spleen, lymph node and CNS organs. In brief, spleens and LNs were incubated with 1 mg/ml collagenase D and 0.1 mg/ml DNaseI for 15 min and then mashed through a 70 μm cell strainer while rinsing with PBS. Erythrocyte lysis (hypotonic lysis buffer: 1 g KHCO.sub.3 and 8.25 g NH.sub.4Cl dissolved in 1 1 H.sub.2O and 200 μl 0.5 M EDTA) was performed on single cell suspension of splenocytes. After an additional washing step with PBS, cells were counted with the Vi-Cell cell counter (Beckman Coulter).

(70) For isolation of CNS infiltrates from brain and spinal cord, mice were anesthetized with Ketamine-Rompun and perfused using 0.9% NaCl through the heart left ventricle. The brain and spinal cord were removed manually, cut into small pieces and digested in PBS++ (PBS with calcium and magnesium, Gibco) containing collagenase D (1 mg/ml) and DNase I (0.1 mg/ml) for 20 min at 37° C. The digested tissue was then homogenized manually by sucking up the tissue pieces into a syringe and pressing out again against the wall of a 15 ml falcon tube. This was performed up to 10 times, until no pieces were visible. The single cell suspension was resuspended in 70% Percoll and layered under a 30:37 Percoll gradient. The final Percoll gradient was 30:37:70% and was centrifuged at 300 g for 40 min at room temperature. The mononuclear cell layer lying at the interphase between 70% and 37% Percoll was washed with 2% FCS/PBS before further analyses.

(71) Statistical Analysis

(72) Statistical significance was evaluated using GraphPad Prism 7 software (Graphpad Software, Inc.) employing one-way ANOVA corrected with Tukey's-comparison test factor when more than two groups were compared. EAE development curves were compared by calculating the area under the disease development curve (AUC) for single mice. Values of p≤0.05 were considered to be statistically significant; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. The employed tests are referred to in the respective figure legends.

Example 2: Examination of Non-Immunogenic mRNA Regarding Activation of Splenocytes

(73) In order to analyze the activation of splenocytes and translation efficiency of the used non-immunogenic mRNA, BALB/c mice were injected intravenously into the retro-orbital plexus with Luciferase-mRNA (10 μg) complexed with F12 liposomes. Luciferase activities were assessed via in vivo imaging 6, 24, 48 and 72 hours after RNA-LPX injection and representative mice are shown in FIG. 1C. Immunization of mice with non-immunogenic mRNA led to a lasting high translation of the LUC-mRNA. Consequently LUC protein expression could be detected up to 72 hours after mRNA immunization.

(74) Furthermore, as seen in FIG. 1A, immunization of mice with non-immunogenic mRNA led to no upregulation of activation markers CD86 on DCs and CD69 on lymphocytes like in untreated control mice. In mice immunized with non-immunogenic LUC-mRNA also no IFNα could be detected in the blood 6 hours after mRNA immunization (FIG. 1B).

Example 3: Characterization of Non-Immunogenic mRNA

(75) Non-immunogenic mRNA is used to deliver specific disease relevant antigens to dendritic cells to ensure antigen-presentation without immune activation in therapeutic applications. It has been shown that the incorporation of 1-methylpseudouridine into mRNA enhances the protein expression in the cells and reduces the immunogenicity of the mRNA in mammalian cell lines as well as in vivo in mice (Andries et al., 2015, J Control Release. 217, 337-344). This effect relies most likely on the increased ability of the mRNA to evade activation of endosomal Toll-like receptors and downstream innate immune signaling (Andries et al., 2015). Additionally to the use of 1-methylpseudouridine instead of the nucleoside uridine for the incorporation into the mRNA during in vitro transcription, HPLC purification of the synthetic mRNA eliminates furthermore immune activation and improves the translation of the nucleoside-modified, protein-encoding mRNA (Kariko et al., 2011, Nucleic Acids Res. 39, e142). By HPLC purification remaining double-stranded mRNA contaminants are removed after mRNA in vitro transcription, resulting in mRNA that does not induce Interferon signaling and inflammatory cytokines (Kariko et al., 2011). An alternative method for the purification of the nucleoside-purified mRNA is the Cellulose-purification (PCT/EP2016/059056).

(76) To investigate whether the mRNA used for therapeutic application is really non-immunogenic, Bioanalyzer and Dotblot analysis were performed to ensure the integrity and the purity of the mRNA. FIG. 2 shows Dotblot analysis of non-immunogenic MOG35-55 mRNA purified by either Cellulose treatment or HPLC purification. The J2 antibody, specific for dsRNA, showed no signal after Cellulose or HPLC purification of the analyzed MOG35-55 mRNA in a range of 40 ng up to 1 μg of used mRNA. This quality control ensures non-immunogenic mRNA characteristics as for both mRNA batches, positive therapeutic effects could be achieved in the EAE treatment. EAE was actively induced in C57BL/6 mice and mice were treated at day 7 and day 10 after disease induction with antigen-specific MOG35-55 coding non-immunogenic mRNA purified either by Cellulose or HPLC method. Both groups were completely resistant to EAE induction (FIG. 3). More detailed description about therapeutic vaccination with non-immunogenic antigen-specific mRNA will be explained in the following examples.

Example 4: Effects of Immunization of Non-Immunogenic MOG35-55 mRNA on the Proliferation of Antigen-Specific T Cells

(77) To study whether the immunization of non-immunogenic MOG35-55 mRNA-LPX results in the presentation of MOG35-55 antigen by DCs to T cells, the proliferation of antigen-specific CD4.sup.+ T cells (CD4.sup.+ 2D2 T cells) that harbor the myelin oligodendrocyte glycoprotein (MOG)-specific T cell receptor, was investigated. Cell-Trace-Violet (CTV)-labeled Thy1.1V MOG35-55-specific CD4.sup.+ 2D2 T cells were transferred into Thy1.2.sup.+ C57BL/6 mice. 24 hours later the recipient mice were immunized with different concentrations (10, 20 or 40 μg of mRNA) of non-immunogenic mRNA coding for the MOG35-55 epitope and another 4 days later the MOG35-55-specific CD4.sup.+ 2D2 T cells were analyzed for proliferation. Control mice received either 20 μg of non-immunogenic irrelevant mRNA or saline. 2D2 T cells proliferated when mice were treated with non-immunogenic MOG35-55 mRNAs in comparison to the control mice (FIG. 4), indicating that antigen presenting cells (APCs) translate and process the antigen-coding non-immunogenic mRNA in the correct manner and present the MOG35-55 peptide. Furthermore immunization of mice with non-immunogenic MOG35-55 mRNA with increasing doses from 10 to 40 μg showed also an increase in the antigen-specific T cell population.

Example 5: Immunization with Antigen-Specific Non-Immunogenic mRNA Induces the Development of Regulatory T Cells in Naïve Mice

(78) Tregs are fundamental in controlling various immune responses and they are important for tolerance induction and maintenance. Preclinical studies in animal models show that adoptive transfer of Tregs can prevent or cure several T cell-mediated diseases, including autoimmune diseases, by restoring immune tolerance to self antigens. Several CD4.sup.+ regulatory T cells have been described (Shevach, E. M., 2006, Immunity 25, 195-201) and categorized into two major subgroups: natural and inducible Tregs, which are generated in the periphery under various tolerogenic conditions. Treg cells regulate the priming of autoreactive T cells by limiting their expansion and differentiation (Sakaguchi, S. et al., 2008, Cell 133, 775-87).

(79) To further investigate the effects of immunization with antigen-specific non-immunogenic mRNA on the corresponding T cell phenotype, a 2D2-Foxp3-eGFP fusion protein has been transfected into the T cells of Th1.1 positive mice. These mice were repetitively immunized (d0, d3, d6 and d9) with non-immunogenic and immunogenic MOG35-55 mRNA, as well as non-immunogenic irrelevant mRNA or saline. Immunization of the mice with non-immunogenic MOG35-55 mRNA led to the development of Foxp3.sup.+ cells in the mice, analyzed 4 days after the last mRNA treatment (d13). Mice treated with immunogenic antigen-specific mRNA or irrelevant non-immunogenic mRNA showed no increase in Foxp3 cell population and had a frequency of Foxp3 positive cells like the saline treated control mice (FIGS. 5A and B).

(80) Whether the induced Foxp3.sup.+ cells act suppressive was assessed by an in vitro suppression assay (FIG. 5C). Therefore, total CD4.sup.+ cells of immunized mice were labeled with CFSE and co-cultured for 72 hours in different suppressor to responder ratios with naïve MOG35-55-specific CD4.sup.+ 2D2 T cells that were additionally labeled with CTV. Importantly, induced regulatory cells from mice immunized with non-immunogenic MOG35-55 mRNA mediated a dose-dependent suppression of the 2D2 effector T cell proliferation in presence of MOG35-55 peptide-loaded BMDCs. In contrast, all other groups exerted no suppressive activity.

(81) Taken together, these results indicate that APC-specific antigen presentation under non-inflammatory conditions mediated the development of functional regulatory T cells.

Example 6: Influence of Therapeutic Vaccination with Non-Immunogenic MOG35-55 mRNA in the Disease Model EAE

(82) To elucidate the tolerogenic effect of mRNA immunization with antigen-specific non-immunogenic mRNA in a disease model, EAE was actively induced in C57BL/6 mice and mice were treated at day 7 and day 10 after disease induction with antigen-specific MOG35-55 coding non-immunogenic mRNA, as well as non-immunogenic irrelevant mRNA. The treatment was started at a time point at which strong DC activation and maturation signals as well as the first T cell priming had already taken place. RNA-immunization with non-immunogenic antigen-specific mRNA completely blocked any signs of EAE (FIG. 6) and no single mouse out of 6 mRNA immunized mice developed the disease.

(83) At the same time, treatment of mice with non-immunogenic mRNA coding for an irrelevant epitope, did not protect the mice from the disease and 5/6 mice developed an EAE (FIG. 6). These findings show that MOG presentation by immature DCs can downmodulate EAE and only animals treated with non-immunogenic antigen-specific mRNA are completely resistant to EAE induction.

(84) Furthermore, it was investigated whether EAE can be treated during later time points of disease progression, when symptoms of the disease are visible and mice reached a disease score of 1-2. Remarkably, it could be shown that after RNA-immunization with non-immunogenic MOG35-55-coding mRNA EAE mice did not develop a severe EAE and the symptoms could be reduced when treated at score 1 (FIG. 7). Even when mice were treated at a score of 2 a fast disease progression could be stopped. The mice only showed a maximal disease score of 2-3, whereas nearly all untreated control mice developed a full EAE with a maximal score of 4. In comparison to EAE mice treated with non-immunogenic MOG35-55 coding mRNA, therapeutic treatment with non-immunogenic irrelevant mRNA had no positive effect on the disease progression and the animals developed EAE similar to the untreated control mice.

(85) To study the molecular mechanism of mRNA-treatment induced tolerance and to see whether the mRNA-immunization with non-immunogenic antigen-specific mRNA and thereby the presentation of MOG35-55 by DCs directly influences effector T cells (Teff), EAE was again induced in C57BL/6 mice, animals were treated with non-immunogenic MOG35-55-coding mRNA at day 7 and day 10 after disease induction and 6 days after the last mRNA-treatment spleen, LN, brain and spinal cord were isolated. Cells from the different organs were reactivated in vitro with MOG35-55 peptide for 6 hours and then gated on CD40 ligand (CD40L) Teff cells. CD40L is rapidly expressed on T cells after activation and was therefore used as a marker for MOG-specific Teff cells. As seen in FIG. 8, administration of non-immunogenic MOG35-55 mRNA leads to significant decrease in the frequency of antigen-specific CD4.sup.+ T cells in the groups treated with non-immunogenic antigen-specific mRNA in brain and spinal cord. On the functional level it could be shown that CD4.sup.+ T cells secrete significantly less Interferon-γ (IFNγ) in the brain and Interleukin-17A (IL-17A) in the spinal cord in mice treated with non-immunogenic MOG35-55 coding mRNA compared to untreated control mice (FIG. 9). These data indicate that mRNA-treatment with non-immunogenic MOG35-55 mRNA can inactivate memory T and Teff cells, leading to a reduction of these cells.

Example 7: Influence of mRNA-Purity on the Treatment-Efficiency of EAE

(86) Experiments with combinations of immunogenic mRNA containing uridine (U) and non-immunogenic mRNA containing 1-methylpseudouridine and being purified by cellulose purification (m1Y) revealed that activation of splenocytes (examined by upregulation of the activation marker CD86 on DCs and CD69 on lymphocytes) as well as IFNα secretion correlates with the amount of used immunogenic mRNA. Nearly no activation was achieved with non-immunogenic mRNA (20 μg m1Y MOG35-55 mRNA) and the activation increased steadily by addition of immunogenic mRNA (U MOG35-55 mRNA) (FIG. 10). 5 μg U mRNA+15 μg m1Y mRNA showed only slightly less activation than 20 μg U mRNA, whereas 20 μg m1Y mRNA again did not activate the immune system at all. Corresponding Dotblot analysis of the different mRNA mixtures also revealed that only 100% of non-immunogenic (m1Y) mRNA show no signal for dsRNA (FIG. 11).

(87) To further investigate the influence of the purity of used mRNA on the treatment efficacy of EAE the same mRNA mixtures were used in order to treat an ongoing EAE. Again, EAE was actively induced in C57BL/6 mice and mice were treated at day 7 and day 10 after disease induction with antigen-specific mRNA. In this experiment the same mRNA mixtures of non-immunogenic and immunogenic MOG35-55 mRNA were used as in the previous “activation” experiment. Unlike 100% non-immunogenic (m1Y) mRNA, mixture of non-immunogenic and immunogenic mRNA (2.5 μg U+17.5 μg m1Y MOG35-55 mRNA as well as 5 μg U+15 μg m1Y MOG35-55 mRNA) cannot prevent the disease development completely and some mice start to develop the disease similar to mice treated with 100% immunogenic mRNA (FIG. 12). These results show that for an effective treatment of EAE pure non-immunogenic (m1Y) mRNA is necessary.

(88) An additional comparison in terms of splenic activation of unmodified and modified mRNA, which was either or not purified by HPLC, was performed. As depicted in FIG. 13, nucleoside-modifications such as pseudouridine (pU) alone, does not induce a loss of recognition of the mRNA by the immune system, but the purification process makes the difference. Non-purified pU-mRNA is still immunogenic and it works like regular mRNA (U-mRNA) in terms of splenic activation (strong upregulation of CD86 on DCs, upregulation of CD69 on B cells, T cells and NK cells) and IFNα secretion. In contrast, pU-mRNA that was additionally purified by HPLC in order to get rid of dsRNA (pU-HPLC mRNA) prevents DC, B cell, T cell and NK cell activation and consequently the secretion of IFNα like in untreated control mice. These results underline that exclusively the combination of Uridine-modification and additional purification of the IVT mRNA minimizes the immunogenicity of the mRNA to zero as observed in naïve mice, a level where tolerance can be induced. Thus, non-immunogenic mRNA (uridine-modified in combination with mRNA-purification) is the optimal mRNA for the purpose of tolerance induction.

Example 8: Treatment with Non-Immunogenic MOG35-55-Specific mRNA does not Induce De Novo Priming of Antigen-Specific CD4.SUP.+ T Cells but Results in the Expansion of Anergic T Cells in Naïve Mice

(89) The maintenance and induction of peripheral tolerance is dependent on the presentation of self-antigen by APCs that express low levels of co-stimulatory molecules, like CD86 or CD40, on their surface. The absence of co-stimulatory-molecule expression and cytokine production by APCs upon treatment with non-immunogenic mRNA was shown in Example 2, FIG. 1. Consequently, upon mRNA treatment, APCs process and present the epitope encoded by the non-immunogenic mRNA in the absence of activating stimuli. TCR engagement in the absence of costimulatory signals does not result in the expansion of effector T cells, rather in apoptosis or T cell anergy (Mueller, D. L., 2010, Nat. Immunol. 11, 21-7). In contrast, during effector T cell priming, DCs release specific cytokines, which together with antigen presentation and the expression of costimulatory molecules polarize effector immune responses by driving the differentiation of CD4.sup.+ T cells into Th1, Th2 or Th17 cells. Indeed, generation of effector T cells upon non-immunogenic antigen-specific mRNA treatment is not intended in an autoimmune disease, as more effector T cells might even lead to a more severe course of the disease instead of a curative treatment.

(90) To investigate the generation of antigen directed T cell responses, naïve C57BL/6 mice were repetitively treated (day 0, 3, 7 and 10) with 20 μg non-immunogenic MOG35-55 coding mRNA, 20 μg non-immunogenic irrelevant mRNA or saline. After four mRNA immunizations, CD4.sup.+ T cells were isolated on day 13 from the spleens and an IFNγ ELISpot was performed. FIG. 14A shows that mice treated with non-immunogenic MOG35-55 coding mRNA did not develop IFNγ.sup.+ CD4.sup.+ effect T cells responding to antigen-recognition upon antigen-specific mRNA treatment.

(91) In accordance with these results, also restimulation of splenic CD4.sup.+ T cells with different concentrations of MOG35-55 peptide (0, 5, 10, 20 and 100 μg) for 48 h did not result in the secretion of other inflammatory cytokines such as TNFα and GM-CSF, as measured in the supernatants by ELISA (FIG. 14B). Only the secretion of the anti-inflammatory cytokine IL-10 was detected in the mice treated with non-immunogenic antigen-specific mRNA, but not with non-immunogenic irrelevant mRNA (FIG. 14C). Furthermore, only in this group, the secretion of Th2 cytokines like IL-4 and IL-5 could be detected (data not shown). The production of IL-10 in response to non-immunogenic antigen-specific mRNA provides evidence of antigen-specific regulatory T cells (Tregs) being activated in response to the treatment Tregs are fundamental in controlling various immune responses. Many different subsets of inducible Tregs have been reported and especially CD25.sup.+Foxp3.sup.+ and Tr1 cells are characterized by the production of high levels of IL-10. These regulatory T cells regulate the priming of autoreactive T cells by limiting their expansion and differentiation.

(92) To investigate the CD4 phenotype after non-immunogenic antigen-specific mRNA treatment, C57BL/6 mice were repetitively immunized with 20 μg non-immunogenic antigen-specific MOG35-55 mRNA, 20 μg non-immunogenic irrelevant mRNA or saline on day 0, 3, 7 and 10. The expression of TIGIT, Tim-3, PD-1 and Lag-3 on CD4.sup.+ MOG35-55 tetramer.sup.+ cells from mice treated with non-immunogenic MOG35-55 coding mRNA was compared to total CD4.sup.+ cells of non-immunogenic irrelevant and untreated control mice. Repetitive immunization with non-immunogenic antigen-specific mRNA results in an upregulation of TIGIT, Tim-3, PD-1 and Lag-3 in comparison to mice treated with non-immunogenic irrelevant mRNA (FIG. 15).

(93) The upregulation of these markers correlates with an anergic T cell phenotype. Anergy is an acquired state of functional unresponsiveness and the consequence of the engagement of the T cell receptor (TCR) without costimulation through CD28 (Jenkins, M. K. et al., 1987, Proc. Natl. Acad. Sci. U.S.A 84, 5409-13; Quill, H. & Schwartz, R H., 1987, J. Immunol. 138, 3704-3712; Jenkins, M. K. et al., 1990, J. Immunol. 144, 16-22). Additionally, it constitutes one means of imposing peripheral tolerance. Anergic T cells are functionally inactive and unable to initiate a productive immune response even when the antigen is encountered in the presence of full costimulation. Anergic CD4.sup.+ T cells with distinct phenotypic and gene-expression programs can even convert into regulatory T cells that, in turn, can promote anergy of pathogenic CD4.sup.+ T cells and inhibit autoimmunity (Kalekar, L. A. et al., 2016, Nat. Immunol. 17, 304-14). Lag-3, Tim-3, ICOS as well as TIGIT mediate co-inhibitory functions on effector T cells and an overexpression of these molecules is associated with T cell exhaustion (Anderson, A. C. et al., 2016, Immunity 44, 989-1004). The accumulation of multiple co-inhibitory receptors on the surface of T cells is even associated with increased dysfunction (Blackburn, S. D. et al., 2009, Nat. Immunol. 10, 29-37). TIGIT itself acts as a co-inhibitory molecule by directly down regulating the proliferation of T cells, but also by preventing DCs maturation, decreasing IL-12 secretion and inducing the production of the immunosuppressive cytokine IL-10 (Yu, X. et al., 2009, Nat. Immunol. 10, 48-57; Joller, N. et al., 2011, J. Immunol. 186, 1338-1342). Lag-3 plays a role in the modulation of T cell homeostasis and effector T cell responses (Workman, C. J. et al., 2002, J. Immunol. 169, 5392-5395; Workman, C. J. & Vignali, D. A. A., 2003, Eur. J. Immunol. 33, 970-979; Workman, C. J. & Vignali, D. A. A., 2005, J. Immunol. 174, 688-695).

(94) Furthermore, immune checkpoint molecules like programmed death 1 receptor (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) are co-inhibitory molecules involved in the negative regulation of immune responses and consequently the maintenance of peripheral self-tolerance. Co-inhibitory molecules are in general upregulated upon T cell activation and constrain the effector response through feedback inhibition. PD-1 and its ligands, PD-L1 and PD-L2 for example deliver inhibitory signals that regulate the balance between T cell activation and tolerance (Keir, M. E. et al., 2008, Annu. Rev. Immunol. 26, 677-704). Immune responses to self-antigens require specific and balanced responses to maintain tolerance, which is mediated by the different co-inhibitory receptors and its ligand.

(95) By an upregulation of PD-1effector T cell responses will be shut off and tissues are in turn protected from immune cell-mediated damage.

(96) All these co-inhibitory receptors play a central role in regulating autoimmune diseases and deficiency in some of these molecules even leads to autoimmunity.

Example 9: More Detailed Characterization of MOG35-55 Specific CD4.SUP.+ T Cells Upon Non-Immunogenic Antigen-Specific mRNA Treatment in the Disease Model EAE

(97) In accordance with the results shown in Example 6, FIGS. 8 and 9 also adoptive cell transfer experiments of MOG35-55 specific CD4.sup.+ T cells showed the same results. MOG35-55 specific Thy1.1.sup.+CD4.sup.+ T cells of 2D2 TCR transgenic mice were transferred into Thy1.2.sup.+ C57BL/6 mice and 24 h later EAE was induced in the recipient mice. On day 7 and 10 after disease induction, mice were treated with 20 μg non-immunogenic MOG35-55-coding mRNA, 20 μg non-immunogenic irrelevant mRNA or saline. Six days after the last mRNA treatment, brain and spinal cord were isolated. Cells from these tissues were reactivated in vitro with MOG35-55 peptide for 6 h and then gated on Thy1.1.sup.+ CD4.sup.+ T cells. As depicted in FIG. 16, administration of non-immunogenic MOG35-55 coding mRNA led to significant decrease in the frequency of MOG35-55-specific CD4.sup.+ T cells in the brain and spinal cord. On the functional level, CD4.sup.+ T cells secrete significantly less IFNγ and Interleukin-17A (IL-17A) in the brain and spinal cord of mice treated with non-immunogenic MOG35-55 coding mRNA compared to control mice (FIG. 17). These data again indicate that mRNA treatment with non-immunogenic MOG35-55 mRNA can inactivate Teff cells, leading to a reduced infiltration of these cells into the brain and spinal cord.

(98) To investigate the antigen-specific CD4 T cell phenotype after successful treatment, EAE was actively induced and on day 7 and day 10 after disease induction animals were treated with 20 μg non-immunogenic MOG35-55 coding mRNA, 20 μg non-immunogenic irrelevant mRNA or saline. At the peak of the disease (day 16 after disease induction), the CD4 T cell phenotype in the spleen was investigated by MOG35-55 tetramer staining. In accordance with the experiments shown in Example 5 and 8, also in the EAE disease setting, the antigen-specific tolerance induced by non-immunogenic antigen-specific mRNA is mediated by the upregulation of co-inhibitory molecules Lag-3, ICOS, TIGIT and Tim-3 on MOG35-55 specific CD4.sup.+ T cells in the spleen (FIG. 18A). Besides the cell-intrinsic mechanism mediating immune tolerance like anergy and negative costimulation, also cell-extrinsic (Treg cell mediated tolerance) mechanisms can mediate the non-immunogenic antigen-specific mRNA treatment effect. As already discussed, Tregs play a crucial role in antigen-specific tolerance (Sakaguchi, S. et al., 1985, J. Exp. Med. 161, 72-87). FACS analysis of MOG35-55 specific CD4.sup.+ T cells furthermore showed significant expansion of Treg populations (FIG. 18B). Additionally, Tregs in the spleens of mice treated with antigen-specific non-immunogenic mRNA express high levels of CD69, indicating that they are highly activated.

(99) In order to determine whether the expression of negative costimulatory molecules was required for tolerance induction and maintenance, PD-1 or CTLA-4 were blocked during mRNA treatment of EAE. EAE treatment at day 7 and day 10 after disease induction with non-immunogenic MOG35-55 coding mRNA in combination with anti-PD-1 or anti-CTLA-4 antibody led to reversal of protection from EAE in comparison to mice treated with non-immunogenic MOG35-55 coding mRNA in combination with IgG controls (FIG. 19). This demonstrates that PD-1 and CTLA-4 are crucial for long-term maintenance of antigen-specific tolerance induced by non-immunogenic antigen-specific mRNA, as antibody blockage of PD-1 and CTLA-4 in parallel to mRNA treatment resulted in a marked reduction in the efficacy of tolerance induction mediated by non-immunogenic antigen-specific mRNA treatment.

(100) Taken together, the depicted experiments investigated the mode of action of this new therapeutic approach of antigen-specific tolerance induction by treatment with antigen-specific non-immunogenic mRNA. The results show that antigen-specific non-immunogenic mRNA treatment is an effective method of altering harmful immune responses in autoimmunity to confer protection. The experiments shown in naïve as well as in disease-afflicted mice demonstrate that the treatment with non-immunogenic antigen-specific mRNA results in the induction of anergic and regulatory T cells that mediate tolerance in the periphery, leading to a successful treatment of an ongoing autoimmune disease. It was shown that the therapeutic effect relies on already well-described mechanisms for tolerance induction as described in the literature discussed in each example. These mechanisms are important and fundamental for tolerance induction independent of the type of autoimmune disease. The mechanism of tolerance mediated by non-immunogenic antigen-specific mRNA treatment can thus be transferred to any other type of autoimmune disease.

Example 10: Antigen-Specific Tolerance Induction by Treatment with Non-Immunogenic Antigen-Specific mRNA is Independent of the EAE Disease Model

(101) All previously described EAE experiments were performed in C57BL/6 mice induced with MOG35-55 peptide resulting in a monophasic disease progression. Another model of EAE is characterized by recurrent disease due to a different relevant epitope (PLP139-151) leading to a relapsing-remitting disease course in SJL mice. This model was employed to demonstrate tolerance induction in a more clinically relevant disease setting. FIG. 20A shows that mRNA treatment 2× per week starting from day 7 and day 10 after EAE induction with non-immunogenic PLP139-151 coding mRNA in the relapsing-remitting disease model has a positive effect on the EAE development as these mice showed a much weaker disease progression than mice treated with non-immunogenic irrelevant control mRNA. To even better simulate the situation in MS, a complex EAE associated with multiple pathogenic autoreactive T cell clones, was used. Since upon a definite diagnosis of MS, the disease is likely already associated with complex anti-myelin autoreactivity, due to “epitope spread” the efficacy of multi-epitope treatment approach was investigated. As shown in FIG. 20B, the complex EAE was successfully treated by a mix of four different non-immunogenic disease-epitope coding mRNAs coding for MOG35-55, PLP139-151, PLP178-191 and MBP84-104. Of each non-immunogenic disease-epitope coding mRNA 10 μg was used, resulting in 40 μg for the efficient treatment. None of the single epitope treatments was able to confer similar treatment outcomes, as shown for 20 μg non-immunogenic MOG35-55 coding mRNA.

(102) These results show that the antigen-specific tolerance induction observed in the different experiments is independent of a specific epitope and can be mediated with different disease relevant epitopes. Furthermore, the data underline that even a complex ongoing disease with a complex pathogenic autoimmune process can be successfully treated by administration of multiple non-immunogenic myelin-specific mRNA constructs.

Example 11: Co-Delivery of Immunomodulatory Cytokines Improves the Tolerogenic Effect of Non-Immunogenic Antigen-Specific mRNA-LPX Treatment

(103) Several immunoregulatory molecules, such as IL-10 can support Treg function. Furthermore the anti-inflammatory cytokine IL-10 is known to induced tolerogenic DCs (Torres-Aguilar, H. et al., 2010, Autoimmun. Rev. 10, 8-17; Torres-Aguilar, H. et al., 2010, J. Immunol. 184, 1765-1775; Steinbrink K. et al., 1997, J. Immunol. 159, 4772-4780) and to suppress T cell proliferation and cytokine responses (Gu, Y. et al., 2008, Eur. J. Immunol. 38, 1807-1813; Guo, B., 2016, J. Clin. Cell Immunol. 7, 1-16). Also IL-27 is a cytokine know to have anti-inflammatory properties and in EAE studies, it has been shown that this cytokine dampens the severity of the disease (Mascanfroni, I. D. et al., 2013, Nat. Immunol. 14, 1054-63; Thom, R. et al., 2017, Front. Immunol. 8, 1-14). In a set of EAE experiments the co-delivery of non-immunogenic antigen-specific mRNA with immunomodulatory cytokines like IL-10 and IL-27 encoded by non-immunogenic mRNA should be investigated in regard to modulate the immune response and improve the efficiency of tolerance induction.

(104) The ability of co-delivery of immunomodulatory cytokines to enhance the tolerogenicity of single-antigen-specific mRNA-LPX treatment was evaluated using a ‘subtherapeutic’ treatment of EAE mice. Non-immunogenic antigen-specific mRNA-LPX treatment has demonstrated robust tolerance induction in the previous experiments, therefore the combinatorial approach of additional non-immunogenic cytokine-coding mRNA treatment was investigated in the EAE model at subtherapeutic doses to identify therapeutic contributions.

(105) To investigate the beneficial effect of non-immunogenic cytokine-coding mRNA treatment on non-immunogenic antigen-specific mRNA in the disease model, EAE was actively induced in C57BL/6 mice and mice were treated at day 7 and day 10 after disease induction with non-immunogenic antigen-specific MOG35-55 coding mRNA, non-immunogenic MOG35-55 coding mRNA in combination with non-immunogenic mIL-10 (FIG. 21A) or mIL-27 (FIG. 21B) coding mRNA, non-immunogenic cytokine-coding mRNA alone, as well as non-immunogenic irrelevant mRNA.

(106) The results depicted in FIG. 21 suggest that co-delivery of immunomodulatory cytokines encoded by non-immunogenic mRNA enhances tolerance in an antigen-specific context. Furthermore, it does not broadly suppress the immune response as single cytokine-coding mRNA treatment showed no changes on EAE progression in comparison to control mice.