OPTIMIZED NUCLEIC ACID MOLECULES

Abstract

The present invention provides optimized nucleic acid molecules, methods for optimization of nucleic acid molecules and uses of optimized nucleic acid molecules. A modular design principle is provided that is suitable to generate a nucleic acid, particularly mRNA, which is tailored for a respective application. The nucleic acid molecules of the present invention can be obtained by the versatile combination of multiple modules on nucleic acid level. Such nucleic acid, e.g. mRNA, can be tailored by combining one or more modules, comprising (i) a nucleic acid moiety encoding a polypeptide of interest (e.g. a protein potentially producing a therapeutic outcome) and (ii) at least one further coding or non-coding nucleic acid moiety, e.g. selected among nucleic acid moieties encoding a polypeptide element, such as a secretory signal peptide (SSP), a multimerization element (dimerization, trimerization, tetramerization and oligomerization), a virus like particle (VLP) forming element, a transmembrane element, a dendritic cell targeting element, an immunological adjuvant element, an element promoting antigen presentation; a 2A peptide; a peptide linker element, elements that extend protein half-life, and/or any other polypeptide or protein. Non-coding nucleic acid moieties may be selected e.g. from the group comprising 3-UTR, 5-UTR, IRES element, miRNA moiety, histone stem loop, poly(C) sequence, polyadenylation signal, polyA-sequence. The optimized nucleic acid molecule can further be characterized by the presence of at least one modified nucleoside. The versatility of the present invention allows for rational design of a large variety of different nucleic acid molecules with desired properties.

Claims

1. A method of treating or preventing disease in a subject comprising administering to the subject an effective amount of a purified ribonucleic acid (RNA) molecule comprising: (i) a first module encoding an infectious disease antigen, or an antigenic fragment thereof; and (ii) a second module encoding a multimerization element, wherein the multimerization element is encoded in the same reading frame as the infectious disease antigen, and wherein the purified RNA molecule is formulated in a lipid carrier.

2. The method of claim 1, wherein the RNA is a messenger RNA (mRNA).

3. The method of claim 1, wherein the coding sequence of the RNA has an increased G/C content relative to wild type sequence encoding the infectious disease antigen and/or multimerization element.

4. The method of claim 1, wherein the RNA comprising at least one non-coding moiety, selected from the group consisting of one or more untranslated regions (UTRs), one or more miRNA moieties, one or more IRES moieties, a histone stem loop, a 5-Cap, a poly(C) sequence, a polyadenylation signal and a poly(A) sequence.

5. The method of claim 4, wherein the RNA is a mRNA comprising a 5-Cap, a poly(A) sequence, 5 UTR and a 3 UTR.

6. The method of claim 1, wherein the RNA comprises at least one chemical modification selected from a sugar modification, a backbone modification, a base modification, a lipid modification and/or a modification of the 5-end of the nucleic acid molecule.

7. The method of claim 6, wherein the RNA comprises at least one 1-methyl-pseudouridine.

8. The method of claim 1, wherein the infectious disease antigen comprises a viral envelope protein or a viral capsid protein.

9. The method of claim 1, wherein the infectious disease antigen comprises a viral envelope protein.

10. The method of claim 1, wherein the infectious disease antigen comprises a viral antigen from a Influenza virus, respiratory syncytial virus (RSV), Herpes simplex virus (HSV), human Papilloma virus (HPV), Human immunodeficiency virus (HIV), Dengue virus, Cytomegalovirus (CMV), Hepatitis B virus (HBV), Rabies virus, Yellow Fever Virus West Nile virus or corona virus.

11. The method according to claim 1, wherein the multimerization element is a dimerization element, a trimerization element, a tetramerization element or an oligomerization element.

12. The method according to claim 11, wherein the multimerization element is a trimerization element.

13. The method according to claim 11, wherein the multimerization element is an engineered leucine zipper or fibritin foldon domain.

14. The method of claim 13, wherein the multimerization element is from enterobacteria phage T4, GCN4pII, GCN4-pLI, or p53.

15. The method of claim 14, wherein the fibritin foldon domain is the trimerization domain from enterobacteria phage T4.

16. The method of claim 11, wherein the multimerization element is from ferritin.

17. The method of claim 11, wherein the multimerization element comprises a sequence at least 90% identical to a sequence of SEQ ID NOs: 1116-1167.

18. The method of claim 17, wherein the multimerization element comprises a trimerization element comprising a sequence at least 90% identical to a sequence according to SEQ ID Nos: 1121-1145.

19. The method of claim 1, wherein the lipid carrier comprises a cationic lipid.

20. The method of claim 5, the RNA comprising molecule comprising: (i) a first module a viral envelope protein viral antigen from an Influenza virus, RSV, HSV, HIV, Dengue virus, CMV, HBV, Rabies virus, Yellow Fever Virus, West Nile virus or coronavirus; and (ii) a second module encoding a multimerization element at least 90% identical to a sequence according to SEQ ID Nos: 1121-1145.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0585] FIGS. 1A-B: shows a western blot to detect HA proteins in cell lysates (A) and cell culture supernatant (B) using an anti HA (H1N1) protein specific antibody. M: protein marker lane; 1: recombinant HA protein (positive control); 2: HA.sub.TM-SGG-ferritin; 3: HA.sub.TM; 4: negative control; 5: HA.sub.TM-C3d_P28; 6: HA.sub.TM-foldon. The size ladder (kDa) of the protein marker is shown on the left of panel (A). See Example 2.

[0586] FIGS. 2A-D: shows IgG1 and IgG2a titers of mice immunized with the indicated formulated HA mRNA vaccines. RiLa served as a negative control. Antibody titers were measured at day 21 and day 28. (A) and (B) shows HA-specific IgG1 antibody titers; (C) and (D) shows HA-specific IgG2a antibody titers. The horizontal bar indicates the median. Every data point represents one individual specimen. See Example 4.

[0587] FIG. 3: shows HI titers of mice immunized with the indicated formulated HA mRNA vaccines. RiLa served as a negative control. HI titers were determined at day 28 (1 week after boost immunization). HI titers of >40 are associated with a protection from influenza virus infection (indicated by dashed line). Every data point represents one individual specimen. See Example 5.

[0588] FIGS. 4A-D: shows IgG1 and IgG2a titers of mice immunized with the indicated formulated HA mRNA vaccines. RiLa served as a negative control. Antibody titers were measured at day 35 and day 49. (A) and (B) shows HA-specific IgG1 antibody titers; (C) and (D) shows HA-specific IgG2a antibody titers. The horizontal bar indicates the median. Every data point represents one individual specimen. See Example 6.

[0589] FIG. 5: shows HI titers of mice immunized with the indicated formulated HA mRNA vaccines. RiLa served as a negative control. HI titers were determined at day 49 (4 weeks after boost immunization). HI titers of >40 are associated with a protection from influenza virus infection (indicated by dashed line). Every data point represents one individual specimen. See Example 7.

[0590] FIG. 6: shows HI titers of mice immunized with the indicated formulated HA mRNA vaccines. RiLa served as a negative control. HI titers were determined at day 14 (2 weeks after boost immunization (pB)). HI titers of >40 are associated with a protection from influenza virus infection (indicated by dashed line). Every data point represents one individual specimen. See Example 8.

EXAMPLES

Example 1: Preparation of mRNA HA Constructs for In Vitro and In Vivo Experiments

[0591] 1.1. Explanation of the HA Constructs:

[0592] For the present example, the target protein was the antigen hemagglutinin of Influenza A virus (A/Netherlands/602/2009(H1N1); GI:228860929). The C-terminal transmembrane domain (TM) of the protein was removed (amino acids 531-566), hereinafter referred to as HAo. To potentially promote oligomerization, a non-heme ferritin of Helicobacter pylori was fused to the C-terminus of HA.sub.TM, separated by a SGG spacer sequence, hereinafter referred as HA.sub.TM-SGG-ferritin. To potentially promote trimerization, a foldon domain of the fibritin/foldon protein of the bacteriophage T4T was fused to the C-terminus of HA.sub.TM, hereinafter referred to as HA.sub.TM-foldon. As immunologic adjuvant element, C3d_P28 was fused to the C-terminus of HA.sub.TM, hereinafter referred to as HA.sub.TM-C3d_P28. To potentially promote multimerization, a human IgG1 Fc domain was fused to the C-terminus of HAo, hereinafter referred to as HA.sub.TM-IgG FC. To obtain targeting of dendritic cells, a CD40 ligand domain was fused to a HA.sub.TM. additionally comprising a GCN4pII for trimerization, hereinafter referred to as HA.sub.TM-GCN4pII-CD40L). The fusion constructs used in the present example as well as the control constructs are listed with their respective SEQ ID NOs in Table 1.

[0593] 1.2. Preparation of DNA and mRNA Constructs

[0594] DNA sequences encoding the target element HA.sub.TM fused to respective additional elements were prepared and used for subsequent RNA in vitro transcription reactions. The constructs are listed in Table 1.

TABLE-US-00001 TABLE 1 Prepared mRNA HA-fusion constructs Protein construct Protein SEQ mRNA SEQ description ID NO ID NO HA.sub.TM 1667 1663 HA.sub.TM-SGG-ferritin 1670 1666 HA.sub.TM-foldon 1669 1665 HA.sub.TM-C3d_P28 1668 1664 HA membrane bound 1735 1732 HA.sub.TM-IgG FC 1736 1733 HA.sub.TM-GCN4pII-CD40L 1737 1734

[0595] The DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization. Sequences were introduced into a derived pUC19 vector and modified to comprise stabilizing UTR sequences derived from alpha-globin-3-UTR (muag (mutated alpha-globin-3-UTR)), a histone-stem-loop structure, and a stretch of 70 adenosine at the 3-terminal end.

[0596] 1.3. RNA In Vitro Transcription

[0597] The respective DNA plasmids were transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a CAP analog (m7GpppG) and a nucleotide mixture. Subsequently, the in vitro transcribed mRNA was purified using PureMessenger (CureVac, Tubingen, Germany; WO2008/077592A1). The obtained mRNA (naked, unformulated mRNA) was used for in vitro expression analysis.

[0598] 1.4. Preparation of Protamine Complexed mRNA Vaccine

[0599] Prior to use in in vivo vaccination experiments, naked mRNA constructs were complexed with protamine. The mRNA complexation consisted of a mixture of 50% naked mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, naked mRNA was added, and the final concentration of the vaccine was adjusted with Ringer's lactate solution. The obtained formulated mRNA vaccine was used for in vivo experiments.

Example 2: Expression of HA Constructs in HEK 293T Cells and Analysis Using Western Blot

[0600] The aim of these experiments was to analyse the expression of the HA mRNA constructs (see Table 1) and to determine the release of the HA protein into the supernatant of transfected HEK 293T cells. All HA mRNA vaccine candidates contained an endogenous secretory signal peptide (N-terminus of the HA protein) that should promote the release from the producing cells into the supernatant. Cell lysates were also analyzed for HA protein expression.

[0601] 2.1. Transfection of HEK 293T Cells

[0602] HEK 293T cells were seeded in a 24-well plate at a density of 70,000 cells/well in cell culture medium (DMEM complete), 48h prior to transfection. Cells were transfected with and 5 g naked, unformulated mRNA HA constructs (see Table 1) using Lipofectamine 2000 (Invitrogen). 48 hours post transfection, transfection supernatants were collected. Additionally, cells were harvested and lysed with RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% TritonX-100, 0.1% SDS). The respective supernatants and cell lysates were stored at 20 C.

[0603] 2.2. Analysis for HA Expression Using Western Blot

[0604] An SDS-PAGE was performed with supernatants and whole cell lysates from all samples with Mini-PROTEAN TGX Precast Mini Gels 7.5% (Bio-Rad). Untransfected cells were used as a negative control. 0.5 g recombinant H1N1 HA (A/California/04/2009; Sino Biological) was used as a positive control. The blotting on a nitrocellulose membrane was performed for 2h in the presence of a blotting buffer. After blocking the membrane in a respective buffer, antibody incubation (primary and secondary antibodies) and signal detection (LI-COR measurement) was performed. The presence of HA was analyzed using a commercially available mouse monoclonal anti influenza A virus H1N1 specific antibody (Clone 2C10H2, Sino Biological, C) in combination with a goat anti mouse IgG1 IRDye 800 coupled secondary antibody (LI-COR Biosciences). The presence of tubulin was analyzed either in cell lysates as a loading control or in supernatants to check for cellular contamination using a rabbit anti / tubulin antibody (Cell signaling Technology) in combination with a goat anti rabbit IgG IRDye 680 coupled secondary antibody (LI-COR Biosciences). The approximate protein sizes (without taking post-translational protein modifications into account) are shown in table 2. Western blot results are shown in FIG. 1.

TABLE-US-00002 TABLE 2 Expected protein sizes (approximations) of the HA monomers Protein construct description Protein size monomer [kDa] HA.sub.TM 59.4 HA.sub.TM-SGG-ferritin 80.7 HA.sub.TM-foldon 64.6 HA.sub.TM-C3d_P28 75

[0605] Results:

[0606] For all four tested mRNA constructs, HA protein monomers were detected in cell lysates and/or supernatants (see FIG. 1), showing that mRNA constructs were translated into protein. The band sizes were in accordance to the expected band sizes. The majority of protein for all four mRNA constructs was detected in the respective supernatants (see FIG. 1B). Since no tubulin protein was detectable in the analyzed supernatants (data not shown), the presence of HA protein was considered to be mediated by secretion triggered by the endogenous secretory signal peptide and not via release by cell death associated with the transfection method. Taken together, all tested mRNA constructs were translated and secreted in HEK 293T cells.

Example 3: Vaccination of Mice with HA Constructs

[0607] Immunization

[0608] Female BALB/c mice were injected intramuscularly (i.m.) with formulated mRNAs vaccines encoding HA protein constructs with doses indicated in Table 3. As a negative control, one group of mice was vaccinated with buffer (ringer lactate, RiLa). All animals received boost injections on day 21. Blood samples were collected on day 21 and 28 for the analysis of the immune response in the effector phase (see Examples 4-5) and additionally on day 35 and 49 for the analysis of the immune response in the memory phase (see Examples 6-7).

TABLE-US-00003 TABLE 3 Vaccination regimen for indicated animal groups Number of Injected mRNA HA Vaccination Group mice construct dose on day 1 8 HA.sub.TM 40 g 0/21 2 8 HA.sub.TM-SGG-ferritin 40 g 0/21 3 8 HA.sub.TM-foldon 40 g 0/21 4 8 HA.sub.TM-C3d_P28 40 g 0/21 5 8 RiLa buffer 0/21

Example 4: ELISA Analysis of an Antigen Specific Humoral Immune Response in the Effector Phase

[0609] The aim of this experiment was to assess the antigen specific humoral immune response in vaccinated mice for the used mRNA vaccines and to compare the detected immune response evoked by HA fusion constructs (with additional element) with the immune response evoked by the target HA antigen without additional element. HA protein specific IgG1 and IgG2a antibodies were detected by ELISA using sera obtained at day 21 and day 28 (effector phase).

[0610] Determination of Anti HA Protein Specific IgG1 and IgG2a Antibodies by ELISA:

[0611] Assessment of an antigen specific immune response was carried out by detecting HA protein specific IgG1 and IgG2 antibodies. MaxiSorp plates (Nalgene Nunc International) were coated with HA protein (Charles River Laboratories). After blocking with 1PBS containing 0.05% Tween-20 and 1% BSA the coated plates were incubated with respective mouse serum dilutions. Binding of specific antibodies to the HA antigens was detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. For the analysis of an antigen specific immune response in the effector phase, sera obtained at day 21 (three weeks after prime vaccination) and day 28 (one week after boost vaccination) were used. Vaccination was performed according to Example 3. The results are shown in FIG. 2.

[0612] Results:

[0613] Assessment of the humoral immune response after immunizations revealed that 40 g of the respective mRNA vaccines induced HA specific IgG1 and IgG2a antibody titers for the HA.sub.TM-SGG-ferritin and the HA.sub.TM-foldon constructs. The HA.sub.TM (without additional element) vaccine induced IgG1 antibodies but not IgG2a antibodies at day 28 (FIG. 2B). The HA.sub.TM-C3d_P28 vaccine did not induce substantial antibody titers.

[0614] Taken together, the addition of ferritin and foldon elements to HA.sub.TM substantially improved the induction of a HA specific humoral immune response in the effector phase, whereas the addition of a C3d_P28 element to HA.sub.TM had no positive effect.

Example 5: Hemagglutination Inhibition Assay to Determine Virus Neutralizing Titers in the Effector Phase

[0615] The aim of this experiment was to determine virus neutralizing titers in the collected mice sera (see Example 3) and to compare virus neutralizing titers of mice vaccinated with HA fusion constructs to the titers of mice vaccinated with the target HA antigen without additional element.

[0616] Hemagglutination Inhibition Assay (HI)

[0617] In a 96-well plate, the obtained sera were mixed with HA H1N1 antigen (A/California/07/2009 (H1N1); NIBSC) and red blood cells (4% erythrocytes; Lohmann Tierzucht). In the presence of HA neutralizing antibodies, an inhibition of hemagglutination of erythrocytes can be observed. The lowest level of titered serum that resulted in a visible inhibition of hemagglutination was the assay result. For the analysis of an antigen specific immune response in the effector phase, sera obtained at day 28 (one week after boost vaccination) were used. Vaccination was performed according to Example 3. The results are shown in FIG. 3.

[0618] Results:

[0619] The results show that potentially protective virus neutralizing titers (>40) were detected for mice vaccinated with the ferritin (2 out of 8 mice) and foldon (3 out of 8 mice) HA fusion constructs, indicating that vaccination with these constructs induced protective neutralizing antibodies in the effector phase. The HA vaccine without additional element and the HA vaccine with a C3d_P28 could not induce virus neutralizing titers.

[0620] Taken together, the addition of ferritin and foldon elements to HAo substantially increased the protective antibody titers in the effector phase, whereas the addition of a C3d_P28 element to HA.sub.TM had no positive effect.

Example 6: ELISA Analysis of an Antigen Specific Humoral Immune Response in the Memory Phase

[0621] Determination of Anti HA Protein Specific IgG1 and IgG2a Antibodies by ELISA:

[0622] ELISA was performed according to example 4. Vaccination was performed according to example 3. For the analysis of an antigen specific immune response in the memory phase, sera obtained at day 35 (two week after boost vaccination) and day 49 (four weeks after boost vaccination) were used. The results are shown in FIG. 4.

[0623] Results:

[0624] Assessment of the humoral immune response after immunizations revealed that 40 g of the respective mRNA vaccines induced strong HA specific IgG1 and IgG2a antibody titers for the HA.sub.TM-SGG-ferritin and the HA.sub.TM-foldon constructs. The HA.sub.TM vaccine (without additional element) and also the HA.sub.TM-C3d_P28 vaccine only induced IgG1 antibodies in few mice. Taken together, the addition of ferritin and foldon elements to HA.sub.TM substantially improved the induction of a HA specific humoral immune response in the memory phase, whereas the addition of a C3d_P28 element to HA.sub.TM had no positive effect.

Example 7: Hemagglutination Inhibition Assay (HI) to Determine Virus Neutralizing Titers in the Memory Phase

[0625] The HI assay was performed according to example 5. Vaccination was performed according to example 3. For the analysis virus neutralizing titers in the memory phase, sera obtained at day 49 (four weeks after boost vaccination) was used. The results are shown in FIG. 5.

[0626] Results:

[0627] The results show that potentially protective virus neutralizing titers (>40) were detected for mice vaccinated with the ferritin (4 out of 8 mice) and foldon (4 out of 8 mice) fusion HA mRNA constructs. As the measurement was performed 4 weeks after boost vaccination, the results suggest that the ferritin and foldon HA fusion constructs were able to induce protective neutralizing titers in the memory phase. Protective HI titers were not detected in HA.sub.TM-C3d_P28 and the HA.sub.TM vaccinated mice.

[0628] Taken together, the addition of ferritin and foldon elements to HA.sub.TM could substantially increase the protective antibody titers in the effector phase, whereas the addition of a C3d_P28 element to HA.sub.TM had no positive effect.

Example 8: Hemagglutination Inhibition Assay to Determine Functional Antibody Titers

[0629] The Hemagglutination inhibition assay (HI) assay was performed according to example 5. Vaccination was performed according to example 3 on day 0 and day 21. For the HI assay, sera obtained 14 days after boost vaccination were used (day 35). In the experiment, mice were vaccinated with HA.sub.TM-IgG1 FC and HA.sub.TM-GCN4pII-CD40L, both in combination with a membrane-bound HA. As control, mice were vaccinated only with the membrane-bound HA. The vaccination schemes as well as the used concentrations are provided in Table 4. The results are shown in FIG. 6.

TABLE-US-00004 TABLE 4 Vaccination regimen for indicated animal groups Number HA fusion construct membrane bound HA Vaccination Group of mice (20 g) (20 g) on day A 8 HA.sub.TM-IgG FC Membrane bound HA 0/21 B 8 HA.sub.TM-GCN4pII- Membrane bound HA 0/21 CD40L C 8 Membrane bound HA 0/21 D 8 RiLa buffer control 0/21

[0630] Results:

[0631] The results show that potentially protective antibody titers (>40) were detected for mice vaccinated with the HATM-IgG1 FC (6 out of 8 mice) and HATM-GCN4pII-CD40L (5 out of 8 mice) fusion mRNA constructs in combination with membrane-bound HA (Groups A and B). Compared with a single treatment with membrane bound HA vaccine (4 out of 8) this led to an increase in protective antibody titers (see FIG. 6).

Example 9: EPO Half-Life Extension Using EPO Fusion Constructs

[0632] 9.1. Explanation of the EPO Constructs:

[0633] For the present example, the target protein is the mice EPO protein (MmEPO; GI: 21389309; NM_007942.2; Uniprot ID P07321; SEQ ID NO: 1738). To extend the half life of the EPO protein, several elements that extend the half life of the protein are C-terminally fused to the EPO protein. The fusion constructs of the present example as well as the control constructs are listed with their respective SEQ ID NOs (fusion proteins and respective RNA coding sequences) in Table 5.

[0634] 9.2. Preparation of EPO DNA and mRNA Constructs

[0635] The DNA sequences encoding the target EPO (SEQ ID NO: 1771) fused to respective additional half life extension elements are prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence and/or codon optimized sequence for stabilization and optimized expression. Sequences were introduced into a vector and modified to additionally comprise stabilizing UTR sequences (3 UTR and 5 UTR), a histone-stem-loop structure, a poly-A stretch, and a poly-C stretch at the 3-terminal end.

[0636] The DNA constructs are used as templates for subsequent RNA in vitro transcription reactions (see Example 1). Subsequently, the in vitro transcribed mRNA is purified using PureMessenger (CureVac, Tubingen, Germany; WO2008/077592A1).

TABLE-US-00005 TABLE 5 Prepared EPO-fusion constructs SEQ ID NOs of SEQ ID NOs of RNA coding EPO-fusion sequences Protein construct description proteins (cds) MmEPO-CgB 1739 1772 MmEPO-Xten 1740 1773 MmEPO-PAS600 1741 1774 MmEPO-PAS200 1742 1775 MmEPO-HAP200 1743 1776 MmEPO-ELP 1744 1777 MmEPO-MmAlb(25-608) 1745 1778 MmEPO-HsAlb(25-609) 1746 1779 MmEPO-HsAlb(25-609_K597P) 1747 1780 MmEPO-linkerG4S-MmAlb(404-608) 1748 1781 MmEPO-ABP-SA21 1749 1782 MmEPO-SSG148_ABD_SpG_high 1750 1783 MmEPO-HsIgG1 1751 1784 MmEPO-MmIgG1 1752 1785 MmEPO-MmIgG2 1753 1786 MmEPO-MmTf(20-697) 1754 1787 MmEPO-HsTf(20-698) 1755 1788 MmEPO-Sa_SpA (121-270) 1756 1789 MmEPO-Hs_monoIgG1 1757 1790 MmEPO-Hs_2x-monoIgG1 1758 1791 MmEPO-IgBD 1759 1792 MmEPO-linkerG4S-IgBP 1760 1793 MmEPO-E-XTEN 1761 1794 MmEPO-ELP(420) 1762 1795 MmEPO-ABP SA15 1763 1796 MmEPO-ABD SPG 1764 1797 MmEPO-HsAlbDIII (P02768; 404-609) 1765 1798 MmEPO-monomeric Mm Fc 1766 1799 MmEPO-tandem monomeric MmFc 1767 1800 MmEPO-HsIgG2 1768 1801 MmEPO-MmIgG2b 1769 1802 MmEPO-HsIgG4 1770 1803

[0637] 9.3. Expression of EPO Constructs in HEK 293T Cells and HeLa Cells and Analysis Using Western blot

[0638] To characterize the expression of EPO mRNA constructs and to determine the release of the EPO protein into the supernatant of transfected HEK 293T cells and transfected HeLa cells, in vitro expression analysis is performed. A detailed description of the experimnets is provided below.

[0639] 9.3.1. Transfection of Cells

[0640] HEK 293T cells and HeLa cells are seeded in a 24-well plate at a density of 70,000 cells/well in cell culture medium 48h prior to transfection. Cells are transfected with 5 g naked, unformulated mRNA EPO constructs (see Table 4) using Lipofectamine 2000 (Invitrogen). As a control, full length EPO mRNA construct is used (without half-life extending element). 24 hours post transfection, cell culture supernatants are collected. Additionally, cells are harvested and lysed with RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% TritonX-100, 0.1% SDS) or harvested using SDS lysis buffer.

[0641] 9.3.2. Analysis of EPO Expression and Secretion Using Western Blot and EPO-ELISA

[0642] An SDS-PAGE is performed with supernatants and whole cell lysates from all samples with Mini-PROTEAN TGX Precast Mini Gels 4-20% (Gradient gel; Bio-Rad). Untransfected cells are used as a negative control. The blotting on a nitrocellulose membrane is performed for 2h in the presence of a blotting buffer. After blocking the membrane in a respective buffer, antibody incubation (primary and secondary antibodies) and signal detection (LI-COR measurement) is performed. The presence of EPO is analyzed using a commercially available anti EPO specific antibody in combination with a suitable IgG1 IRDye 800 coupled secondary antibody (LI-COR Biosciences). Additionally, EPO levels in the culture medium are quantitatively measured 24 hours post transfection using a commercially available mouse EPO ELISA kit (R&D Systems, Wiesbaden, Germany). The constructs showing suitable expression and secretion characteristics are used in in vivo experiments.

[0643] 9.3. In Vivo Characterization of TransIT Formulated EPO Constructs

[0644] To characterize the half-life of the generated EPO-fusion proteins, mRNA encoding said fusion proteins is formulated for in vivo application using TransIT and injected intraperitoneal or intravenously into female BALB/c mice in equimolar amounts. As control, EPO protein and TransIT formulated EPO mRNA (without half-life extending element) are used. 6 hours, 1 day, 4 days and 7 days after injection, a few microliters of blood are collected, heparinized, and centrifuged. EPO levels in the supernatant are measured using a mouse EPO ELISA kit (R&D Systems). In addition, reticulocytes are analysed using a commercially available Retic-COUNT kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer's instructions. Stained cells are analyzed on a FACS Canto (BD Biosciences). Reticulocyte levels are given as percentage of total red blood cells.